JP2023154669A - microscope - Google Patents

Abstract

To provide a microscope which can rapidly acquire an image of a high-quality sample.SOLUTION: A microscope includes: an illumination optical system for irradiating a sample with illumination light; a detection optical system for receiving detection light from the sample; and a phase modulation unit 60 arranged in the illumination optical system. The phase modulation unit 60 includes: a phase modulation element 65 having a plurality of irradiation regions which can be irradiated with illumination light, the phase modulation element providing a predetermined phase distribution of the illumination light according to the irradiation positions of the illumination light in the plurality of radiation regions; and a first phase modulation scanner 63 for changing the irradiation positions of the illumination light in the plurality of radiation regions.SELECTED DRAWING: Figure 3

Description

The present invention relates to a microscope.

BACKGROUND ART Microscopes have been proposed that are capable of correcting aberrations caused by a sample by changing the phase of light using a phase modulation element, and that are capable of obtaining high-quality images of the sample. Additionally, a microscope has been proposed that can obtain images of a sample in a short time by using multiple illumination lights to illuminate the sample (see, for example, Patent Document 1 and Patent Document 2). . As described above, microscopes are required to obtain high-quality images of samples in a short time.

Japanese Patent Application Publication No. 2013-34127 JP2021-89430A

The microscope according to the present invention includes an illumination optical system that irradiates a sample with illumination light from a light source, a detection optical system that receives detection light from the sample, and a detection optical system disposed in at least one of the illumination optical system and the detection optical system. The phase modulation unit has a plurality of irradiation areas that can be irradiated with at least one of the illumination light and the detection light, and the phase modulation unit has a plurality of irradiation areas that can be irradiated with at least one of the illumination light and the detection light; a phase modulation element that imparts a predetermined phase distribution to the at least one of the lights according to the irradiation position; and a first scanning unit that relatively moves the irradiation position and the plurality of irradiation areas. .

FIG. 1 is an explanatory diagram showing a microscope according to a first embodiment. FIG. 2 is an explanatory diagram showing an example of an image acquisition range. FIG. 2 is an explanatory diagram showing a phase modulation unit according to the first embodiment. FIG. 3 is an explanatory diagram showing a state in which the irradiation area of illumination light is changed in the phase modulation unit according to the first embodiment. FIG. 3 is an explanatory diagram showing an irradiation area of illumination light in the phase modulation unit according to the first embodiment. FIG. 3 is an explanatory diagram showing an observation area in the first embodiment. 3 is a flowchart showing the flow of a sample image acquisition method. It is a flowchart which shows the flow of processing which measures an aberration. It is a flow chart which shows the flow of processing which measures aberration concerning a modification. FIG. 7 is an explanatory diagram showing a modification of the phase modulation unit according to the first embodiment. It is an explanatory view showing a microscope concerning a 2nd embodiment. It is an explanatory view showing a microscope concerning a 3rd embodiment. It is an explanatory view showing a microscope concerning a 4th embodiment. It is an explanatory view showing a microscope concerning a 5th embodiment. It is an explanatory view showing a phase modulation unit concerning a 6th embodiment. FIG. 7 is an explanatory diagram showing an irradiation area of illumination light in a phase modulation unit according to a sixth embodiment. It is an explanatory view showing a modification of a phase modulation unit concerning a 6th embodiment. It is an explanatory view showing a microscope concerning an 11th embodiment. It is an explanatory view showing a phase modulation unit concerning an 11th embodiment. FIG. 7 is an explanatory diagram showing an irradiation area of illumination light in a phase modulation unit according to an eleventh embodiment. It is an explanatory view showing an observation field in an 11th embodiment. It is an explanatory view showing a modification of a phase modulation unit concerning an 11th embodiment. It is an explanatory view showing a phase modulation unit concerning a 12th embodiment. FIG. 9 is an explanatory diagram showing an irradiation area of illumination light in a phase modulation unit according to a twelfth embodiment. It is an enlarged view showing a part of irradiation area in a phase modulation unit concerning a 12th embodiment. FIG. 7 is an explanatory diagram regarding a partial region of an irradiation region in a phase modulation unit according to a twelfth embodiment. It is an explanatory view showing an observation field in a 12th embodiment. It is an explanatory view showing the 1st modification of the phase modulation unit concerning a 12th embodiment. It is an explanatory view showing the 2nd modification of the phase modulation unit concerning a 12th embodiment. It is an explanatory view showing the 3rd modification of the phase modulation unit concerning a 12th embodiment. It is an explanatory view showing the 4th modification of the phase modulation unit concerning a 12th embodiment. It is an explanatory view showing a phase modulation unit concerning a 16th embodiment. FIG. 7 is an explanatory diagram showing an irradiation area of illumination light in a phase modulation unit according to a sixteenth embodiment. FIG. 7 is an explanatory diagram regarding a partial region of an irradiation region in a phase modulation unit according to a sixteenth embodiment. It is an explanatory view showing the 1st modification of the phase modulation unit concerning a 16th embodiment. It is an explanatory view showing the 1st modification of the phase modulation unit concerning a 16th embodiment. It is an explanatory view showing a phase modulation unit concerning a 20th embodiment.

[First embodiment]
Hereinafter, a microscope according to each embodiment will be described. First, a microscope 1 according to a first embodiment will be described with reference to FIG. In the following description, the coordinate axis extending in the optical axis direction of the objective lens 28 of the microscope 1 will be referred to as the z-axis. Further, coordinate axes extending in directions orthogonal to each other in a plane perpendicular to the z-axis are referred to as an x-axis and a y-axis, respectively. The direction in which the z-axis extends may be referred to as the z direction, the direction in which the x-axis extends may be referred to as the x-direction, and the direction in which the y-axis extends may be referred to as the y-direction. Note that the +z direction is an upward direction in the case of an inverted microscope, and a downward direction in the case of an upright microscope. Further, a position in the z direction may be referred to as a z position, a position in the x direction may be referred to as an x position, and a position in the y direction may be referred to as a y position.

The microscope 1 according to the first embodiment is also referred to as a scanning microscope. The microscope 1 according to the first embodiment includes a stage 11, a light source unit 16, an illumination optical system 21, a detection optical system 31, a detector 41, a wavefront sensor 46, a calculation device 51, and a storage section 52. , an interface section 53, and a microscope control section 54. The stage 11 is formed into a plate shape with an opening in the center. The stage 11 supports a sample TP to be observed via a holding member CG such as a cover glass. Examples of the sample TP include a biological sample (not shown), beads (not shown), and the like. A biological sample is a thick sample such as a cell or a biological tissue. The beads are minute spheres (for example, about 0.2 μm in diameter) made of polystyrene. The sample TP may be stained with a fluorescent dye or the like. Furthermore, fluorescently dyed beads or metal particles may be introduced into the sample TP.

The stage 11 is provided with a stage drive section 12 . The stage drive unit 12 is configured using an electric motor, a piezo element, or the like. The stage drive unit 12 moves the stage 11 in a plane (in the xy plane) perpendicular to the z-axis (optical axis of the objective lens 28). By moving the stage 11 in a plane perpendicular to the z-axis by the stage drive unit 12, it is possible to acquire a wide-range image of the sample TP supported by the stage 11 via the holding member CG. Note that the stage drive unit 12 may move the stage 11 in the z direction.

The light source unit 16 emits illumination light (excitation light) for exciting the fluorescent substance contained in the sample TP. The light source unit 16 includes a light source 17 and a shutter 18. As the light source 17, for example, a laser light source that can emit laser light (excitation light) in a predetermined wavelength range as illumination light is used. The shutter 18 is configured to either pass the illumination light emitted from the light source 17 or block it.

The illumination optical system 21 illuminates the sample TP with illumination light (excitation light) emitted from the light source unit 16. The illumination optical system 21 includes, in order from the light source unit 16 side, a collimator lens 22, a phase modulation unit 60, a dichroic mirror 24, a sample scanning section 25, a scan lens 26, a second objective lens 27, and an objective lens 28. and has. The collimator lens 22 converts the illumination light emitted from the light source unit 16 into substantially parallel light. The phase modulation unit 60 performs phase modulation on the illumination light emitted from the light source unit 16, and corrects at least part of the aberrations generated in the sample TP, the illumination optical system 21, and the detection optical system 31. The dichroic mirror 24 has a characteristic that the illumination light (excitation light) emitted from the light source unit 16 is transmitted therethrough, and light in a predetermined wavelength band (for example, fluorescence) of the light generated by the sample TP on the stage 11 is reflected. have Note that the dichroic mirror 24 may be configured so that the illumination light emitted from the light source unit 16 is reflected and light in a predetermined wavelength band among the light generated by the sample TP is transmitted.

The sample scanning unit 25 is arranged at a position optically conjugate with the pupil position of the objective lens 28 or in the vicinity thereof. The sample scanning unit 25 is configured using a mirror (for example, a galvano mirror, a resonant mirror, etc.) that is held so that the direction (azimuth) of a reflecting surface changes. The sample scanning unit 25 changes the traveling direction of the illumination light reflected by the reflecting surface by changing the direction of the reflecting surface. Thereby, the sample scanning section 25 scans the sample TP using the illumination light from the light source unit 16 in two directions, the x direction and the y direction. Note that the sample scanning unit 25 is configured using not only a mirror but also a transmissive deflector such as an acousto-optic deflector (AOD) or an electro-optic crystal (KTN crystal) to change the traveling direction of the illumination light. You can do it like this.

The scan lens 26 collects the illumination light from the light source unit 16. The second objective lens 27 converts the illumination light from the light source unit 16 into substantially parallel light again. The objective lens 28 is arranged near the bottom of the stage 11. The objective lens 28 faces the sample TP on the stage 11 via the opening of the stage 11 and a holding member CG such as a cover glass. The gap between the objective lens 28 and the holding member CG may be filled with the immersion liquid IM, or may be filled with a gas such as air.

Further, the objective lens 28 is attached to the casing (not shown) of the microscope 1 via an objective lens holder 29. The objective lens holder 29 is configured using, for example, a drive device (not shown) such as an electric motor. The objective lens holder 29 moves the objective lens 28 up and down in the z direction together with a revolver (not shown). When the objective lens 28 is moved in the z direction by the objective lens holder 29, the relative position of the objective lens 28 with respect to the sample TP changes, and the sample TP
The focal position of the objective lens 28 relative to the object changes in the z direction. By displacing the focal position of the objective lens 28 in the z-direction by the objective lens holder 29, it is possible to acquire images of cross sections inside the sample TP at different positions in the z-direction. Hereinafter, images of multiple cross sections of the sample TP at different positions in the z direction will also be referred to as "z stack images." Note that by moving the stage 11 in the z-direction using the stage drive unit 12, the focal position of the objective lens 28 may be displaced in the z-direction to obtain a z-stack image.

FIG. 2 is a diagram schematically showing an example of a z-stack image of the sample TP. FIG. 2 shows a first two-dimensional image TI1 of the xy section of the sample TP, a second two-dimensional image TI2 of the xy section of the sample TP whose z position is different from the first two-dimensional image TI1, and a first two-dimensional image TI1 of the xy section of the sample TP. A third two-dimensional image TI3 of the xy section of the sample TP whose z position is different from the two-dimensional image TI1 and the second two-dimensional image TI2 is shown. In this way, the first two-dimensional image TI1, the second two-dimensional image TI2, and the third two-dimensional image TI3 are two-dimensional images of the xy cross section of the sample TP at different z positions. The z-stack image includes first to third two-dimensional images TI1 to TI3, that is, two-dimensional images of the xy cross section at different z positions of the sample TP. Note that the number of two-dimensional images is not limited to three as shown in FIG. 2, but may be any number.

The detection optical system 31 receives light generated by the sample TP as detection light. The detection optical system 31 includes, in order from the sample TP side, an objective lens 28, a second objective lens 27, a scan lens 26, a sample scanning section 25, and a dichroic mirror 24. Further, the detection optical system 31 includes, in order from the dichroic mirror 24 side, a detection filter 32, a condensing lens 33, an insertion/removal mirror 34, and a detection pinhole plate 35. Further, the detection optical system 31 includes a measurement pinhole plate 36 and a measurement relay lens 37. In this way, the objective lens 28, the second objective lens 27, the scan lens 26, the sample scanning section 25, and the dichroic mirror 24 are included in both the illumination optical system 21 and the detection optical system 31.

The detection filter 32 transmits light in a predetermined wavelength band (for example, fluorescence) among the light generated by the sample TP. The detection filter 32 blocks at least a portion of, for example, illumination light reflected by the sample TP, external light, stray light, and the like. The condensing lens 33 condenses the detection light from the sample TP. The insertion/removal mirror 34 is arranged to be removable from the optical path between the condenser lens 33 and the detection pinhole plate 35 . When the insertion/removal mirror 34 is removed from the optical path between the condenser lens 33 and the detection pinhole plate 35, the detection light from the sample TP reaches the detection pinhole plate 35. When the insertion/removal mirror 34 is inserted into the optical path between the condenser lens 33 and the detection pinhole plate 35, the detection light from the sample TP is reflected by the insertion/removal mirror 34 and passes through the measurement pinhole plate 36. reach

The detection pinhole plate 35 is formed into a plate shape with a pinpole (not shown) in the center, and is arranged at a position optically conjugate with the sample TP. The detector 41 detects the detection light that has passed through the detection pinhole plate 35. As the detector 41, for example, a photomultiplier tube, a photodiode, an avalanche photodiode, or the like is used.

The measurement pinhole plate 36 is formed into a plate shape having a pinpole (not shown) in the center, and is arranged at a position optically conjugate with the sample TP. The measurement relay lens 37 converts the detection light from the sample TP into substantially parallel light. The wavefront sensor 46 measures the aberration of the wavefront of the detection light from the sample TP. As the wavefront sensor 46, for example, a Shack-Hartmann wavefront sensor or the like is used.

The arithmetic device 51 includes a CPU (Central Processing Unit), and controls the microscope 1 including the microscope control unit 54 based on a program stored in the storage unit 52. The storage unit 52 includes a storage medium such as a memory element or a hard disk, and temporarily stores signal data of the detection light detected by the detector 41 in addition to the above-mentioned program.

The interface section 53 includes an input section (not shown) that can be operated by a user and includes at least one of a mouse, a keyboard, a touch pad, a trackball, etc., and a display section (not shown) such as a liquid crystal display. . The input section of the interface section 53 detects an operation by the user and outputs the detection result to the arithmetic device 51 as input data input by the user. The arithmetic unit 51 causes a GUI (graphical user interface) necessary for operating the microscope 1 and an image of the sample TP generated by the microscope control unit 54 to be displayed on the display unit. The interface unit 53 communicates data with a server or the like located outside the microscope 1 via a network line.

The microscope control unit 54 transmits a control signal to the sample scanning unit 25 to control the traveling direction of the illumination light and detection light that are changed by the sample scanning unit 25. The microscope control unit 54 transmits a control signal to the stage drive unit 12 to control the position of the stage 11 driven by the stage drive unit 12. The microscope control section 54 transmits a control signal to the objective lens holding section 29 to control the position of the objective lens 28 held by the objective lens holding section 29 . The microscope control unit 54 transmits a control signal to the shutter 18 to control opening and closing of the shutter 18. The microscope control unit 54 transmits a control signal to the insertion/removal mirror 34 to control insertion/removal of the insertion/removal mirror 34 .

Further, the microscope control unit 54 generates a two-dimensional image of the sample TP based on the detection signal transmitted from the detector 41. The microscope control unit 54 corrects at least one of the aberrations generated in the sample TP, the illumination optical system 21, and the detection optical system 31 based on the detection signal transmitted from the detector 41 and the measurement signal transmitted from the wavefront sensor 46. Get data about the department. The microscope control unit 54 transmits a control signal to the phase modulation unit 60 to control phase modulation of the illumination light by the phase modulation unit 60.

Illumination light (excitation light) emitted from the light source 17 of the light source unit 16 passes through the collimator lens 22 of the illumination optical system 21 and becomes substantially parallel light. The illumination light transmitted through the collimator lens 22 enters the phase modulation unit 60. When the illumination light enters the phase modulation unit 60, the phase modulation unit 60 performs phase modulation on the input illumination light and emits the phase modulated illumination light. The substantially parallel illumination light emitted from the phase modulation unit 60 enters the dichroic mirror 24 . The illumination light that has entered the dichroic mirror 24 passes through the dichroic mirror 24 and enters the sample scanning section 25 . The illumination light that has entered the sample scanning section 25 passes through the sample scanning section 25, passes through the scan lens 26 and the second objective lens 27, and enters the objective lens 28. The illumination light incident on the objective lens 28 passes through the objective lens 28 and is focused on the focal plane of the objective lens 28. A portion of the sample TP where the illumination light is focused (that is, a portion that overlaps with the focal plane of the objective lens 28) is two-dimensionally scanned in two directions, the x direction and the y direction, by the sample scanning unit 25.

Fluorescence is emitted as detection light from a fluorescent substance contained in a portion of the sample TP where illumination light (excitation light) is focused. Note that the detection light is not limited to fluorescence, and may be, for example, scattered light or reflected light. The detection light may be a light wave that exhibits a nonlinear response to the intensity of the illumination light, such as fluorescence emitted by multiphoton excitation, second harmonics, third harmonics, etc. good. Alternatively, waves other than light generated from the sample may be detected; for example, acoustic waves generated from the sample may be detected by a transducer placed facing the sample via an immersion liquid or the like. Good too. Since the detection light generated from the sample is the same as that described in all embodiments hereinafter, a description thereof will be omitted.

Detection light (fluorescence) generated by the sample TP enters an objective lens 28 as a detection optical system 31. The detection light incident on the objective lens 28 is transmitted through the objective lens 28 and is passed through the second objective lens 28.
7 and the scan lens 26 and enters the sample scanning section 25. The detection light incident on the sample scanning section 25 passes through the sample scanning section 25 and enters the dichroic mirror 24 . Since the detection light (fluorescence) that has entered the dichroic mirror 24 has a different wavelength from the illumination light (excitation light), it is reflected by the dichroic mirror 24 and reaches the detection filter 32 . The fluorescence that has reached the detection filter 32 passes through the detection filter 32 and is transmitted through the condenser lens 33 .

When the insertion/removal mirror 34 is removed from the optical path between the condenser lens 33 and the detection pinhole plate 35, the detection light transmitted through the condenser lens 33 reaches the detection pinhole plate 35. The detection light collected at a position conjugate with the focal position of the objective lens 28 passes through the detection pinhole plate 35 and enters the detector 41 . The detector 41 performs photoelectric conversion of the light (detection light) that has entered the detector 41, and generates data corresponding to the amount of light (brightness) of the light as a light detection signal. The detector 41 transmits the generated data to the microscope control section 54. The microscope control unit 54 performs a process of integrating the data transmitted from the detector 41 at predetermined sampling intervals to form one pixel worth of data, and arranging the data in synchronization with the two-dimensional scanning by the sample scanning unit 25. As a result, one image data in which data for a plurality of pixels are arranged two-dimensionally (in two directions) is generated and stored in the storage unit 52. In this way, the microscope control unit 54 generates a two-dimensional image of the sample TP, thereby acquiring an image of the sample TP. Hereinafter, the two-dimensional image generation processing by the microscope control unit 54 described in all the embodiments is the same, so the explanation will be omitted.

Note that since the sample TP is scanned by the illumination light passing through the sample scanning section 25, the detection light generated at the sample TP may deviate from the optical axis AX2 of the detection optical system 31. However, when the detection light generated by the sample TP passes through the objective lens 28, the second objective lens 27, and the scan lens 26 and passes through the sample scanning section 25, it proceeds along the optical axis AX2 of the detection optical system 31. Become. Hereinafter, the process in which light that has deviated from the optical axis travels along the optical axis may be referred to as descanning. Despite the fact that the traveling direction of the illumination light is changed by the sample scanning unit 25 due to the descanning of the detection light in the sample scanning unit 25, the detection light that has passed through the condenser lens 33 is focused at the position of the detection pinhole plate 35. The light can pass through the detection pinhole plate 35. However, light generated from a portion of the sample TP that is shifted in the z direction from the condensing position of the illumination light (that is, the focal position of the objective lens 28) cannot pass through the detection pinhole plate 35. Therefore, in this embodiment, even when detecting detection light that responds linearly to the intensity of illumination light, such as reflected light and scattered light, it is necessary to It is possible to remove the light generated from the part.

When the removable mirror 34 is inserted into the optical path between the condenser lens 33 and the detection pinhole plate 35, the detection light transmitted through the condenser lens 33 is reflected by the removable mirror 34 and passes through the measurement pin. It reaches the hole plate 36. The detection light condensed at a position conjugate with the focal position of the objective lens 28 passes through the measurement pinhole plate 36, passes through the measurement relay lens 37, and enters the wavefront sensor 46. The wavefront sensor 46 measures the aberration of the wavefront of the detection light incident on the wavefront sensor 46, and transmits data regarding the wavefront aberration as a measurement signal to the microscope control unit 54. Based on the measurement signal transmitted from the wavefront sensor 46, the microscope control unit 54 acquires data regarding at least part of the aberrations occurring in the sample TP, the illumination optical system 21, and the detection optical system 31.

Note that even though the traveling direction of the illumination light is changed by the sample scanning unit 25 due to the descanning of the detection light in the sample scanning unit 25, the detection light that has passed through the condenser lens 33 remains at the position of the measurement pinhole plate 36. The light can be focused and passed through the measurement pinhole plate 36. However, light generated from a portion of the sample TP that is shifted in the z direction from the focal point of the illumination light cannot pass through the measurement pinhole plate 36. Therefore, as described above, it is possible to remove the so-called background light, which is light generated from areas other than the condensing position of the illumination light. The measurement pinhole plate 36 also functions as a low-pass filter, and by blocking higher-order aberration components of the wavefront of the detection light, it is possible to improve the measurement accuracy of the wavefront aberration by the wavefront sensor 46.

In the microscope 1 according to the first embodiment, a beam splitter (not shown) is provided in place of the insertion/removal mirror 34, and a portion of the detection light incident on the beam splitter is transmitted through the beam splitter to the detector 41. The other part of the detection light that reaches the beam splitter and enters the beam splitter may be configured so that it is reflected by the beam splitter and reaches the wavefront sensor 46.

In the microscope 1 according to the first embodiment, the collimator lens 22, the scan lens 26, the second objective lens 27, the objective lens 28, the condenser lens 33, and the measurement relay lens 37 may be composed of a plurality of lenses. , and may include a reflecting mirror. Hereinafter, regardless of the configuration of the microscope according to all embodiments, the lens included in the microscope may be configured from a plurality of lenses or may be configured to include a reflecting mirror, so a description thereof will be omitted. . Furthermore, in the microscope 1 according to the first embodiment, the light source unit 16, the illumination optical system 21, the detection optical system 31, the detector 41, and the wavefront sensor 46 are arranged below the stage 11 and the sample TP. However, it may be placed above the stage 11 and the sample TP. Hereinafter, each part of the microscope according to all embodiments may be arranged above the stage 11 and the sample TP, so a description thereof will be omitted.

[Configuration of phase modulation unit]
Next, the phase modulation unit 60 according to the first embodiment will be described with reference to FIG. 3. As shown in FIG. 3, the phase modulation unit 60 according to the first embodiment includes a first relay lens 61, a first mirror 62, a first phase modulation scanner 63, a second relay lens 64, and a phase modulation unit 60. It includes an element 65, a second phase modulation scanner 66, a second mirror 67, and a third relay lens 68. The first relay lens 61 collects the illumination light that has passed through the collimator lens 22 and has become substantially parallel light. The first mirror 62 reflects the illumination light from the first relay lens 61 toward the first phase modulation scanner 63 .

The first phase modulation scanner 63 is arranged at a position B1 that is optically conjugate with the sample TP. The first phase modulation scanner 63 is configured using a mirror (for example, a galvano mirror, a resonant mirror, etc.) held so that the direction (azimuth angle) of the reflecting surface changes by uniaxial or biaxial rotation. . The first phase modulation scanner 63 changes the traveling direction of the illumination light reflected by the first mirror 62. Thereby, the first phase modulation scanner 63 scans the phase modulation element 65 with the illumination light from the collimator lens 22, as shown in FIGS. 3 and 4. Note that the first phase modulation scanner 63 is not limited to a mirror, but may be configured using a transmissive deflector such as an acousto-optic deflector (AOD) or an electro-optic crystal (KTN crystal), and may be configured using a transmission type deflector such as an acousto-optic deflector (AOD) or an electro-optic crystal (KTN crystal), may be changed. Note that the same applies to the phase modulation scanners described in all embodiments hereinafter, so the explanation will be omitted.

The second relay lens 64 is arranged facing the phase modulation element 65. The second relay lens 64 converts the illumination light that has passed through the first phase modulation scanner 63 into substantially parallel light. The second relay lens 64 collects the illumination light reflected by the phase modulation element 65.

The phase modulation element 65 is arranged at a position H1 that is optically conjugate with the pupil position of the objective lens 28. The phase modulation element 65 is configured using, for example, a reflective liquid crystal element or a transmissive liquid crystal element, and displays a plurality of phase modulation patterns arranged vertically and horizontally or in any one direction on the display surface. In each embodiment, a phase pattern, a compensation wavefront, etc. (phase distribution) applied to illumination light or detection light is referred to as a phase modulation pattern. Note that the phase modulation element 65 may be configured using a spatial phase modulator (SLM), a MEMS (micro electro mechanical system)-SLM, or a deformable mirror. Note that the same applies to the phase modulation elements described in all the embodiments hereinafter, so the explanation will be omitted. Phase modulation element 6
The display surface 5 is provided with a plurality of irradiation areas that can be irradiated with illumination light. The plurality of irradiation areas on the display surface of the phase modulation element 65 are arranged in parallel in the vertical and horizontal directions or in any one direction corresponding to the plurality of phase modulation patterns.

FIG. 5 shows an example of arrangement of a plurality of irradiation areas. In the example of FIG. 5, 16 square-shaped irradiation areas that can be irradiated with illumination light are provided corresponding to 16 phase modulation patterns displayed on the display surface of the phase modulation element 65. Note that the number of phase modulation patterns and the number of irradiation areas of the phase modulation element 65 can be arbitrarily determined depending on the sample TP. Note that the number of phase modulation patterns and the number of irradiation areas are the same in all embodiments hereinafter, and therefore their explanation will be omitted. Among the 16 irradiation areas, the first row (from the top of FIG. 5) includes, in order from the left, a first irradiation area SA1, a second irradiation area SA2, a third irradiation area SA3, and a fourth irradiation area SA4. and is provided. Among the 16 irradiation areas, the second row (from the top of FIG. 5) includes, in order from the left, a fifth irradiation area SA5, a sixth irradiation area SA6, a seventh irradiation area SA7, and an eighth irradiation area SA8. and is provided. Among the 16 irradiation areas, in the third row (from the top of FIG. 5), from the left side, there are 9th irradiation area SA9, 10th irradiation area SA10, 11th irradiation area SA11, and 12th irradiation area SA12. and is provided. Among the 16 irradiation areas, in the fourth row (from the top of FIG. 5), from the left side, there are 13th irradiation area SA13, 14th irradiation area SA14, 15th irradiation area SA15, and 16th irradiation area SA16. and is provided.

The first to sixteenth irradiation areas SA1 to SA16 are arranged side by side so as not to overlap each other. When any one of the first to sixteenth irradiation areas SA1 to SA16 is irradiated with illumination light, the phase of the illumination light changes (modulates). Specifically, the illumination light is given a phase distribution according to a phase modulation pattern set in the irradiation area irradiated with the illumination light. In addition, by changing the irradiation area (that is, phase modulation pattern) on which the illumination light is irradiated in the phase modulation element 65, the phase distribution imparted to the illumination light (when the detection light is irradiated to the irradiation area, the phase distribution imparted to the detection light phase distribution).

The second phase modulation scanner 66 is arranged at a position B1 that is optically conjugate with the sample TP. The second phase modulation scanner 66 is configured similarly to the first phase modulation scanner 63. The second phase modulation scanner 66 is configured such that the illumination light from the second relay lens 64 travels toward the second mirror 67, that is, the illumination light from the second relay lens 64 is aligned with the optical axis AX1 of the illumination optical system 21. The direction of illumination light is changed so that the illumination light travels along the same direction. The second mirror 67 reflects the illumination light that has passed through the second phase modulation scanner 66 toward the third relay lens 68 . The third relay lens 68 converts the illumination light reflected by the second mirror 67 into substantially parallel light. Note that instead of the first phase modulation scanner 63 and the second phase modulation scanner 66, a single phase modulation scanner may be used.

The illumination light that has passed through the collimator lens 22 of the illumination optical system 21 enters the first relay lens 61 of the phase modulation unit 60 . The illumination light that has entered the first relay lens 61 is transmitted through the first relay lens 61 and reflected by the first mirror 62 . The illumination light reflected by the first mirror 62 enters the first phase modulation scanner 63 and is once focused at a position B1 that is optically conjugate with the sample TP. The first phase modulation scanner 63 scans the phase modulation element 65 with the illumination light that has entered the first phase modulation scanner 63 . The illumination light that has entered the first phase modulation scanner 63 passes through the first phase modulation scanner 63 and is transmitted through the second relay lens 64 . The illumination light transmitted through the second relay lens 64 becomes substantially parallel light, and is irradiated onto one of the plurality of irradiation areas in the phase modulation element 65.

When the illumination light is irradiated to any one of the plurality of irradiation areas (for example, the first to 16th irradiation areas SA1 to SA16) in the phase modulation element 65, the illumination light is set to the irradiation area that has been irradiated. The phase distribution of the illumination light changes (modulates) according to the phase modulation pattern (phase distribution) created by the illumination light. The illumination light irradiated onto any one of the plurality of irradiation areas in the phase modulation element 65 is reflected at the irradiation area. Note that the first phase modulation scanner 63 scans the phase modulation element 65 with the illumination light incident on the first phase modulation scanner 63, so that the illumination light irradiates within the plurality of irradiation areas in the phase modulation element 65. It is possible to change the irradiated area.

The illumination light that is irradiated onto any one of the plurality of irradiation areas in the phase modulation element 65 and reflected is transmitted through the second relay lens 64 . The illumination light that has passed through the second relay lens 64 enters the second phase modulation scanner 66 and is once focused at a position B1 that is optically conjugate with the sample TP. The second phase modulation scanner 66 is configured such that the illumination light from the second relay lens 64 travels toward the second mirror 67, that is, the illumination light from the second relay lens 64 is aligned with the optical axis AX1 of the illumination optical system 21. The direction of illumination light is changed so that the illumination light travels along the same direction. The illumination light incident on the second phase modulation scanner 66 passes through the second phase modulation scanner 66 and is reflected by the second mirror 67 . The illumination light reflected by the second mirror 67 passes through the third relay lens 68, becomes substantially parallel light, and enters the dichroic mirror 24 of the illumination optical system 21. In this way, the phase modulation unit 60 performs phase modulation on the illumination light.

Note that since the phase modulation element 65 is scanned by the illumination light passing through the first phase modulation scanner 63, the illumination light reflected from any one of the plurality of irradiation areas in the phase modulation element 65 is transmitted to the illumination optical system. 21 may deviate from the optical axis AX1. However, when the illumination light reflected from any one of the plurality of irradiation areas passes through the second relay lens 64 and passes through the second phase modulation scanner 66, the illumination light is reflected along the optical axis AX1 of the illumination optical system 21. You will be able to move forward. By descanning the illumination light in the second phase modulation scanner 66, the illumination light transmitted through the third relay lens 68 even though the first phase modulation scanner 63 changes the traveling direction of the illumination light and changes the irradiation area. can be incident on the dichroic mirror 24 of the illumination optical system 21 as substantially parallel light.

As described above, the first phase modulation scanner 63 scans the phase modulation element 65 with the illumination light incident on the first phase modulation scanner 63, thereby illuminating the plurality of irradiation areas of the phase modulation element 65. It is possible to change the irradiation area where the light is irradiated. The first phase modulation scanner 63 changes the irradiation area onto which the illumination light is irradiated in the phase modulation element 65 when the observation area on the sample TP where microscopic observation is performed is displaced. Note that a phase modulation pattern for correcting at least part of the aberrations generated between the sample TP, the illumination optical system 21, and the detection optical system 31, that is, the sample TP and the illumination optical system 21, is created for each of the plurality of observation areas on the sample TP. A predetermined phase distribution for correcting at least part of the aberrations generated in the detection optical system 31 is determined in advance. The phase modulation element 65 displays, in each irradiation area on the display surface of the phase modulation element 65, a phase modulation pattern determined in advance for the corresponding observation area. As a result, the first phase modulation scanner 63 changes the irradiation area in the phase modulation element 65 for each observation area, so that the phase of the illumination light is adjusted according to the phase modulation pattern (phase distribution) appropriately determined for each observation area. can be changed (modulated). Therefore, it is possible to obtain a high-quality image of the sample TP in a short time, in which at least some of the aberrations caused by the sample TP, the illumination optical system 21, and the detection optical system 31 are corrected.

FIG. 6 shows an example of the arrangement of a plurality of observation areas in the sample TP. In the example of FIG. 6, a plurality of observation areas (for example, 16 observation areas) in the sample TP are set within the field of view of the microscope 1. Among the 16 observation areas, in the first row (from the top of FIG. 6), from the left side, there are a first observation area KA1, a second observation area KA2, a third observation area KA3, and a fourth observation area KA4. is set. Among the 16 observation areas, in the second row (from the top of FIG. 6), from the left side, there are a fifth observation area KA5, a sixth observation area KA6, a seventh observation area KA7, and an eighth observation area KA8. is set. Among the 16 observation areas, in the third row (from the top of FIG. 6), from the left side, there are a 9th observation area KA9, a 10th observation area KA10, an 11th observation area KA11, and a 12th observation area KA12. is set. Among the 16 observation areas, in the fourth row (from the top of FIG. 6), from the left side, there are a 13th observation area KA13, a 14th observation area KA14, a 15th observation area KA15, and a 16th observation area KA16. is set. Note that the number of observation areas can be arbitrarily determined depending on the sample TP. Note that the number of observation areas is the same in all embodiments hereinafter, so the explanation will be omitted.

In the example shown in FIGS. 5 and 6, the phase modulation element 65 displays, in the first irradiation area SA1 of the phase modulation element 65, a phase modulation pattern determined in advance for the first observation area KA1. The phase modulation element 65 displays, in the second irradiation area SA2 of the phase modulation element 65, a phase modulation pattern determined in advance for the second observation area KA2. The phase modulation element 65 displays a phase modulation pattern previously determined for the third observation area KA3 in the third irradiation area SA3 of the phase modulation element 65. The phase modulation element 65 displays, in the fourth irradiation area SA4 of the phase modulation element 65, a phase modulation pattern determined in advance for the fourth observation area KA4. Similarly, the phase modulation element 65 displays phase modulation patterns previously determined for the fifth to sixteenth observation areas KA5 to KA16 in the fifth to sixteenth irradiation areas SA5 to SA16 of the phase modulation element 65.

When observing the first to sixteenth observation areas KA1 to KA16 in the sample TP, that is, when acquiring images of the first to sixteenth observation areas KA1 to KA16, first, an image of the first observation area KA1 is acquired. When acquiring an image of the first observation area KA1, the first phase modulation scanner 63 of the phase modulation unit 60 changes the irradiation area of the illumination light onto the phase modulation element 65 to the first irradiation area SA1. The second phase modulation scanner 66 operates in synchronization with the first phase modulation scanner 63, and directs the traveling direction of the illumination light LA1 irradiated onto the first irradiation area SA1 and reflected to the optical axis AX1 of the illumination optical system 21. in the same direction. The sample scanning unit 25 of the illumination optical system 21 performs raster scanning in the first observation area KA1 using the illumination light LA1 irradiated onto the first irradiation area SA1 of the phase modulation element 65. Therefore, it is possible to obtain a high-quality image of the first observation area KA1 in which aberrations caused by the first observation area KA1 in the sample TP are corrected.

After acquiring an image of the first observation area KA1, an image of the second observation area KA2 is acquired. When acquiring an image of the second observation area KA2, the first phase modulation scanner 63 of the phase modulation unit 60 changes the irradiation area of the illumination light onto the phase modulation element 65 from the first irradiation area SA1 to the second irradiation area SA2. . The second phase modulation scanner 66 operates in synchronization with the first phase modulation scanner 63, and directs the traveling direction of the illumination light LA2 irradiated onto the second irradiation area SA2 and reflected to the optical axis AX1 of the illumination optical system 21. in the same direction. The sample scanning unit 25 of the illumination optical system 21 performs raster scanning in the second observation area KA2 using the illumination light LA2 irradiated onto the second irradiation area SA2 of the phase modulation element 65. Therefore, it is possible to obtain a high-quality image of the second observation area KA2 in which aberrations caused by the second observation area KA2 in the sample TP are corrected.

Similarly, after acquiring the image of the second observation area KA2, images of the third to sixteenth observation areas KA3 to KA16 are acquired in order. Note that when the observation area in the sample TP is displaced to the third to sixteenth observation areas KA3 to KA16, the first phase modulation scanner 63 of the phase modulation unit 60 shifts the irradiation area of the illumination light onto the phase modulation element 65 to the third to sixteenth observation areas KA3 to KA16. The irradiation area is changed to the irradiation area SA3 to the 16th irradiation area SA16. This makes it possible to obtain a high-quality image of the sample TP in a short time, in which aberrations caused by the first to sixteenth observation areas KA1 to KA16 in the sample TP are individually corrected.
[Image acquisition method]

Next, a method for acquiring an image of the sample TP using the microscope 1 will be described with reference to FIG. FIG. 7 is a flowchart showing the flow of the method for acquiring an image of the sample TP. To acquire an image of the sample TP, first, image acquisition conditions for the sample TP are set (step ST10). In the process of setting image acquisition conditions, the computing device 51 displays a screen such as a GUI on the display unit (not shown) of the interface unit 53 to prompt the user to set the image acquisition range IR (see FIG. 2). Display. The image acquisition range IR is a range in which images of the sample TP are acquired. For example, as shown in FIG. 2, the image acquisition range IR includes a range Rx in the x direction, a range Ry in the y direction, and a range Rz in the z direction.

When the user inputs a provisional image acquisition range into the input section (not shown) of the interface section 53, the microscope control section 54 transmits a control signal to the shutter 18 and controls the shutter 18 to open. The microscope control unit 54 also sends a control signal to the stage drive unit 12 based on the provisional image acquisition range input to the input unit, and controls the stage drive unit 12 to move the stage 11 to a predetermined position. I do. The microscope control unit 54 transmits a control signal to the sample scanning unit 25 to control the traveling direction of the illumination light and detection light that are changed by the sample scanning unit 25.

At this time, as described above, the portion of the sample TP where the illumination light is focused is two-dimensionally scanned in two directions, the x direction and the y direction, by the sample scanning unit 25. Detection light (fluorescence) generated by the sample TP enters the detector 41 via the detection optical system 31. The detector 41 performs photoelectric conversion of the light (detection light) that has entered the detector 41 and transmits a light detection signal to the microscope control unit 54 . The microscope control unit 54 generates a two-dimensional image of the sample TP based on the detection signal transmitted from the detector 41, and stores data of the generated two-dimensional image in the storage unit 52. At this time, the microscope control section 54 transmits a control signal to the objective lens holding section 29 to control the objective lens holding section 29 to displace the focal position of the objective lens 28 in the z direction. Thereby, the microscope control unit 54 generates two-dimensional images of a plurality of cross sections of the sample TP at different positions in the z direction, that is, a z stack image. For example, as shown in FIG. 2, a z-stack image including first to third two-dimensional images TI1 to TI3 at different positions in the z-direction is displayed on the display section of the interface section 53.

The user uses the GUI while viewing the z-stack image of the sample TP displayed on the display section of the interface section 53 to input the x and y positions of the stage 11 and the objective lens 28 into the input section of the interface section 53. Input an instruction to move the focal position and set the desired image acquisition range IR. Further, the computing device 51 causes the display unit of the interface unit 53 to display a screen such as a GUI that prompts the user to set the number of times to acquire two-dimensional images or z-stack images and the time interval for acquiring images. Using the GUI, the user inputs and sets the number of times the two-dimensional image or z-stack image is to be acquired and the time interval for acquiring the images in the input section of the interface unit 53.

Note that, as a method of setting the image acquisition range IR, for example, the range in the x direction and the range in the y direction of the image acquisition range IR is specified by the number of pixels in the image, and the scan pitch, which is the pitch of pixels in the image, is specified. Thus, the range Rx in the x direction and the range Ry in the y direction of the image acquisition range IR may be set. For example, input the number of two-dimensional images of multiple cross-sections of the sample TP at different positions in the z-direction, which constitute the z-stack image to be acquired, and the distance in the z-direction between the two-dimensional images of the multiple cross-sections. By doing so, the range Rz in the z direction of the image acquisition range IR may be set. Note that the method of setting the image acquisition range IR is not limited to this; for example, by specifying the lengths of the rectangular parallelepiped area in which the image of the sample TP is to be acquired in the x direction, y direction, and z direction, An image acquisition range IR may be set.

Next, an aberration measurement position and an aberration estimation position are determined (step ST20). In the process of determining the aberration measurement position and the aberration estimation position, NM aberration measurement positions for measuring at least part of the aberrations occurring in the sample TP, the illumination optical system 21, and the detection optical system 31 within the image acquisition range IR. , NE aberration estimation positions where at least part of the aberrations occurring in the sample TP, the illumination optical system 21, and the detection optical system 31 are estimated from calculation are determined. For example, as shown in FIG. 2, the image acquisition range IR is divided into a plurality of grid-like regions. Then, among the plurality of divided regions, the coordinate position of the region PRm where the black circle is drawn (for example, the coordinate position of the center of the region PRm) is determined as the aberration measurement position, and the coordinate position of the region PRe where the white circle is drawn ( For example, the coordinate position of the center of the region PRe) may be determined as the estimated aberration position. Here, the size of each divided region PRm, PRe in the x direction is Px, the size of each region PRm, PRe in the y direction is Py, and the size of each region PRm, PRe in the z direction is Pz. do. It is desirable to set the size (Px, Py, Pz) of each region PRm, PRe to a size that allows the aberrations to be considered to be the same within the region.

As a method for determining the aberration measurement position, for example, the calculation device 51 displays an image showing the entire image acquisition range IR on the display section of the interface section 53, and the user uses the input section of the interface section 53 to perform aberration measurement. The location may also be specified. Specifically, when the user inputs a command to start determining the aberration measurement position into the input unit, the microscope control unit 54 moves the stage 11 to the above-mentioned predetermined position and sets the aberration measurement position set in step ST10. Control is performed to operate the sample scanning section 25 based on the image acquisition range IR. The microscope control unit 54 generates an image of the sample TP within the image acquisition range IR based on the detection signal transmitted from the detector 41. The arithmetic device 51 causes the display section of the interface section 53 to display an image in which lines indicating the boundaries of the regions PRm and PRe are superimposed on the image of the sample TP generated by the microscope control section 54 . The image showing the entire image acquisition range IR may be, for example, the aforementioned z-stack image (two-dimensional images of multiple cross sections of the sample TP at different positions in the z direction).

The user specifies the aberration measurement position using the GUI while viewing the image of the sample TP within the image acquisition range IR displayed on the display section of the interface section 53. For example, by moving the mouse cursor to any of the regions PRm in the image of the sample TP displayed on the display section of the interface section 53 and clicking (inputting) the mouse button that constitutes the input section of the interface section 53, , the aberration measurement position may be specified. The microscope control unit 54 causes the storage unit 52 to store the coordinate positions (ie, x position, y position, and z position) of the region PRm input to the input unit by clicking a mouse button as an aberration measurement position. The user specifies a plurality of aberration measurement positions by repeatedly clicking the mouse button on a plurality of regions PRm in the image of the sample TP, and the storage unit 52 stores the coordinates of the plurality of aberration measurement positions specified by the user. remember. Here, it is assumed that the number of aberration measurement positions designated by the user is NM.

Among the plurality of regions obtained by dividing the image acquisition range IR into a grid, the coordinate position of the remaining region PRe that has not been determined as the aberration measurement position is determined as the aberration estimation position. By measuring only the aberrations in some of the divided regions PRm and estimating the aberrations in the remaining region PRe, it is possible to shorten the time required to measure the aberrations. Thereby, it is possible to shorten the operating time of the microscope 1, and it is also possible to reduce phototoxicity to the sample TP.

Note that the NM aberration measurement positions are designated by the user, but are not limited to this, and may be determined automatically. For example, when the user inputs a command to start determining the aberration measurement position into the input unit, the microscope control unit 54 generates a z-stack image (where the position in the z direction is (two-dimensional images of a plurality of cross sections of different samples TP) may be generated. In the z-stack image generated by the microscope control unit 54, the arithmetic device 51 measures the aberration of, for example, NM regions in descending order of brightness among a plurality of regions obtained by dividing the image acquisition range IR into a grid pattern. The coordinate positions of the selected NM areas may be selected as the positions and stored in the storage unit 52 as the aberration measurement positions. Note that at this time, the range in the z direction in the z stack image may be made narrower than the range Rz in the z direction of the image acquisition range IR, or the scan pitch in the z stack image may be made larger than a set value.

Next, movement is performed to an aberration measurement position where aberrations are not being measured (step ST30). In the process of moving to the aberration measurement position, the microscope control unit 54 controls the position of the stage 11 driven by the stage drive unit 12 to move the convergence point or center point of the illumination light to each aberration measurement position ( x position, y position, and z position). The microscope control unit 54 may move the convergence point or center point of the illumination light to each aberration measurement position by controlling the traveling direction of the illumination light, which is changed by the sample scanning unit 25. In addition, the microscope control unit 54 controls the position of the objective lens 28 held by the objective lens holding unit 29 to adjust the focal position of the objective lens 28 (i.e., the condensing point or center point of the illumination light) for each aberration. It may be moved to the measurement position (z position).

Next, at each aberration measurement position, aberrations occurring in the sample TP, the illumination optical system 21, and the detection optical system 31 are measured (step ST40). Details of the process for measuring aberrations will be described later.

Next, it is determined whether or not aberrations are being measured at all NM aberration measurement positions (step ST50). If the determination is NO, that is, if there is an aberration measurement position where aberrations are not measured, the process returns to step ST30. On the other hand, if the determination is YES, that is, if aberrations are being measured at all aberration measurement positions, the process advances to step ST60.

In the next step ST60, aberrations occurring in the sample TP, the illumination optical system 21, and the detection optical system 31 are estimated at each aberration estimation position. As a method for estimating aberrations, for example, a weighted average of aberrations can be used, in which distances between the aberration estimation position of interest and NM aberration measurement positions are weighted. Among the NM aberration measurement positions, the phase distribution of the aberration measured in the area near the m-th aberration measurement position is defined as φ _m (x _p , y _p ). Among the NE aberration estimation positions, the aberration phase distribution in the region of the k-th aberration estimation position is ψ _k (x _p , y _p ). Let d _mk be the distance between the m-th aberration measurement position and the k-th aberration estimation position. Here, (x _p , y _p ) represents the (x, y) coordinates on the pupil plane (a plane perpendicular to the optical axis at a position optically conjugate with the pupil position of the objective lens 28). At this time, the aberration phase distribution ψ _k (x _p , y _p ) in the region of the k-th aberration estimation position may be calculated using the following equation (1).

Note that the method for estimating aberrations is not limited to the method described above. For example, based on the aberrations measured at NM aberration measurement positions, the parameters of an aberration model that expresses aberrations that change depending on the position of the visual field are determined, and based on the determined parameters of the aberration model, NE The aberration at the estimated aberration position may be calculated.

Next, a two-dimensional image of the sample TP is acquired (step ST70). In the process of acquiring a two-dimensional image of the sample TP, the microscope control unit 54 controls the sample TP and the illumination optical system 21 based on the aberrations measured in steps ST30 to ST50 and the aberrations estimated in step ST60. A phase modulation pattern to be applied to the illumination light is determined in order to correct at least part of the aberrations caused by the detection optical system 31. The microscope control unit 54 controls the phase modulation element 65 to display the obtained phase modulation pattern. Further, the microscope control unit 54 controls the position of the objective lens 28 held by the objective lens holding unit 29 to displace the focal position of the objective lens 28 to the z position where a two-dimensional image is acquired. Thereafter, the microscope control unit 54 generates a two-dimensional image of the sample TP as described in the above embodiment.

Next, two-dimensional images of the sample TP are acquired at all z positions in the image acquisition range IR, and it is determined whether a z-stack image of the sample TP has been generated (step ST80). If the determination is NO, that is, if there is a z position of the sample TP for which a two-dimensional image has not been acquired, the process returns to step ST70. On the other hand, if the determination is YES, that is, if two-dimensional images of the sample TP are acquired at all z positions and a z-stack image of the sample TP is generated, the steps are performed at the time interval set in the previous step ST10. Proceed to ST90.

In the next step ST90, it is determined whether or not z-stack images of the sample TP have been acquired a set number of times set in the previous step ST10 at the time intervals set in the previous step ST10. If the determination is NO, that is, if z-stack images of the sample TP have not been acquired the set number of times, the process returns to step ST70. On the other hand, if the determination is YES, that is, if z-stack images of the sample TP have been acquired a set number of times, the process ends. In this way, a z-stack image of the sample TP can be obtained. Furthermore, it is possible to generate a type-lapse video of the sample TP, which is made up of z-stack images of the sample TP acquired at predetermined time intervals.

[Measurement of aberration]
Next, a process for measuring aberrations will be described with reference to FIG. 8. FIG. 8 is a flowchart showing the flow of processing for measuring aberrations. In the process of measuring aberrations, first, the removable mirror 34 is inserted into the optical path between the condenser lens 33 and the detection pinhole plate 35 (step ST401). In the process of inserting the insertion/removal mirror 34, the microscope control unit 54 performs control to insert the insertion/removal mirror 34 into the optical path between the condenser lens 33 and the detection pinhole plate 35.

Next, the wavefront sensor 46 receives detection light (step ST402). At this time, the microscope control unit 54 transmits a control signal to the shutter 18 to control the shutter 18 to open. Thereby, the illumination light emitted from the light source unit 16 is focused on the aberration measurement position on the sample TP via the illumination optical system 21. When the removable mirror 34 is inserted into the optical path between the condensing lens 33 and the detection pinhole plate 35, the detection light (fluorescence) generated at the aberration measurement position in the sample TP is transmitted through the insertion, as described above. It is reflected by the demirror 34 and enters the wavefront sensor 46 . The wavefront sensor 46 measures the aberration of the wavefront of the detection light incident on the wavefront sensor 46, and transmits data regarding the wavefront aberration as a measurement signal to the microscope control unit 54. The microscope control unit 54 acquires data regarding at least part of the aberrations occurring in the sample TP and the detection optical system 31 based on the measurement signal transmitted from the wavefront sensor 46 .

At this time, the microscope control section 54 may transmit a control signal to the sample scanning section 25 to control the traveling direction of the illumination light so as to illuminate the brightest part in the area near the aberration measurement position. While the wavefront sensor 46 is receiving light, the microscope control unit 54 sends a control signal to the sample scanning unit 25 to control the traveling direction of the illumination light so as to two-dimensionally scan the area near the aberration measurement position. Good too.

Next, the insertion/removal mirror 34 is removed from the optical path between the condenser lens 33 and the detection pinhole plate 35 (step ST403), and the process ends. In the process of removing the insertion/removal mirror 34, the microscope control unit 54 controls the insertion/removal mirror 34 to be removed from the optical path between the condenser lens 33 and the detection pinhole plate 35.

As described above, according to the first embodiment, the phase modulation unit 60 has a plurality of irradiation areas that can be irradiated with illumination light, and the phase modulation unit 60 has a plurality of irradiation areas that can be irradiated with illumination light, and the phase modulation unit 60 has a plurality of irradiation areas that can be irradiated with illumination light. It has a phase modulation element 65 that imparts a phase distribution to the illumination light, and a first phase modulation scanner 63 that changes the irradiation area to which the illumination light is irradiated. If the phase modulation pattern that changes the phase of the illumination light is changed by changing the irradiation area of the illumination light on the phase modulation element 65 by the first phase modulation scanner 63, the phase displayed by the phase modulation element 65 can be changed. The phase modulation pattern can be changed in a shorter time than when changing the modulation pattern. Therefore, by changing the irradiation area of the illumination light on the phase modulation element 65 when the observation area on the sample TP is displaced, a high-quality sample can be obtained in which aberrations caused by a plurality of observation areas on the sample TP are individually corrected. It becomes possible to acquire images of TP in a short time.

Further, the phase modulation unit 60 keeps the traveling direction of the illumination light whose phase has changed by being irradiated onto the irradiation area constant, even though the first phase modulation scanner 63 changes the irradiation area of the illumination light on the phase modulation element 65. (direction along the optical axis AX1 of the illumination optical system 21). Thereby, only the phase of the illumination light can be changed without changing the optical path of the illumination light.

Moreover, the plurality of irradiation areas in the phase modulation element 65 are arranged so that they do not overlap with each other. Thereby, an appropriate phase modulation pattern can be provided for each image acquisition range of the sample TP.

[Modified example of aberration measurement]
Although the microscope 1 according to the first embodiment is provided with a wavefront sensor 46, an insertion/removal mirror 34, a measurement pinhole plate 36, and a measurement relay lens 37, the present invention is not limited thereto. For example, the sample TP and the illumination optical system 21 can be detected based on the detection light incident on the detector 41 without providing the wavefront sensor 46, the insertion/removal mirror 34, the measurement pinhole plate 36, and the measurement relay lens 37. Aberrations occurring in the optical system 31 may also be measured.

Next, a modification of the process of measuring aberrations will be described with reference to FIG. In the process of measuring aberrations according to the modified example, the phase modulation pattern displayed on the phase modulation element 65 is changed, and the aberrations are determined based on the detection light detected in the plurality of phase modulation patterns. In the process of measuring aberrations according to the modification, a phase modulation pattern on the pupil plane is expressed by a linear sum of basis functions.

In this modification, the coordinates in the x direction and y direction on the pupil plane are (xP, yP). Let the m-th term in the basis function be h _m (xP, yP). Let b _m be the coefficient of the m-th term in the basis function. Further, the m-th component of the basis function is also referred to as "mode m" of the basis function. As the basis function, for example, a Zernike polynomial, a Legendre polynomial, a trigonometric function, a wavelet function, etc. can be used. When the processes of steps ST451 to ST462, which will be described later, are executed, the values of ND coefficients b ₁ , b ₂ , . . . b _ND are stored in the storage unit 52.

FIG. 9 is a flowchart showing the flow of processing for measuring aberrations according to a modification. In the process of measuring aberrations, first, initial settings are performed (step ST451). In the process of performing initial settings, the microscope control unit 54 sets the values of the coefficients of each mode of the basis functions to initial values, and stores them in the storage unit 52. Specifically, b _m =0 (m=1, 2, . . . ND) is set. Further, the number of repetitions itr (itr=1, 2, . . . NI) of aberration measurement is set to itr=1. Among the modes of the basis functions, the mode m'(m'=1, 2, . . . ND) of interest is set to m'=1. Phase modulation pattern setting number j (j=1, 2,...NJ)
, set j=1.

Next, the coefficients of the mode m' of interest among the modes of the basis functions are set (step ST452). Specifically, the microscope control unit 54 sets the values of NJ coefficients b _m' (j) (j=1, 2, . . . NJ) of mode m' of the basis function. Coefficient b _m' (
The value of j) may be set, for example, by dividing a predetermined numerical range into equal intervals. Also,
The above numerical range may be changed depending on the number of repetitions itr. For example, by narrowing the numerical range each time the number of repetitions itr increases, it is possible to accurately determine the value of the coefficient b _m of each mode m of the basis function. Further, the microscope control unit 54 causes the storage unit 52 to store the value of the coefficient b _m of the mode m of the current basis function as b_old.

Next, a phase modulation pattern to be displayed on the phase modulation element 65 is set (step ST453). In the process of setting a phase modulation pattern, the microscope control unit 54 sets NJ phase modulation patterns Ψ _j (xP, yP) (j=1, 2, . . . NJ). The j-th phase modulation pattern Ψ _j (xP, yP) is a phase modulation pattern in which only the value of the coefficient b _m' (j) of the mode m' of interest among the modes of the basis function is changed, and is expressed by the following formula ( 2).

In equation (2), the sum of b _m h _m (xP, yP) is the sum from m=1 to NJ, excluding the case where m=m'. As the value of the coefficient b _m , the value (b_old) of the coefficient b _m of the mode m of the current basis function stored in the storage unit 52 is used.

Next, the phase modulation pattern displayed on the phase modulation element 65 is changed (step ST454). In the process of changing the phase modulation pattern, the microscope control unit 54 changes the phase modulation pattern displayed on the phase modulation element 65 to the j-th phase modulation pattern Ψ _j (xP, yP).

Next, detection light is detected by the detector 41 (step ST455). At this time, the microscope control unit 54 transmits a control signal to the shutter 18 to control the shutter 18 to open. Thereby, the illumination light emitted from the light source unit 16 is focused on the aberration measurement position on the sample TP via the illumination optical system 21. Detection light (fluorescence) generated at the aberration measurement position in the sample TP enters the detector 41 via the illumination optical system 21. The detector 41 performs photoelectric conversion of the light (detection light) that has entered the detector 41 and transmits a light detection signal to the microscope control unit 54 .

The microscope control unit 54 calculates the evaluation value V(j) based on the detection signal transmitted from the detector 41, and stores the data of the calculated evaluation value V(j) in the storage unit 52. For example, an integrated value of detection signals transmitted from the detector 41 may be used as the evaluation value V(j).

For example, while the wavefront sensor 46 is receiving light, the microscope control unit 54 sends a control signal to the sample scanning unit 25 to change the traveling direction of the illumination light so as to two-dimensionally scan the area near the aberration measurement position. May be controlled. In this case, the microscope control unit 54 generates a two-dimensional image of the area near the aberration measurement position based on the detection signal transmitted from the detector 41, and calculates the evaluation value V(j ) may be calculated. As the evaluation value V(j) based on the image, for example, at least one of the contrast of the image, the maximum brightness value, the standard deviation of the brightness value, or the integrated value of the power spectrum in a predetermined frequency domain when the image is Fourier transformed. You may also use one. Further, as the image-based evaluation value V(j), a correlation value between a plurality of images obtained by scanning a region near the aberration measurement position a plurality of times may be used. As the image-based evaluation value V(j), the cutoff frequency of the image was calculated based on the known document "Fourier ring correlation simplifies image restoration in fluorescence microscopy, nature communications 10, Article number: 3103 (2019)". Good too.

Next, it is determined whether j=NJ (step ST456). If the determination is NO, that is, if j≠NJ, the process advances to step ST457. In step ST457, j=j+1 is set, and the process returns to step ST454. Thereby, evaluation values V(j) are calculated for the remaining phase modulation patterns among the NJ phase modulation patterns set in the previous step ST453.

On the other hand, if the determination in step ST456 is YES, that is, if j=NJ, the process advances to step ST458. In step ST458, the coefficient value b _m * of mode m of the basis function is determined based on the evaluation value V(j) calculated for NJ phase modulation patterns. When the coefficient value b _m * is determined, the value of the coefficient b m of mode m of the basis function stored in the storage unit 52 is updated to the coefficient value b _{m *} .

As the coefficient value b _m *, for example, the value of the coefficient b m' (j) of mode _m ' corresponding to j when the evaluation value V (j) is maximum may be selected. Also, as the coefficient value b _m *, the evaluation value V (j) is expressed as a function using the coefficient b _m' (j) of mode m' as an argument, and interpolation is performed using the data points of the evaluation value V (j), The value of the coefficient b _m' (j) of mode m' when the evaluation value V(j) becomes the maximum may be determined. As a method for interpolating the data points of the evaluation value V(j), for example, Lagrange interpolation, Newton interpolation, Hermitian interpolation, spline interpolation, etc. can be used. Further, as the coefficient value b _m *, a coefficient value when the evaluation value V(j) becomes the maximum may be calculated based on a function approximation of the evaluation value V(j). For example, it is possible to use a quadratic function, a Gaussian function, or the like as a function for performing functional approximation of the evaluation value V(j).

In the next step ST459, it is determined whether m'=ND. If the determination is NO, that is, if m'≠ND, the process advances to step ST460. In step ST460, m'=m'+1 is set, and the process returns to step ST452. As a result, the value of the coefficient b _m of the remaining mode m among each mode m of the basis function is calculated.

On the other hand, if the determination in step ST459 is YES, that is, if m'=ND, the process proceeds to step ST461. In step ST461, it is determined whether itr=NI. If the determination is NO, that is, itr≠NI, the process advances to step ST462. In step ST462, itr=itr+1 is set, and the process returns to step ST452. Thereby, by determining the coefficient value b _m * of each mode m multiple times, the value of the coefficient b _m of each mode m of the basis function can be set to a more appropriate value.

On the other hand, if the determination in step ST462 is YES, that is, if itr=NI, the process ends. Aberrations (phase modulation patterns) can also be measured using this method. Note that the method for measuring aberrations is not limited to the above two methods, and for example, the method disclosed in US Pat. No. 8,866,107 may be used.

[Modified example of phase modulation unit]
Next, a modification of the phase modulation unit in the first embodiment will be described with reference to FIG. 10. As shown in FIG. 10, the phase modulation unit 80 according to the modification of the first embodiment includes a beam splitter 86, a first relay lens 81, a mirror 82, a phase modulation scanner 83, and a second relay lens 84. and a phase modulation element 65. The ratio of transmittance and reflectance of the beam splitter 86 is set to, for example, 1:1. A part of the light that enters the beam splitter 86 from the collimator lens 22 of the illumination optical system 21 passes through the beam splitter 86 and enters the first relay lens 81 . A portion of the light that has entered the beam splitter 86 from the first relay lens 81 is reflected by the beam splitter 86 and enters the dichroic mirror 24 of the illumination optical system 21 .

The first relay lens 81 collects the illumination light that has entered the first relay lens 81 from the beam splitter 86 . The first relay lens 81 converts the illumination light that has entered the first relay lens 81 from the mirror 82 into substantially parallel light. The mirror 82 reflects the illumination light that has entered the mirror 82 from the first relay lens 81 toward the phase modulation scanner 83 . The mirror 82 reflects the illumination light that has entered the mirror 82 from the phase modulation scanner 83 toward the first relay lens 81 .

The phase modulation scanner 83 is arranged at a position B2 that is optically conjugate with the sample TP. The phase modulation scanner 83 is configured using a mirror (for example, a galvano mirror, a resonant mirror, etc.) held so that the direction (azimuth angle) of a reflecting surface changes by one-axis rotation or two-axis rotation. The phase modulation scanner 83 changes the traveling direction of the illumination light that has entered the phase modulation scanner 83 from the mirror 82 . Thereby, the phase modulation scanner 83 scans the phase modulation element 65 with the illumination light from the collimator lens 22.

Further, the phase modulation scanner 83 is configured such that the illumination light incident on the phase modulation scanner 83 from the second relay lens 84 travels toward the mirror 82, that is, the illumination light from the second relay lens 84 is directed to the illumination optical system 21. The traveling direction of the illumination light is changed so that it travels along the optical axis AX1.

The second relay lens 84 converts the illumination light that has passed through the phase modulation scanner 83 into substantially parallel light. The second relay lens 84 collects the illumination light reflected by the phase modulation element 65. The phase modulation element 65 is arranged at a position H2 that is optically conjugate with the pupil position of the objective lens 28. The phase modulation element 65 has the same configuration as the phase modulation element 65 according to the first embodiment, is given the same reference numeral as in the first embodiment, and detailed description thereof will be omitted.

The illumination light that has passed through the collimator lens 22 of the illumination optical system 21 enters the beam splitter 86 of the phase modulation unit 80 . A part of the illumination light that has entered the beam splitter 86 from the collimator lens 22 passes through the beam splitter 86 and enters the first relay lens 81 . The illumination light incident on the first relay lens 81 is transmitted through the first relay lens 81 and reflected by the mirror 82. The illumination light reflected by the mirror 82 enters the phase modulation scanner 83 and is once focused at a position B2 that is optically conjugate with the sample TP. The phase modulation scanner 83 scans the phase modulation element 65 with the illumination light that has entered the phase modulation scanner 83 . The illumination light that has entered the phase modulation scanner 83 from the mirror 82 passes through the phase modulation scanner 83 and is transmitted through the second relay lens 84 . The illumination light transmitted through the second relay lens 84 becomes substantially parallel light, and is irradiated onto one of the plurality of irradiation areas in the phase modulation element 65.

When the illumination light is irradiated onto any one of the plurality of irradiation areas (for example, the first to 16th irradiation areas SA1 to SA16) in the phase modulation element 65, phase modulation occurs in the irradiation area irradiated with the illumination light. The phase of the illumination light changes depending on the pattern. The illumination light irradiated onto any one of the plurality of irradiation areas in the phase modulation element 65 is reflected at the irradiation area. Note that the phase modulation scanner 83 scans the phase modulation element 65 with the illumination light incident on the phase modulation scanner 83, thereby determining an irradiation area to which the illumination light is irradiated among a plurality of irradiation areas in the phase modulation element 65. It is possible to change.

The illumination light that is irradiated onto any one of the plurality of irradiation areas in the phase modulation element 65 and reflected is transmitted through the second relay lens 84 . The illumination light that has passed through the second relay lens 84 enters the phase modulation scanner 83 and is once focused at a position B2 that is optically conjugate with the sample TP. The phase modulation scanner 83 is arranged so that the illumination light from the second relay lens 84 travels toward the mirror 82, that is, the illumination light from the second relay lens 84 travels along the optical axis AX1 of the illumination optical system 21. change the direction of illumination light. Illumination light that enters the phase modulation scanner 83 from the second relay lens 84 passes through the phase modulation scanner 83 and is reflected by the mirror 82 . The illumination light reflected by the mirror 82 passes through the first relay lens 81 and enters the beam splitter 86 as substantially parallel light. A part of the illumination light that enters the beam splitter 86 from the first relay lens 81 is reflected by the beam splitter 86 and enters the dichroic mirror 24 of the illumination optical system 21 . Thereby, the phase modulation of the illumination light is performed in the same manner as in the case of the phase modulation unit 60 according to the first embodiment.

[Second embodiment]
Next, referring to FIG. 11, a microscope 101 according to a second embodiment will be described. The microscope 101 according to the second embodiment is also referred to as a scanning microscope. The microscope 101 according to the second embodiment includes a stage 11, a light source unit 16, an illumination optical system 121, a detection optical system 131, a detector 41, a wavefront sensor 46, a calculation device 51, and a storage section 52. , an interface section 53, and a microscope control section 54. The stage 11 and the light source unit 16 have the same configuration as the stage 11 and the light source unit 16 according to the first embodiment, are given the same reference numerals as in the first embodiment, and detailed description thereof will be omitted.

The illumination optical system 121 illuminates the sample TP with illumination light (excitation light) emitted from the light source unit 16. The illumination optical system 121 includes, in order from the light source unit 16 side, a collimator lens 122, a dichroic mirror 123, a phase modulation unit 60, a sample scanning section 125, a scan lens 126, a second objective lens 127, and an objective lens 128. and has. The configurations and functions of the collimator lens 122, dichroic mirror 123, sample scanning unit 125, scan lens 126, second objective lens 127, and objective lens 128 included in the illumination optical system 121 are the same as those in the first embodiment. The collimator lens 22, the dichroic mirror 24, the sample scanning unit 25, the scan lens 26, the second objective lens 27, and the objective lens 28 included in the illumination optical system 21 of the microscope are the same, so the explanation will be omitted.

Further, the detection optical system 131 receives light generated by the sample TP as detection light. The detection optical system 131 includes, in order from the sample TP side, an objective lens 128, a second objective lens 127, a scan lens 126, a sample scanning section 125, a phase modulation unit 60, and a dichroic mirror 123. This embodiment differs from the first embodiment in that the unit 60 is disposed between the sample scanning section 125 and the dichroic mirror 123.

The phase modulation unit 60 has the same configuration as the phase modulation unit 60 according to the first embodiment, is given the same reference numeral as in the first embodiment, and detailed description thereof will be omitted. In the second embodiment, the phase modulation unit 60 performs phase modulation on the illumination light that has passed through the dichroic mirror 123, and also performs phase modulation on the detection light that has passed through the sample scanning section 125. At least part of the aberrations caused by the optical system 121 and the detection optical system 131 is corrected.

Further, the detection optical system 131 includes, in order from the dichroic mirror 123 side, a detection filter 132, a condensing lens 133, an insertion/removal mirror 134, and a detection pinhole plate 135. Further, the detection optical system 131 includes a measurement pinhole plate 136 and a measurement relay lens 137, but each function and configuration thereof are the same as in the first embodiment, so a description thereof will be omitted. In this way, the objective lens 128, the second objective lens 127, the scan lens 126, the sample scanning section 125, the phase modulation unit 60, and the dichroic mirror 123 are used in both the illumination optical system 121 and the detection optical system 131. include.

The detector 41 and the wavefront sensor 46 have the same configuration as the detector 41 and the wavefront sensor 46 according to the first embodiment, are given the same reference numerals as in the first embodiment, and detailed description thereof will be omitted. Further, the arithmetic device 51, the storage section 52, the interface section 53, and the microscope control section 54 have the same configuration as the arithmetic device 51, the storage section 52, the interface section 53, and the microscope control section 54 according to the first embodiment. , are given the same reference numerals as in the first embodiment, and detailed description thereof will be omitted.

In the microscope 101 according to the second embodiment, the illumination light (excitation light) emitted from the light source 17 of the light source unit 16 passes through the collimator lens 122 of the illumination optical system 121 and becomes substantially parallel light, and is then sent to the dichroic mirror 123. This differs from the first embodiment in that the light enters, passes through the dichroic mirror 123, and enters the phase modulation unit 60. Fluorescence is emitted as detection light from a fluorescent substance contained in a portion of the sample TP where illumination light (excitation light) is focused. Further, the detection light (fluorescence) generated in the sample TP passes through the objective lens 128 as the detection optical system 131, the second objective lens 127, the scan lens 126, and the sample scanning section 125, and enters the phase modulation unit 60 at the point where it enters the phase modulation unit 60. This embodiment is also different from the first embodiment. When the detection light enters the phase modulation unit 60, the phase modulation unit 60 performs phase modulation on the incident detection light and emits the phase modulated detection light. The detection light, which is substantially parallel light, emitted from the phase modulation unit 60 enters the dichroic mirror 123.

In the microscope 101 according to the second embodiment, a beam splitter (not shown) is provided in place of the removable mirror 134, and a portion of the detection light incident on the beam splitter is transmitted through the beam splitter to the detector 41. The other part of the detection light that reaches the beam splitter and enters the beam splitter may be configured so that it is reflected by the beam splitter and reaches the wavefront sensor 46.

In the microscope 101 according to the second embodiment, the illumination light transmitted through the dichroic mirror 123 of the illumination optical system 121 enters the first relay lens 61 of the phase modulation unit 60, but the subsequent steps are the same as in the first embodiment. The explanation will be omitted. Note that when the illumination light reflected from any one of the plurality of irradiation areas in the phase modulation element 65 passes through the second relay lens 64 and passes through the second phase modulation scanner 66, it is reflected by the illumination optical system 121. It begins to advance along the optical axis AX11.

Furthermore, the detection light that has passed through the sample scanning section 125 as the detection optical system 131 is incident on the third relay lens 68 of the phase modulation unit 60 . The detection light that has entered the third relay lens 68 passes through the third relay lens 68, the second mirror 67, the second phase modulation scanner 66, and the second relay lens 64 in the reverse order of the illumination light. The same irradiation area as the illumination light is irradiated among the plurality of irradiation areas in the phase modulation element 65.

When the detection light is irradiated onto the same irradiation area as the illumination light in the phase modulation element 65, the phase of the detection light changes according to the phase modulation pattern in the irradiation area. The detection light irradiated onto the same irradiation area as the illumination light in the phase modulation element 65 is reflected at the irradiation area.

The detection light that is irradiated onto the same irradiation area as the illumination light in the phase modulation element 65 and reflected passes through the second relay lens 64 and the first phase modulation scanner 63 and is reflected by the first mirror 62 . The detection light reflected by the first mirror 62 passes through the first relay lens 61, becomes substantially parallel light, and enters the dichroic mirror 123 serving as the detection optical system 131. In this way, the phase modulation unit 60 performs phase modulation on the detection light.

Note that when the detection light reflected in the same irradiation area as the illumination light in the phase modulation element 65 passes through the second relay lens 64 and passes through the first phase modulation scanner 63, it is reflected along the optical axis AX12 of the detection optical system 131. You will be able to move forward. Even though the second phase modulation scanner 66 (and the first phase modulation scanner 63) changes the traveling direction of the detection light and changes the irradiation area by descanning the detection light in the first phase modulation scanner 63, The detection light transmitted through the first relay lens 61 becomes substantially parallel light and can be incident on the dichroic mirror 123 as the detection optical system 131.

The first phase modulation scanner 63 and the second phase modulation scanner 66 scan the phase modulation element 65 with the illumination light and the detection light, so that the illumination light and the detection light are It is possible to change the irradiation area where the light is irradiated. The first phase modulation scanner 63 and the second phase modulation scanner 66 control the irradiation area that is irradiated with illumination light and detection light in the phase modulation element 65 when the observation area in which the microscope observation is performed on the sample TP is displaced. change. Note that a phase modulation pattern for correcting at least part of the aberrations generated between the sample TP, the illumination optical system 121, and the detection optical system 131, that is, the sample TP and the illumination optical system 121, is created for each of the plurality of observation areas on the sample TP. A predetermined phase distribution for correcting at least part of the aberrations generated in the detection optical system 131 is determined in advance. The phase modulation element 65 displays, in each irradiation area on the display surface of the phase modulation element 65, a phase modulation pattern determined in advance for the corresponding observation area. As a result, the first phase modulation scanner 63 and the second phase modulation scanner 66 change the irradiation area on the phase modulation element 65 for each observation area, so that a phase modulation pattern (phase distribution) appropriately determined for each observation area is obtained. ) can change (modulate) the phase of the illumination light. Therefore, it is possible to obtain a high-quality image of the sample TP in a short time, in which at least part of the aberrations caused by the sample TP, the illumination optical system 121, and the detection optical system 131 are corrected.

Further, even when using the microscope 101 according to the second embodiment, it is possible to acquire an image of the sample TP by the image acquisition method described in the first embodiment. Therefore, according to the second embodiment, the same effects as the first embodiment can be obtained. Further, according to the second embodiment, the phase modulation unit 60 performs phase modulation on the detection light in addition to the phase modulation on the illumination light, so it is possible to obtain a higher quality image of the sample TP in a short time. It becomes possible.

In the microscope 101 according to the second embodiment, for example, the wavefront sensor 46, the insertion/removal mirror 134, the measurement pinhole plate 136, and the measurement relay lens 137 are not provided, and based on the detection light incident on the detector 41, , at least part of the aberrations occurring in the sample TP, the illumination optical system 121, and the detection optical system 131 may be measured. In this case, the aberration (phase modulation pattern) may be measured by a modification of the process for measuring the aberration described in the first embodiment.

In the microscope 101 according to the second embodiment, a phase modulation unit 80 according to a modification of the first embodiment may be provided instead of the phase modulation unit 60. Similarly to the second embodiment described above, the phase modulation unit 80 according to the modification of the first embodiment can perform phase modulation on the illumination light and the detection light.

[Third embodiment]
Next, a microscope 201 according to a third embodiment will be described with reference to FIG. 12. The microscope 201 according to the third embodiment is also referred to as a scanning microscope. A microscope 201 according to the third embodiment includes a stage 11, a light source unit 16, an illumination optical system 221, and a detection optical system 2.
31, a detector 41, a wavefront sensor 46, an arithmetic unit 51, a storage section 52, an interface section 53, and a microscope control section 54. The stage 11 and the light source unit 16 have the same configuration as the stage 11 and the light source unit 16 according to the first embodiment, are given the same reference numerals as in the first embodiment, and detailed description thereof will be omitted.

The illumination optical system 221 illuminates the sample TP with illumination light (excitation light) emitted from the light source unit 16. The illumination optical system 221 includes, in order from the light source unit 16 side, a collimator lens 222, a phase modulation unit 60, a first dichroic mirror 224, a sample scanning section 225, a scan lens 226, a second objective lens 227, and a second objective lens 227. It has two dichroic mirrors 228 and an objective lens 229. The configurations and functions of the collimator lens 222, dichroic mirror 224, sample scanning unit 225, scan lens 226, second objective lens 227, and objective lens 229 included in the illumination optical system 221 are the same as those in the first embodiment. The collimator lens 22, the dichroic mirror 24, the sample scanning unit 25, the scan lens 26, the second objective lens 27, and the objective lens 28 included in the illumination optical system 21 of the microscope are the same, so the explanation will be omitted.

The phase modulation unit 60 has the same configuration as the phase modulation unit 60 according to the first embodiment, is given the same reference numeral as in the first embodiment, and detailed description thereof will be omitted. In the third embodiment, the phase modulation unit 60 performs phase modulation on the illumination light that has passed through the collimator lens 222, and corrects at least part of the aberrations that occur between the sample TP and the illumination optical system 221.

The detection optical system 231 receives light generated by the sample TP as detection light. The detection optical system 231 includes, in order from the sample TP side, an objective lens 229, a second dichroic mirror 228, a second objective lens 227, a scan lens 226, a sample scanning section 225, and a first dichroic mirror 224. . Further, the detection optical system 231 includes, in order from the second dichroic mirror 228 side, a first detection relay lens 232, a second detection relay lens 233, and a detection filter 234. Further, the detection optical system 231 includes, in order from the first dichroic mirror 224 side, a measurement filter 235, a condenser lens 236, a measurement pinhole plate 237, and a measurement relay lens 238. The functions and configurations are the same as those in the first embodiment, so descriptions thereof will be omitted. In this way, the objective lens 229, the second dichroic mirror 228, the second objective lens 227, the scan lens 226, the sample scanning section 225, and the first dichroic mirror 224 are connected to the illumination optical system 221 and the detection optical system 231. included in both.

Note that the second dichroic mirror 228 is arranged to be removably inserted into the optical path between the objective lens 229 and the second objective lens 227. When the second dichroic mirror 228 is inserted into the optical path between the objective lens 229 and the second objective lens 227, the illumination light from the light source unit 16 passes through the second dichroic mirror 228 and enters the objective lens 229. , the detection light from the sample TP is reflected by the second dichroic mirror 228 and enters the first detection relay lens 232 . When the second dichroic mirror 228 leaves the optical path between the objective lens 229 and the second objective lens 227, the illumination light from the light source unit 16 enters the objective lens 229, and the detection light from the sample TP enters the optical path between the objective lens 229 and the second objective lens 227. The light enters the second objective lens 227.

The first detection relay lens 232 collects detection light from the sample TP. The second detection relay lens 233 converts the detection light from the sample TP into substantially parallel light again. The detection filter 234 transmits light in a predetermined wavelength band (for example, fluorescence) among the light generated by the sample TP. The detection filter 234 blocks at least a portion of, for example, illumination light reflected by the sample TP, external light, stray light, and the like. Detector 41 detects detection light from sample TP.

The detector 41 and the wavefront sensor 46 have the same configuration as the detector 41 and the wavefront sensor 46 according to the first embodiment, are given the same reference numerals as in the first embodiment, and detailed description thereof will be omitted. Further, the arithmetic device 51, the storage section 52, the interface section 53, and the microscope control section 54 have the same configuration as the arithmetic device 51, the storage section 52, the interface section 53, and the microscope control section 54 according to the first embodiment. , are given the same reference numerals as in the first embodiment, and detailed description thereof will be omitted.

In the third embodiment, the microscope control unit 54 transmits a control signal to the sample scanning unit 225 to control the traveling direction of the illumination light and detection light that are changed by the sample scanning unit 225. The microscope control unit 54 transmits a control signal to the objective lens holding unit 230 to control the position of the objective lens 229 held by the objective lens holding unit 230. The microscope control unit 54 transmits a control signal to the second dichroic mirror 228 to control insertion and removal of the second dichroic mirror 228.

The illumination light (excitation light) emitted from the light source 17 of the light source unit 16 passes through the collimator lens 222 of the illumination optical system 221, the phase modulation unit 60, the first dichroic mirror 224, the sample scanning section 225, the scan lens 226, and the second The point that the light reaches the second dichroic mirror 228 via the objective lens 227 is the same as in the first embodiment.

When the second dichroic mirror 228 is inserted into the optical path between the objective lens 229 and the second objective lens 227, the illumination light passes through the second dichroic mirror 228 and enters the objective lens 229. The illumination light incident on the objective lens 229 passes through the objective lens 229 and is focused on the focal plane of the objective lens 229. A portion of the sample TP where the illumination light is focused (that is, a portion that overlaps with the focal plane of the objective lens 229) is two-dimensionally scanned in two directions, the x direction and the y direction, by the sample scanning unit 225.

Fluorescence is emitted as detection light from a fluorescent substance contained in a portion of the sample TP where illumination light (excitation light) is focused. Detection light (fluorescence) generated by the sample TP enters an objective lens 229 as a detection optical system 231. The detection light that has entered the objective lens 229 passes through the objective lens 229 and enters the second dichroic mirror 228 . Since the detection light (fluorescence) that has entered the second dichroic mirror 228 has a different wavelength from the illumination light (excitation light), it is reflected by the second dichroic mirror 228 and passes through the first detection relay lens 232 and the second detection relay lens. Transmits 233. The fluorescence transmitted through the first detection relay lens 232 and the second detection relay lens 233 passes through the detection filter 234 and enters the detector 41 . The function and configuration of the detector 41 are the same as those in the first embodiment, so their explanation will be omitted.

When the second dichroic mirror 228 is separated from the optical path between the objective lens 229 and the second objective lens 227, the illumination light that has passed through the scan lens 226 and the second objective lens 227 enters the objective lens 229. The light is transmitted and focused on the focal plane of the objective lens 229. The detection light (fluorescence) generated in the sample TP enters and passes through the objective lens 229 as the detection optical system 231, passes through the second objective lens 227 and the scan lens 226, and enters the sample scanning unit 225. Thereafter, the process until it is incident on the wavefront sensor 46 is the same as in the first embodiment, so the explanation will be omitted. The wavefront sensor 46 measures the aberration of the wavefront of the detection light incident on the wavefront sensor 46, and transmits data regarding the aberration of the wavefront to the microscope control unit 54 as a measurement signal. The microscope control unit 54 acquires data regarding at least part of the aberrations occurring in the sample TP and the detection optical system 231 based on the measurement signal transmitted from the wavefront sensor 46.

In the microscope 201 according to the third embodiment, the phase modulation unit 60 performs phase modulation on the illumination light in the same manner as in the first embodiment. Further, even when using the microscope 201 according to the third embodiment, it is possible to acquire an image of the sample TP by the image acquisition method described in the first embodiment. Therefore, according to the third embodiment, the same effects as the first embodiment can be obtained. Furthermore, according to the third embodiment, since the number of optical elements through which the detection light passes before entering the detector 41 is small, it is possible to reduce loss in the amount of detection light.

In the third embodiment, in step ST401 in the process of measuring aberrations described in the first embodiment, the insertion/removal mirror 34 is inserted into the optical path between the condenser lens 33 and the detection pinhole plate 35. Instead, the second dichroic mirror 228 is removed from the optical path between the objective lens 229 and the second objective lens 227. Furthermore, in step ST403, instead of removing the insertion/removal mirror 34 from the optical path between the condenser lens 33 and the detection pinhole plate 35, the second dichroic mirror 228 is inserted between the objective lens 229 and the second objective lens 227. Insert into the optical path between the

In the microscope 201 according to the third embodiment, for example, without providing the wavefront sensor 46, the first dichroic mirror 224, the measurement filter 235, the condenser lens 236, the measurement pinhole plate 237, and the measurement relay lens 238, Based on the detection light incident on the detector 41, aberrations occurring in the sample TP, the illumination optical system 221, and the detection optical system 231 may be measured. In this case, the aberration (phase modulation pattern) may be measured by a modified example of the process for measuring the aberration described in the first embodiment. Further, in this case, the second dichroic mirror 228 may be arranged and fixed on the optical path between the objective lens 229 and the second objective lens 227.

In the microscope 201 according to the third embodiment, a phase modulation unit 80 according to a modification of the first embodiment may be provided instead of the phase modulation unit 60. Similarly to the modification of the first embodiment described above, the phase modulation unit 80 according to the modification of the first embodiment can perform phase modulation on illumination light.

[Fourth embodiment]
Next, a microscope 301 according to a fourth embodiment will be described with reference to FIG. 13. The microscope 301 according to the fourth embodiment is also referred to as a scanning microscope. A microscope 301 according to the fourth embodiment includes a stage 11, a light source unit 16, an illumination optical system 321, a detection optical system 331, a measurement optical system 335, a detector 41, a wavefront sensor 46, and a calculation device 51. , a storage section 52 , an interface section 53 , and a microscope control section 54 . The stage 11 and the light source unit 16 have the same configuration as the stage 11 and the light source unit 16 according to the first embodiment, are given the same reference numerals as in the first embodiment, and detailed description thereof will be omitted.

The illumination optical system 321 illuminates the sample TP with illumination light (excitation light) emitted from the light source unit 16. The illumination optical system 321 includes, in order from the light source unit 16 side, a collimator lens 322, a phase modulation unit 60, a dichroic mirror 324, a sample scanning section 325, a scan lens 326, a second objective lens 327, and an illumination objective. It has a lens 328. The configurations and functions of the collimator lens 322, dichroic mirror 324, sample scanning unit 325, scan lens 326, second objective lens 327, and illumination objective lens 328 included in the illumination optical system 321 are as follows: They are the same as the collimator lens 22, dichroic mirror 24, sample scanning section 25, scan lens 26, second objective lens 27, and objective lens 28 included in the illumination optical system 21 of the microscope in the embodiment, so description thereof will be omitted. do.

The phase modulation unit 60 has the same configuration as the phase modulation unit 60 according to the first embodiment, is given the same reference numeral as in the first embodiment, and detailed description thereof will be omitted.

The detection optical system 331 receives light generated by the sample TP as detection light. The detection optical system 331 includes, in order from the sample TP side, a detection objective lens 332 and a detection filter 334. The detection objective lens 332 is arranged near the top of the stage 11. The detection objective lens 332 faces the sample TP on the stage 11 from the side opposite to the illumination objective lens 328 via a holding member (not shown) such as a cover glass. The gap between the detection objective lens 332 and the holding member may be filled with gas such as air, or may be filled with immersion liquid.

Further, the detection objective lens 332 is attached to the casing (not shown) of the microscope 301 via a detection objective lens holder 333. The detection objective lens holder 333 is configured using, for example, a drive device (not shown) such as an electric motor. The detection objective lens holder 333 moves the detection objective lens 332 up and down in the z direction together with a revolver (not shown). When the detection objective lens 332 is moved in the z direction by the detection objective lens holder 333, the relative position of the detection objective lens 332 with respect to the sample TP changes, and the focal position of the detection objective lens 332 with respect to the sample TP is moved in the z direction. Change. The illumination objective lens holder 329 displaces the focal position of the illumination objective lens 328 in the z direction, and the detection objective lens holder 333 moves the detection objective lens 332 to match the focal position of the illumination objective lens 328. By displacing the focal point position in the z-direction, it is possible to obtain images of cross sections inside the sample TP at different positions in the z-direction, that is, z-stack images.

The detection filter 334 transmits light (for example, fluorescence) in a predetermined wavelength band among the light generated by the sample TP. The detection filter 334 blocks at least a portion of, for example, illumination light, external light, stray light, etc. that have passed through the sample TP. Detector 41 detects detection light from sample TP.

The measurement optical system 335 receives light generated by the sample TP as measurement light. The measurement optical system 335 includes, in order from the sample TP side, an illumination objective lens 328, a second objective lens 327, a scan lens 326, a sample scanning section 325, and a dichroic mirror 324. Further, the measurement optical system 335 includes, in order from the dichroic mirror 324 side, a measurement filter 336, a condensing lens 337, a measurement pinhole plate 338, and a measurement relay lens 339. Each configuration is the same as that in the first embodiment, so description thereof will be omitted. In this way, the illumination objective lens 328, the second objective lens 327, the scan lens 326, the sample scanning section 325, and the dichroic mirror 324 are included in both the illumination optical system 321 and the measurement optical system 335. .

The detector 41 and the wavefront sensor 46 have the same configuration as the detector 41 and the wavefront sensor 46 according to the first embodiment, are given the same reference numerals as in the first embodiment, and detailed description thereof will be omitted. Further, the arithmetic device 51, the storage section 52, the interface section 53, and the microscope control section 54 have the same configuration as the arithmetic device 51, the storage section 52, the interface section 53, and the microscope control section 54 according to the first embodiment. , are given the same reference numerals as in the first embodiment, and detailed description thereof will be omitted.

In the fourth embodiment, the microscope control unit 54 transmits a control signal to the sample scanning unit 325 to control the traveling direction of the illumination light and detection light that are changed by the sample scanning unit 325. The microscope control unit 54 transmits control signals to the illumination objective lens holding unit 329 and the detection objective lens holding unit 333 to determine the position of the illumination objective lens 328 held in the illumination objective lens holding unit 329 and the detection The position of the detection objective lens 332 held by the detection objective lens holder 333 is controlled.

The illumination light (excitation light) emitted from the light source 17 of the light source unit 16 is transmitted to the collimator lens 322 of the illumination optical system 321, the phase modulation unit 60, the dichroic mirror 324, the sample scanning section 325, the scan lens 326, and the second objective lens. The point where the light passes through 327 and enters the illumination objective lens 328 is the same as in the first embodiment. The illumination light that has entered the illumination objective lens 328 passes through the illumination objective lens 328 and is focused on the focal plane of the illumination objective lens 328. A portion of the sample TP where the illumination light is focused (that is, a portion that overlaps with the focal plane of the illumination objective lens 328) is two-dimensionally scanned in two directions, the x direction and the y direction, by the sample scanning unit 325. .

Fluorescence is emitted from a fluorescent substance contained in a portion of the sample TP where the illumination light (excitation light) is focused. A part of the light (fluorescence) generated by the sample TP enters the detection objective lens 332 of the detection optical system 331 as detection light. The detection light incident on the detection objective lens 332 passes through the detection objective lens 332, passes through the detection filter 334, and enters the detector 41.

The other part of the light (fluorescence) generated by the sample TP enters and passes through the illumination objective lens 328 as the measurement optical system 335 as measurement light, and passes through the second objective lens 327 and the scan lens 326. Although the light enters the sample scanning unit 325, the steps from then on until the light enters the wavefront sensor 46 are the same as in the first embodiment, so a description thereof will be omitted. The wavefront sensor 46 measures the aberration of the wavefront of the measurement light incident on the wavefront sensor 46, and transmits data regarding the aberration of the wavefront to the microscope control unit 54 as a measurement signal. The microscope control unit 54 acquires data regarding aberrations occurring in the sample TP and the illumination optical system 321 based on the measurement signal transmitted from the wavefront sensor 46.

In the microscope 301 according to the fourth embodiment, the phase modulation unit 60 performs phase modulation on the illumination light in the same manner as in the first embodiment. Further, even when using the microscope 301 according to the fourth embodiment, it is possible to acquire an image of the sample TP by the image acquisition method described in the first embodiment. Therefore, according to the fourth embodiment, the same effects as the first embodiment can be obtained. Further, according to the fourth embodiment, since the detection objective lens 332 faces the sample TP on the stage 11 from the opposite side to the illumination objective lens 328, the scattered light generated by the sample TP is detected as detection light. In this case, the detection optical system 331 can efficiently detect forward scattered light from the sample TP. Further, the illumination optical system 321 can illuminate the sample TP with illumination light having a plurality of wavelengths, and the detection optical system 331 can detect light of a specific wavelength that has passed through the sample TP as detection light.

Note that in the fourth embodiment, step ST401 and step ST403 in the process of measuring aberrations described in the first embodiment are omitted. Furthermore, in step ST402, the measurement light (fluorescence) generated at the aberration measurement position in the sample TP enters the wavefront sensor 46 via the measurement optical system 335, as described above. The wavefront sensor 46 measures the aberration of the wavefront of the measurement light incident on the wavefront sensor 46, and transmits data regarding the aberration of the wavefront to the microscope control unit 54 as a measurement signal. The microscope control unit 54 acquires data regarding aberrations occurring in the sample TP and the illumination optical system 321 based on the measurement signal transmitted from the wavefront sensor 46.

In the microscope 301 according to the fourth embodiment, for example, the detector is not provided with the wavefront sensor 46, the dichroic mirror 324, the measurement filter 336, the condenser lens 337, the measurement pinhole plate 338, and the measurement relay lens 339. Based on the detection light incident on 41, aberrations occurring in the sample TP, the illumination optical system 321, and the detection optical system 331 may be measured. In this case, the aberration (phase modulation pattern) may be measured by a modified example of the process for measuring the aberration described in the first embodiment.

In the microscope 301 according to the fourth embodiment, a phase modulation unit 80 according to a modification of the first embodiment may be provided instead of the phase modulation unit 60. Similarly to the modification of the first embodiment described above, the phase modulation unit 80 according to the modification of the first embodiment can perform phase modulation on illumination light.

[Fifth embodiment]
Next, a microscope 401 according to a fifth embodiment will be described with reference to FIG. 14. The microscope 401 according to the fifth embodiment is also referred to as an epifluorescence microscope. A microscope 401 according to the fifth embodiment includes a stage 11, a light source unit 416, an illumination optical system 421, a detection optical system 431, a two-dimensional detector 441, a wavefront sensor 46, a calculation device 51, and a storage section. 52, an interface section 53, and a microscope control section 54. The stage 11 has the same configuration as the stage 11 according to the first embodiment, is given the same reference numeral as in the first embodiment, and detailed description thereof will be omitted.

The light source unit 416 emits illumination light (excitation light) for exciting the fluorescent substance contained in the sample TP. Light source unit 416 includes a light source 417 and a shutter 418. As the light source 417, for example, a mercury lamp, an LED (Light Emitting Diode) lamp, or the like is used. The shutter 418 is configured to either pass the illumination light emitted from the light source 417 or block it.

The illumination optical system 421 illuminates the sample TP with illumination light (excitation light) emitted from the light source unit 416. The illumination optical system 421 includes, in order from the light source unit 416 side, a collimator lens 422, an illumination filter 423, an illumination field stop 424, an illumination relay lens 425, a dichroic mirror 426, and an objective lens 427. The structures and functions of the collimator lens 422, dichroic mirror 426, and objective lens 427 included in the illumination optical system 421 are the same as the collimator lens 22 and dichroic mirror 24 included in the illumination optical system 21 of the microscope in the first embodiment. Since this is the same as the objective lens 28, the explanation will be omitted. The illumination filter 423 transmits light in a predetermined wavelength range (excitation light) out of the illumination light from the light source unit 416. The illumination field stop 424 is arranged at a position optically conjugate with the focal position of the objective lens 427. The field stop for illumination 424 limits the area where the sample TP is illuminated by the illumination light. The illumination relay lens 425 collects the illumination light from the light source unit 416.

The detection optical system 431 receives light generated by the sample TP as detection light. The detection optical system 431 includes, in order from the sample TP side, an objective lens 427 and a dichroic mirror 426. Further, the detection optical system 431 includes, in order from the dichroic mirror 426 side, a second objective lens 432, a detection field stop 433, a collimator lens 434, a mirror 435, a phase modulation unit 60, a detection filter 436, and an insert. It has a mirror removal 437 and an imaging lens 438. Further, the detection optical system 431 includes a first measurement relay lens 439 and a second measurement relay lens 440 in order from the insertion/removal mirror 437 side. In this way, the objective lens 427 and the dichroic mirror 426 are included in both the illumination optical system 421 and the detection optical system 431.

The second objective lens 432 collects detection light from the sample TP. The detection field stop 433 is arranged at a position optically conjugate with the focal position of the objective lens 427. The detection field stop 433 limits the field of view of the microscope 401. The collimator lens 434 converts the detection light from the sample TP into substantially parallel light. Mirror 435 reflects the detection light from sample TP toward phase modulation unit 60 .

The phase modulation unit 60 has the same configuration as the phase modulation unit 60 according to the first embodiment, is given the same reference numeral as in the first embodiment, and detailed description thereof will be omitted. In the fifth embodiment, the phase modulation unit 60 performs phase modulation on the detection light reflected by the mirror 435, and corrects at least part of the aberrations generated between the sample TP and the detection optical system 431.

The detection filter 436 transmits light in a predetermined wavelength band (for example, fluorescence) among the light generated by the sample TP. The detection filter 436 blocks at least a portion of, for example, illumination light reflected by the sample TP, external light, stray light, and the like. The insertion/removal mirror 437 is arranged to be removable from the optical path between the detection filter 436 and the imaging lens 438. When the insertion/removal mirror 437 leaves the optical path between the detection filter 436 and the imaging lens 438, the detection light from the sample TP enters the imaging lens 438. When the removable mirror 437 is inserted into the optical path between the detection filter 436 and the imaging lens 438, the detection light from the sample TP is reflected by the removable mirror 437 and enters the first measurement relay lens 439. .

The imaging lens 438 forms an image of the detection light from the sample TP on the detection surface of the two-dimensional detector 441. The two-dimensional detector 441 is arranged at a position optically conjugate with the sample TP. The two-dimensional detector 441 detects the detection light imaged by the imaging lens 438. As the two-dimensional detector 441, for example, a CCD (Charge Coupled Device) camera, a CMOS (Complementary Metal Oxide Semiconductor) camera, or the like is used.

The first measurement relay lens 439 collects the detection light from the sample TP. The second measurement relay lens 440 converts the detection light from the sample TP into substantially parallel light. The wavefront sensor 46 measures the aberration of the wavefront of the detection light from the sample TP.

The wavefront sensor 46 has the same configuration as the wavefront sensor 46 according to the first embodiment, is given the same reference numeral as in the first embodiment, and detailed description thereof will be omitted. Further, the arithmetic device 51, the storage section 52, the interface section 53, and the microscope control section 54 have the same configuration as the arithmetic device 51, the storage section 52, the interface section 53, and the microscope control section 54 according to the first embodiment. , are given the same reference numerals as in the first embodiment, and detailed description thereof will be omitted.

In the fifth embodiment, the microscope control unit 54 transmits a control signal to the objective lens holding unit 428 to control the position of the objective lens 427 held by the objective lens holding unit 428. The microscope control unit 54 transmits a control signal to the shutter 418 to control opening and closing of the shutter 418. The microscope control unit 54 transmits a control signal to the insertion/removal mirror 437 to control insertion/removal of the insertion/removal mirror 437.

Illumination light (excitation light) emitted from the light source 417 of the light source unit 416 passes through the collimator lens 422 of the illumination optical system 421 and becomes substantially parallel light. The illumination light that has passed through the collimator lens 422 passes through an illumination filter 423 and an illumination field diaphragm 424, and is transmitted through an illumination relay lens 425. The illumination light transmitted through the illumination relay lens 425 is reflected by the dichroic mirror 426 while converging, and enters the objective lens 427. The illumination light incident on the objective lens 427 passes through the objective lens 427 and forms an image of the illumination field stop 424 on the focal plane of the objective lens 427.

Fluorescence is emitted as detection light from a fluorescent substance contained in a portion of the sample TP where the illumination light (excitation light) forms an image of the illumination field stop 424 (that is, a portion overlapping with the focal plane of the objective lens 427). Detection light (fluorescence) generated by the sample TP enters an objective lens 427 as a detection optical system 431. The detection light that has entered the objective lens 427 passes through the objective lens 427 and enters the dichroic mirror 426 . Since the detection light (fluorescence) that has entered the dichroic mirror 426 has a different wavelength from the illumination light (excitation light), it passes through the dichroic mirror 426 and enters the second objective lens 432 .

The detection light that has entered the second objective lens 432 passes through the second objective lens 432, passes through the detection field stop 433, and enters the collimator lens 434. The detection light that has entered the collimator lens 434 passes through the collimator lens 434 to become substantially parallel light, is reflected by the mirror 435, and enters the phase modulation unit 60. When the detection light enters the phase modulation unit 60, the phase modulation unit 60 performs phase modulation on the incident detection light and emits the phase modulated detection light.

The detection light, which is substantially parallel light, emitted from the phase modulation unit 60 enters the detection filter 436. When the removable mirror 437 is removed from the optical path between the detection filter 436 and the imaging lens 438, the detection light incident on the detection filter 436 passes through the detection filter 436 and passes through the imaging lens 438, The light enters the dimensional detector 441 to form an image of the sample TP.

The two-dimensional detector 441 performs photoelectric conversion of the light (detected light) that has entered the two-dimensional detector 441, and generates data corresponding to the amount of light (brightness) of the light as a light detection signal. The two-dimensional detector 441 transmits data generated at a plurality of pixels to the microscope control unit 54. Based on the data for multiple pixels transmitted from the two-dimensional detector 441, the microscope control unit 54 generates one image data in which the data for multiple pixels are arranged two-dimensionally (in two directions), and stores the data in the storage unit 52. to be memorized. In this way, the microscope control unit 54 generates a two-dimensional image of the sample TP, thereby acquiring an image of the sample TP.

When the removable mirror 437 is inserted into the optical path between the detection filter 436 and the imaging lens 438, the detection light incident on the detection filter 436 passes through the detection filter 436, is reflected by the removable mirror 437, and is reflected by the removable mirror 437. 1 enters the measurement relay lens 439. The detection light that has entered the first measurement relay lens 439 passes through the first measurement relay lens 439 and the second measurement relay lens 440 and enters the wavefront sensor 46 . The wavefront sensor 46 measures the aberration of the wavefront of the detection light incident on the wavefront sensor 46, and transmits data regarding the aberration of the wavefront to the microscope control unit 54 as a measurement signal. The microscope control unit 54 acquires data regarding at least part of the aberrations occurring in the sample TP, the illumination optical system 421, and the detection optical system 431 based on the measurement signal transmitted from the wavefront sensor 46.

In the microscope 401 according to the fifth embodiment, the detection light reflected by the mirror 435 of the detection optical system 431 enters the third relay lens 68 of the phase modulation unit 60, but after that, the phase with respect to the detection light in the second embodiment is Since this is similar to modulation, the explanation will be omitted.

As described above, the second phase modulation scanner 66 scans the phase modulation element 65 with the detection light incident on the second phase modulation scanner 66, thereby performing detection within the plurality of irradiation areas of the phase modulation element 65. It is possible to change the irradiation area where the light is irradiated. The second phase modulation scanner 66 changes the irradiation area to which the detection light is irradiated in the phase modulation element 65 when the observation area in which the sample TP is subjected to microscopic observation is displaced. In addition, for each of the plurality of observation areas on the sample TP, a phase modulation pattern for correcting at least part of the aberrations generated between the sample TP and the detection optical system 431, that is, among the aberrations generated between the sample TP and the detection optical system 431. , a predetermined phase distribution for correcting at least a portion thereof is determined in advance. The phase modulation element 65 displays, in each irradiation area on the display surface of the phase modulation element 65, a phase modulation pattern determined in advance for the corresponding observation area. As a result, the second phase modulation scanner 66 changes the irradiation area on the phase modulation element 65 for each observation area, so that the phase of the illumination light is adjusted according to the phase modulation pattern (phase distribution) appropriately determined for each observation area. can be changed (modulated). Therefore, it is possible to obtain a high-quality image of the sample TP in a short time, in which at least a portion of the aberrations caused by the sample TP and the detection optical system 431 have been corrected.

Further, even when using the microscope 401 according to the fifth embodiment, it is possible to acquire an image of the sample TP by the image acquisition method described in the first embodiment. Therefore, according to the fifth embodiment, the same effects as the first embodiment can be obtained. Further, according to the fifth embodiment, the two-dimensional detector 441 can detect the detection light generated in one of the first to sixteenth observation areas KA1 to KA16 in the sample TP at a time, for example. , it becomes possible to obtain a high-quality image of the sample TP in a shorter time.

In the microscope 401 according to the fifth embodiment, for example, the wavefront sensor 46, the insertion/removal mirror 437, the first measurement relay lens 439, and the second measurement relay lens 440 are not provided, and the detection that enters the two-dimensional detector 441 is performed. Aberrations occurring in the sample TP, the illumination optical system 421, and the detection optical system 431 may be measured based on light. In this case, the aberration (phase modulation pattern) may be measured by a modified example of the process for measuring the aberration described in the first embodiment.

In the microscope 401 according to the fifth embodiment, a phase modulation unit 80 according to a modification of the first embodiment may be provided instead of the phase modulation unit 60. Similarly to the fifth embodiment described above, the phase modulation unit 80 according to the modification of the first embodiment can perform phase modulation on the detection light.

[Sixth embodiment]
Next, a microscope according to a sixth embodiment will be described with reference to FIG. 15. The microscope according to the sixth embodiment has the same configuration as the microscope 1 according to the first embodiment, except that the phase modulation unit 160 according to the sixth embodiment is provided instead of the phase modulation unit 60, and the microscope according to the sixth embodiment has the same configuration as the microscope 1 according to the first embodiment. Common parts other than the unit 160 are given the same reference numerals as in the first embodiment, and detailed illustrations and explanations are omitted. In the microscope according to the sixth embodiment, the phase modulation unit 160 performs phase modulation on the illumination light that has passed through the collimator lens 22, and eliminates at least aberrations caused by the sample TP, the illumination optical system 21, and the detection optical system 31. Correct some parts. As shown in FIG. 15, the phase modulation unit 160 according to the sixth embodiment includes a first relay lens 161, a first mirror 162, a second relay lens 163, a first cylindrical lens 164, and a second cylindrical lens. 165, a third relay lens 166, a phase modulation scanner 167, a fourth relay lens 168, a phase modulation element 169, a second mirror 170, and a fifth relay lens 171. Note that the portion surrounded by a broken line in FIG. 15 is a diagram of a part of the phase modulation unit 160 viewed from a direction perpendicular to FIG. 15.

The first relay lens 161 collects the illumination light that has passed through the collimator lens 22 and has become substantially parallel light. The first mirror 162 is arranged near a position B11 that is optically conjugate with the sample TP. The first mirror 162 reflects the illumination light from the first relay lens 161 toward the second relay lens 163. The second relay lens 163 converts the illumination light reflected by the first mirror 162 into substantially parallel light. The first cylindrical lens 164 and the second cylindrical lens 165 convert the illumination light from the second relay lens 163 into substantially parallel light having an elliptical cross-section. The third relay lens 166 collects the illumination light from the second cylindrical lens 165.

The phase modulation scanner 167 is arranged at a position B11 that is optically conjugate with the sample TP. The phase modulation scanner 167 is configured using a mirror (for example, a galvano mirror, a resonant mirror, etc.) held so that the direction (azimuth angle) of a reflecting surface changes by uniaxial rotation or biaxial rotation. The phase modulation scanner 167 changes the traveling direction of the illumination light transmitted through the third relay lens 166. Thereby, the phase modulation scanner 167 scans the phase modulation element 169 with the illumination light from the collimator lens 22, as shown in FIG.

Further, the phase modulation scanner 167 is configured such that the illumination light incident on the phase modulation scanner 167 from the fourth relay lens 168 travels toward the third relay lens 166, that is, the illumination light from the fourth relay lens 168 illuminates the phase modulation scanner 167. The traveling direction of the illumination light is changed so that it travels along the optical axis AX1 of the optical system 21.

The fourth relay lens 168 is arranged facing the phase modulation element 169. The fourth relay lens 168 converts the illumination light that has passed through the phase modulation scanner 167 into substantially parallel light (elliptical in cross section). The fourth relay lens 168 collects the illumination light reflected by the phase modulation element 169.

The phase modulation element 169 is arranged at a position H11 that is optically conjugate with the pupil position of the objective lens 28. The phase modulation element 169 is configured using, for example, a reflective liquid crystal element or a transmissive liquid crystal element, and displays a plurality of phase modulation patterns arranged in a direction perpendicular to the longitudinal direction of the elliptical cross section of the illumination light on the display surface. do. The display surface of the phase modulation element 169 is provided with a plurality of irradiation areas that can be irradiated with illumination light. The plurality of irradiation areas on the display surface of the phase modulation element 169 are arranged in a direction perpendicular or parallel to the longitudinal direction of the elliptical cross section of the illumination light, corresponding to the plurality of phase modulation patterns.

FIG. 16 shows an example of arrangement of a plurality of irradiation areas. In the example of FIG. 16, 16 rectangular irradiation areas that can be irradiated with illumination light are provided corresponding to 16 phase modulation patterns displayed on the display surface of the phase modulation element 169. The 16 irradiation areas include (in order from the top of FIG. 16) a first irradiation area SB1, a second irradiation area SB2, a third irradiation area SB3, a fourth irradiation area SB4, a fifth irradiation area SB5, The sixth irradiation area SB6, the seventh irradiation area SB7, the eighth irradiation area SB8, the ninth irradiation area SB9, the tenth irradiation area SB10, the eleventh irradiation area SB11, the twelfth irradiation area SB12, and the third irradiation area SB12. A thirteenth irradiation area SB13, a fourteenth irradiation area SB14, a fifteenth irradiation area SB15, and a sixteenth irradiation area SB16 are provided.

The first to sixteenth irradiation areas SB1 to SB16 are arranged in a direction perpendicular to the longitudinal direction of the elliptical cross section of the illumination light so as not to overlap each other. When any one of the first to sixteenth irradiation areas SB1 to SB16 is irradiated with illumination light, the phase of the illumination light changes (modulates). Specifically, the illumination light is given a phase distribution according to a phase modulation pattern set in the irradiation area irradiated with the illumination light. In addition, by changing the irradiation area (that is, the phase modulation pattern) on which the illumination light is irradiated in the phase modulation element 169, the phase distribution imparted to the illumination light (when the detection light is irradiated to the irradiation area, the phase distribution imparted to the detection light phase distribution).

The second mirror 170 is arranged near a position B11 that is optically conjugate with the sample TP. The second mirror 170 reflects the illumination light from the phase modulation element 169 that has passed through the second relay lens 163 toward the fifth relay lens 171 . The fifth relay lens 171 converts the illumination light reflected by the second mirror 170 into substantially parallel light.

The illumination light transmitted through the collimator lens 22 of the illumination optical system 21 enters the first relay lens 161 of the phase modulation unit 160. The illumination light incident on the first relay lens 161 is transmitted through the first relay lens 161 and reflected by the first mirror 162. The illumination light reflected by the first mirror 162 is transmitted through the second relay lens 163. The illumination light that has passed through the second relay lens 163 passes through the first cylindrical lens 164 and the second cylindrical lens 165, and becomes approximately parallel light that is elliptical in cross section. The illumination light that has passed through the second cylindrical lens 165 passes through the third relay lens 166, enters the phase modulation scanner 167, and is once focused at a position B11 that is optically conjugate with the sample TP. The phase modulation scanner 167 uses the illumination light that has entered the phase modulation scanner 167 to scan the phase modulation element 169 in a direction perpendicular to the longitudinal direction of the elliptical cross section of the illumination light. The illumination light incident on the phase modulation scanner 167 passes through the phase modulation scanner 167 and is transmitted through the fourth relay lens 168 . The illumination light transmitted through the fourth relay lens 168 becomes substantially parallel light having an elliptical cross-sectional view, and is irradiated onto one of the plurality of irradiation areas in the phase modulation element 169.

When the illumination light is irradiated onto any one of the plurality of irradiation areas (for example, the first to 16th irradiation areas SB1 to SB16) in the phase modulation element 169, the phase modulation occurs in the irradiation area irradiated with the illumination light. The phase of the illumination light changes (modulates) according to the pattern (phase distribution). Illumination light irradiated onto any one of the plurality of irradiation areas in the phase modulation element 169 is reflected at the irradiation area. Note that the phase modulation scanner 167 can change the irradiation area to which the illumination light is irradiated among the plurality of irradiation areas by scanning the phase modulation element 169 with the illumination light that enters the phase modulation scanner 167. It is.

The illumination light that is irradiated onto any one of the plurality of irradiation areas in the phase modulation element 169 and reflected is transmitted through the fourth relay lens 168 . The illumination light that has passed through the fourth relay lens 168 enters the phase modulation scanner 167 and is once focused at a position B11 that is optically conjugate with the sample TP. The phase modulation scanner 167 is arranged so that the illumination light from the fourth relay lens 168 travels toward the third relay lens 166, that is, the illumination light from the fourth relay lens 168 is directed along the optical axis AX1 of the illumination optical system 21. The direction of the illumination light is changed so that the illumination light travels along the same direction. The illumination light incident on the phase modulation scanner 167 passes through the phase modulation scanner 167 and is transmitted through the third relay lens 166 . The illumination light that has passed through the third relay lens 166 passes through the second cylindrical lens 165 and the first cylindrical lens 164 and becomes substantially parallel light that is circular in cross-section. The illumination light that has passed through the first cylindrical lens 164 passes through the second relay lens 163 and is reflected by the second mirror 170 . The illumination light reflected by the second mirror 170 passes through the fifth relay lens 171 and becomes substantially parallel light, which enters the dichroic mirror 24 of the illumination optical system 21 . In this way, the phase modulation unit 160 performs phase modulation on the illumination light.

Note that since the phase modulation element 169 is scanned by the illumination light passing through the phase modulation scanner 167, the illumination light reflected from any one of the plurality of irradiation areas in the phase modulation element 169 is reflected by the illumination optical system 21. It may deviate from the optical axis AX1. However, when the illumination light reflected from one of the plurality of irradiation areas passes through the fourth relay lens 168 and passes through the phase modulation scanner 167, it proceeds along the optical axis AX1 of the illumination optical system 21. It becomes like this. By descanning the illumination light in the phase modulation scanner 167, even though the phase modulation scanner 167 changes the traveling direction of the illumination light and changes the irradiation area, the illumination light transmitted through the fifth relay lens 171 remains approximately parallel. The light can be incident on the dichroic mirror 24 of the illumination optical system 21 as light.

As described above, the phase modulation scanner 167 scans the phase modulation element 169 with the illumination light incident on the phase modulation scanner 167, thereby changing the irradiation area to which the illumination light is irradiated among the plurality of irradiation areas. Is possible. The phase modulation scanner 167 changes the irradiation area on which the illumination light is irradiated in the phase modulation element 169 when the observation area on the sample TP where microscopic observation is performed is displaced. In addition, for each of the 16 observation areas on the sample TP, a phase modulation pattern is created for correcting at least part of the aberrations generated between the sample TP, the illumination optical system 21, and the detection optical system 31, that is, the sample TP and the illumination optical system. A predetermined phase distribution for correcting at least part of the aberrations occurring in the detection optical system 31 and the detection optical system 31 is determined in advance. The phase modulation element 169 displays, on each irradiation area on the display surface of the phase modulation element 169, a phase modulation pattern determined in advance for the corresponding observation area. As a result, the phase modulation scanner 167 changes the irradiation area on the phase modulation element 169 for each of the 16 observation areas, thereby adjusting the illumination light according to the phase modulation pattern (phase distribution) appropriately determined for each observation area. The phase can be changed (modulated). Therefore, it is possible to obtain a high-quality image of the sample TP in a short time, in which at least a portion of the aberrations caused by the sample TP, the illumination optical system 21, and the detection optical system 31 have been corrected.

A plurality of observation areas in the sample TP can be set in the same manner as the observation areas illustrated in the first embodiment. Specifically, as shown in FIG. 6, 16 observation areas KA1 to KA16 in the sample TP are set within the field of view of the microscope. The phase modulation element 169 displays a phase modulation pattern previously determined for the first observation area KA1 in the first irradiation area SB1 of the phase modulation element 169. The phase modulation element 169 has a second irradiation area SB of the phase modulation element 169.
2, a phase modulation pattern determined in advance for the second observation area KA2 is displayed. The phase modulation element 169 displays a phase modulation pattern previously determined for the third observation area KA3 in the third irradiation area SB3 of the phase modulation element 169. The phase modulation element 169 displays a phase modulation pattern previously determined for the fourth observation area KA4 in the fourth irradiation area SB4 of the phase modulation element 169. Similarly, the phase modulation element 169 displays phase modulation patterns previously determined for the fifth to sixteenth observation areas KA5 to KA16 on the fifth to sixteenth irradiation areas SB5 to SB16 of the phase modulation element 169.

When observing the first to sixteenth observation areas KA1 to KA16 in the sample TP, that is, when acquiring images of the first to sixteenth observation areas KA1 to KA16, first, an image of the first observation area KA1 is acquired. When acquiring an image of the first observation area KA1, the first phase modulation scanner 167 of the phase modulation unit 160 changes the irradiation area of the illumination light onto the phase modulation element 169 to the first irradiation area SB1. The sample scanning unit 25 of the illumination optical system 21 performs raster scanning in the first observation area KA1 using the illumination light LB1 irradiated onto the first irradiation area SB1 of the phase modulation element 169. Therefore, it is possible to obtain a high-quality image of the first observation area KA1 in which aberrations caused by the first observation area KA1 in the sample TP are corrected.

After acquiring an image of the first observation area KA1, an image of the second observation area KA2 is acquired. When acquiring an image of the second observation area KA2, the first phase modulation scanner 167 of the phase modulation unit 160 changes the irradiation area of the illumination light onto the phase modulation element 169 from the first irradiation area SB1 to the second irradiation area SB2. . The sample scanning unit 25 of the illumination optical system 21 performs raster scanning in the second observation area KA2 using the illumination light LB2 irradiated onto the second irradiation area SB2 of the phase modulation element 169. Therefore, it is possible to obtain a high-quality image of the second observation area KA2 in which aberrations caused by the second observation area KA2 in the sample TP are corrected.

Similarly, after acquiring the image of the second observation area KA2, images of the third to sixteenth observation areas KA3 to KA16 are acquired in order. Note that when the observation area in the sample TP is displaced to the third to sixteenth observation areas KA3 to KA16, the first phase modulation scanner 167 of the phase modulation unit 160 shifts the irradiation area of the illumination light onto the phase modulation element 169 to the third to sixteenth observation areas KA3 to KA16. The irradiation area is changed from SB3 to the 16th irradiation area SB16. This makes it possible to obtain a high-quality image of the sample TP in a short time, in which aberrations caused by the first to sixteenth observation areas KA1 to KA16 in the sample TP are individually corrected.

Further, even when using the microscope according to the sixth embodiment, it is possible to acquire an image of the sample TP by the image acquisition method described in the first embodiment. Therefore, according to the sixth embodiment, the phase modulation unit 160 has a plurality of irradiation areas that can be irradiated with illumination light, and has a phase distribution for the illumination light according to the irradiation position of the illumination light in the plurality of irradiation areas. It has a phase modulation element 169 that provides a phase modulation element 169, and a phase modulation scanner 167 that changes the irradiation area that is irradiated with illumination light. If the phase modulation pattern that changes the phase of the illumination light is changed by changing the irradiation area of the illumination light on the phase modulation element 169 using the phase modulation scanner 167, the phase modulation pattern displayed by the phase modulation element 169 can be changed. The phase modulation pattern can be changed in a shorter time than when switching the phase modulation pattern. Therefore, by changing the irradiation area of the illumination light on the phase modulation element 169 when the observation area on the sample TP is displaced, a high-quality sample can be obtained in which aberrations caused by a plurality of observation areas on the sample TP are individually corrected. It becomes possible to acquire images of TP in a short time.

In addition, the phase modulation unit 160 keeps the traveling direction of the illumination light whose phase has changed by irradiating the irradiation region in a constant direction (the illumination optical system 21 The phase modulation scanner 167 has a phase modulation scanner 167 (direction along the optical axis AX1). Thereby, only the phase of the illumination light can be changed without changing the optical path of the illumination light.

Further, the plurality of irradiation areas in the phase modulation element 169 are arranged so that they do not overlap with each other. Thereby, an appropriate phase modulation pattern can be provided for each image acquisition range of the sample TP.

Further, the phase modulation unit 160 includes a first cylindrical lens 164 and a second cylindrical lens 165 that make the cross-sectional shape of the illumination light irradiated onto one of the plurality of irradiation areas elliptical. This makes it possible to display a phase modulation pattern having a smooth phase distribution in the longitudinal direction of the irradiation area in the phase modulation element 169.

[Modified example of phase modulation unit]
Next, a modification of the phase modulation unit in the sixth embodiment will be described with reference to FIG. 17. As shown in FIG. 17, the phase modulation unit 180 according to the modification of the sixth embodiment includes a beam splitter 191, a first relay lens 181, a mirror 182, a second relay lens 183, and a first cylindrical lens 184. , a second cylindrical lens 185 , a third relay lens 186 , a phase modulation scanner 187 , a fourth relay lens 188 , and a phase modulation element 169 . Note that the part surrounded by a broken line in FIG. 17 is a diagram of a part of the phase modulation unit 180 viewed from a direction perpendicular to FIG. 17.

The ratio of transmittance and reflectance of the beam splitter 191 is set to, for example, 1:1. A part of the light that enters the beam splitter 191 from the collimator lens 22 of the illumination optical system 21 passes through the beam splitter 191 and enters the first relay lens 181 . A part of the light that has entered the beam splitter 191 from the first relay lens 181 is reflected by the beam splitter 191 and enters the dichroic mirror 24 of the illumination optical system 21 .

The first relay lens 181 collects the illumination light that has passed through the collimator lens 22 and has become substantially parallel light. The mirror 182 is placed near a position B12 that is optically conjugate with the sample TP. Mirror 182 reflects the illumination light from first relay lens 181 toward second relay lens 183 . The second relay lens 183 converts the illumination light reflected by the mirror 182 into substantially parallel light. The first cylindrical lens 184 and the second cylindrical lens 185 convert the illumination light from the second relay lens 183 into substantially parallel light having an elliptical cross-sectional view. The third relay lens 186 collects the illumination light from the second cylindrical lens 185.

The phase modulation scanner 187 is arranged at a position B12 that is optically conjugate with the sample TP. The phase modulation scanner 187 is configured using a mirror (for example, a galvano mirror, a resonant mirror, etc.) held so that the direction (azimuth angle) of a reflecting surface changes by uniaxial rotation or biaxial rotation. The phase modulation scanner 187 changes the traveling direction of the illumination light transmitted through the third relay lens 186. Thereby, the phase modulation scanner 187 scans the phase modulation element 169 with the illumination light from the collimator lens 22, as shown in FIG.

Further, the phase modulation scanner 187 is configured such that the illumination light incident on the phase modulation scanner 187 from the fourth relay lens 188 travels toward the third relay lens 186, that is, the illumination light from the fourth relay lens 188 illuminates the phase modulation scanner 187. The traveling direction of the illumination light is changed so that it travels along the optical axis AX1 of the optical system 21.

The fourth relay lens 188 is arranged facing the phase modulation element 169. The fourth relay lens 188 converts the illumination light that has passed through the phase modulation scanner 187 into substantially parallel light (elliptical in cross section). The fourth relay lens 188 collects the illumination light reflected by the phase modulation element 169.

The phase modulation element 169 is arranged at a position H12 that is optically conjugate with the pupil position of the objective lens 28. The phase modulation element 169 has the same configuration as the phase modulation element 169 according to the sixth embodiment, is given the same reference numeral as in the sixth embodiment, and detailed description thereof will be omitted.

The illumination light transmitted through the collimator lens 22 of the illumination optical system 21 enters the beam splitter 191 of the phase modulation unit 180. A part of the illumination light that has entered the beam splitter 191 from the collimator lens 22 passes through the beam splitter 191 and enters the first relay lens 181 . The illumination light incident on the first relay lens 181 is transmitted through the first relay lens 181 and reflected by the mirror 182. The illumination light reflected by the mirror 182 is transmitted through the second relay lens 183. The illumination light that has passed through the second relay lens 183 passes through the first cylindrical lens 184 and the second cylindrical lens 185 and becomes approximately parallel light having an elliptical cross-sectional view. The illumination light that has passed through the second cylindrical lens 185 passes through the third relay lens 186, enters the phase modulation scanner 187, and is once focused at a position B12 that is optically conjugate with the sample TP. The phase modulation scanner 187 uses the illumination light that has entered the phase modulation scanner 187 to scan the phase modulation element 169 in a direction perpendicular or parallel to the longitudinal direction of the elliptical cross section of the illumination light. The illumination light incident on the phase modulation scanner 187 passes through the phase modulation scanner 187 and is transmitted through the fourth relay lens 188 . The illumination light transmitted through the fourth relay lens 188 becomes substantially parallel light having an elliptical cross-sectional view, and is irradiated onto one of the plurality of irradiation areas in the phase modulation element 169.

When the illumination light is irradiated onto any one of the plurality of irradiation areas (for example, the first to 16th irradiation areas SB1 to SB16) in the phase modulation element 169, phase modulation occurs in the irradiation area irradiated with the illumination light. The phase of the illumination light changes depending on the pattern. Illumination light irradiated onto any one of the plurality of irradiation areas in the phase modulation element 169 is reflected at the irradiation area. Note that the phase modulation scanner 187 can change the irradiation area to which the illumination light is irradiated among the plurality of irradiation areas by scanning the phase modulation element 169 with the illumination light that enters the phase modulation scanner 187. It is.

The illumination light that is irradiated onto any one of the plurality of irradiation areas in the phase modulation element 169 and reflected is transmitted through the fourth relay lens 188 . The illumination light that has passed through the fourth relay lens 188 enters the phase modulation scanner 187 and is once focused at a position B12 that is optically conjugate with the sample TP. The phase modulation scanner 187 is arranged so that the illumination light from the fourth relay lens 188 travels toward the third relay lens 186, that is, the illumination light from the fourth relay lens 188 is directed along the optical axis AX1 of the illumination optical system 21. The direction of the illumination light is changed so that the illumination light travels along the same direction. The illumination light incident on the phase modulation scanner 187 passes through the phase modulation scanner 187 and is transmitted through the third relay lens 186. The illumination light that has passed through the third relay lens 186 passes through the second cylindrical lens 185 and the first cylindrical lens 184 and becomes approximately parallel light that is circular in cross-section. The illumination light that has passed through the first cylindrical lens 184 passes through the second relay lens 183 and is reflected by the mirror 182. The illumination light reflected by the mirror 182 passes through the first relay lens 181, becomes substantially parallel light, and enters the beam splitter 191. A part of the illumination light that has entered the beam splitter 191 from the first relay lens 181 is reflected by the beam splitter 191 and enters the dichroic mirror 24 of the illumination optical system 21 . Thereby, the phase modulation of the illumination light is performed in the same manner as in the case of the phase modulation unit 160 according to the sixth embodiment.

[Seventh embodiment]
Next, a microscope according to a seventh embodiment will be described. The microscope according to the seventh embodiment has the same configuration as the microscope 101 according to the second embodiment, except that the phase modulation unit 160 according to the sixth embodiment is provided instead of the phase modulation unit 60, and Common parts other than the unit 160 are given the same reference numerals as in the second embodiment, and detailed illustrations and explanations are omitted. In the microscope according to the seventh embodiment, the phase modulation unit 160 performs phase modulation on the illumination light that has passed through the dichroic mirror 123 of the illumination optical system 121, and also performs phase modulation on the illumination light that has passed through the sample scanning unit 125 as the detection optical system 131. Phase modulation is performed on the detection light to correct at least part of the aberrations that occur in the sample TP, the illumination optical system 121, and the detection optical system 131.

The illumination light that has passed through the dichroic mirror 123 of the illumination optical system 121 is incident on the first relay lens 161 of the phase modulation unit 160, but the subsequent steps are the same as in the sixth embodiment, so the explanation will be omitted.

Further, the detection light that has passed through the sample scanning section 125 as the detection optical system 131 is incident on the fifth relay lens 171 of the phase modulation unit 160. The detection light incident on the fifth relay lens 171 is transmitted to the fifth relay lens 171, the second mirror 170, the second relay lens 163, the first cylindrical lens 164, and the second cylindrical lens in the reverse order of the illumination light. The illumination light passes through the lens 165, the third relay lens 166, the phase modulation scanner 167, and the fourth relay lens 168, and becomes a substantially parallel light having an elliptical cross-section. The same irradiation area is irradiated.

When the detection light is irradiated onto the same irradiation area in the phase modulation element 169 as the illumination light, the phase of the illumination light changes according to the phase modulation pattern in the irradiation area. The detection light irradiated onto the same irradiation area as the illumination light in the phase modulation element 169 is reflected at the irradiation area.

The detection light that is irradiated onto the same irradiation area as the illumination light in the phase modulation element 169 and reflected is transmitted to the fourth relay lens 168, the phase modulation scanner 167, the third relay lens 166, the second cylindrical lens 165, and the first The light passes through the cylindrical lens 164 and the second relay lens 163 and is reflected by the first mirror 162. The detection light reflected by the first mirror 162 passes through the first relay lens 161, becomes substantially parallel light, and enters the dichroic mirror 123 serving as the detection optical system 131. In this way, the phase modulation unit 160 performs phase modulation on the detection light.

Note that the detection light reflected from the same irradiation area as the illumination light in the phase modulation element 169 passes through the fourth relay lens 168 and passes through the phase modulation scanner 167, and then proceeds along the optical axis AX12 of the detection optical system 131. It becomes like this. Although the phase modulation scanner 167 changes the traveling direction of the detection light and changes the irradiation area by descanning the detection light in the phase modulation scanner 167, the detection light transmitted through the first relay lens 161 remains approximately parallel. The light becomes light and can be incident on the dichroic mirror 123 as the detection optical system 131.

The phase modulation scanner 167 scans the phase modulation element 169 with the illumination light and detection light incident on the phase modulation scanner 167, so that the illumination light and detection light irradiate within a plurality of irradiation areas in the phase modulation element 169. It is possible to change the irradiated area. The phase modulation scanner 167 changes the irradiation area to which the illumination light and detection light are irradiated in the phase modulation element 169 when the observation area in which the sample TP is subjected to microscopic observation is displaced. Note that, for each of the 16 observation areas on the sample TP, a phase modulation pattern is created for correcting at least part of the aberrations generated between the sample TP, the illumination optical system 121, and the detection optical system 131, that is, the sample TP and the illumination optical system. A predetermined phase distribution for correcting at least part of the aberrations caused by the detection optical system 121 and the detection optical system 131 is determined in advance. The phase modulation element 169 displays, in each irradiation area on the display surface of the phase modulation element 169, a phase modulation pattern determined in advance for the corresponding observation area. As a result, the phase modulation scanner 167 changes the irradiation area on the phase modulation element 169 for each of the 16 observation areas, thereby adjusting the illumination light according to the phase modulation pattern (phase distribution) appropriately determined for each observation area. The phase can be changed (modulated). Therefore, it is possible to obtain a high-quality image of the sample TP in a short time, in which at least part of the aberrations caused by the sample TP, the illumination optical system 121, and the detection optical system 131 are corrected.

Further, even when using the microscope according to the seventh embodiment, it is possible to acquire an image of the sample TP using the image acquisition method described in the first embodiment, similarly to the microscope 101 according to the second embodiment. Therefore, according to the seventh embodiment, the same effects as the sixth embodiment and the second embodiment can be obtained.

In the microscope according to the seventh embodiment, a phase modulation unit 180 according to a modification of the sixth embodiment may be provided instead of the phase modulation unit 160. Similarly to the seventh embodiment described above, the phase modulation unit 180 according to the modification of the sixth embodiment can perform phase modulation on the illumination light and the detection light.

[Eighth embodiment]
Next, a microscope according to an eighth embodiment will be described. The microscope according to the eighth embodiment has the same configuration as the microscope 201 according to the third embodiment, except that the phase modulation unit 160 according to the sixth embodiment is provided instead of the phase modulation unit 60. Common parts other than the unit 160 are given the same reference numerals as in the third embodiment, and detailed illustrations and explanations are omitted.

In the microscope according to the eighth embodiment, the phase modulation unit 160 performs phase modulation on the illumination light transmitted through the collimator lens 222 of the illumination optical system 221, and eliminates at least aberrations occurring between the sample TP and the illumination optical system 221. Correct some parts. At this time, the phase modulation unit 160 performs phase modulation on the illumination light in the same manner as in the sixth embodiment. Further, even when using the microscope according to the eighth embodiment, it is possible to acquire an image of the sample TP using the image acquisition method described in the first embodiment, similarly to the microscope 201 according to the third embodiment. Therefore, according to the eighth embodiment, the same effects as the sixth embodiment and the third embodiment can be obtained.

In the microscope according to the eighth embodiment, a phase modulation unit 180 according to a modification of the sixth embodiment may be provided instead of the phase modulation unit 160. Similarly to the modification of the sixth embodiment described above, the phase modulation unit 180 according to the modification of the sixth embodiment can perform phase modulation on illumination light.

[Ninth embodiment]
Next, a microscope according to a ninth embodiment will be described. The microscope according to the ninth embodiment has the same configuration as the microscope 301 according to the fourth embodiment, except that the phase modulation unit 160 according to the sixth embodiment is provided instead of the phase modulation unit 60. Common parts other than the unit 160 are given the same reference numerals as in the fourth embodiment, and detailed illustrations and explanations are omitted.

In the microscope according to the ninth embodiment, the phase modulation unit 160 performs phase modulation on the illumination light transmitted through the collimator lens 322 of the illumination optical system 321, and eliminates at least aberrations occurring between the sample TP and the illumination optical system 321. Correct some parts. At this time, the phase modulation unit 160 performs phase modulation on the illumination light in the same manner as in the sixth embodiment. Further, even when using the microscope according to the ninth embodiment, it is possible to acquire an image of the sample TP using the image acquisition method described in the first embodiment, similarly to the microscope 301 according to the fourth embodiment. Therefore, according to the ninth embodiment, the same effects as the sixth embodiment and the fourth embodiment can be obtained.

In the microscope according to the ninth embodiment, a phase modulation unit 180 according to a modification of the sixth embodiment may be provided instead of the phase modulation unit 160. Similarly to the modification of the sixth embodiment described above, the phase modulation unit 180 according to the modification of the sixth embodiment can perform phase modulation on illumination light.

[Tenth embodiment]
Next, a microscope according to a tenth embodiment will be described. The microscope according to the tenth embodiment has the same configuration as the microscope 401 according to the fifth embodiment, except that the phase modulation unit 160 according to the sixth embodiment is provided instead of the phase modulation unit 60. Common parts other than the unit 160 are given the same reference numerals as in the fifth embodiment, and detailed illustrations and explanations are omitted. In the microscope according to the tenth embodiment, the phase modulation unit 160 performs phase modulation on the detection light reflected by the mirror 435 of the detection optical system 431, and eliminates at least one of the aberrations generated between the sample TP and the detection optical system 431. Correct the part.

The detection light reflected by the mirror 435 of the detection optical system 431 enters the fifth relay lens 171 of the phase modulation unit 160, but the rest is the same as in the seventh embodiment, so the explanation will be omitted.

As described above, the phase modulation scanner 167 scans the phase modulation element 169 with the detection light incident on the phase modulation scanner 167, thereby changing the irradiation area to which the detection light is irradiated among the plurality of irradiation areas. Is possible. The phase modulation scanner 167 changes the irradiation area to which the detection light is irradiated in the phase modulation element 169 when the observation area of the sample TP where microscopic observation is performed is displaced. Note that, for each of the 16 observation areas on the sample TP, a phase modulation pattern is created for correcting at least part of the aberrations generated between the sample TP, the illumination optical system 421, and the detection optical system 431, that is, the sample TP and the illumination optical system. A predetermined phase distribution for correcting at least part of the aberrations caused by the detection optical system 421 and the detection optical system 431 is determined in advance. The phase modulation element 169 displays, in each irradiation area on the display surface of the phase modulation element 169, a phase modulation pattern determined in advance for the corresponding observation area. As a result, the phase modulation scanner 167 changes the irradiation area on the phase modulation element 169 for each of the 16 observation areas, and the detection light is adjusted according to the phase modulation pattern (phase distribution) appropriately determined for each observation area. The phase can be changed (modulated). Therefore, it is possible to obtain a high-quality image of the sample TP in a short time, in which at least part of the aberrations caused by the sample TP, the illumination optical system 421, and the detection optical system 431 have been corrected.

Further, even when using the microscope according to the tenth embodiment, it is possible to acquire an image of the sample TP by the image acquisition method described in the first embodiment, similarly to the microscope 401 according to the fifth embodiment. Therefore, according to the tenth embodiment, the same effects as the sixth and fifth embodiments can be obtained.

In the microscope according to the tenth embodiment, a phase modulation unit 180 according to a modification of the sixth embodiment may be provided instead of the phase modulation unit 160. Similarly to the tenth embodiment described above, the phase modulation unit 180 according to the modification of the sixth embodiment can perform phase modulation on the detection light.

[Eleventh embodiment]
Next, referring to FIG. 18, a microscope 501 according to an eleventh embodiment will be described. The microscope 501 according to the eleventh embodiment is also referred to as a scanning microscope. A microscope 501 according to the eleventh embodiment includes a stage 11, a light source unit 16, an illumination optical system 521, a detection optical system 531, an array type detector 541, a wavefront sensor 46, a calculation device 51, and a storage section. 52, an interface section 53, and a microscope control section 54. The stage 11 and the light source unit 16 have the same configuration as the stage 11 and the light source unit 16 according to the first embodiment, are given the same reference numerals as in the first embodiment, and detailed description thereof will be omitted.

The illumination optical system 521 illuminates the sample TP with illumination light (excitation light) emitted from the light source unit 16. The illumination optical system 521 includes, in order from the light source unit 16 side, a collimator lens 522, a phase modulation unit 560, a dichroic mirror 524, a sample scanning section 525, a scan lens 526, a second objective lens 527, and an objective lens 528. and has. The collimator lens 522 converts the illumination light emitted from the light source unit 16 into substantially parallel light, and the phase modulation unit 560 performs phase modulation on the illumination light to eliminate at least aberrations occurring between the sample TP and the illumination optical system 521. Correct some parts. The configurations and functions of the collimator lens 522, dichroic mirror 524, sample scanning unit 525, scan lens 526, second objective lens 527, and objective lens 528 included in the illumination optical system 521 are the same as those in the first embodiment. The collimator lens 22, dichroic mirror 24, sample scanning unit 25, scan lens 26, second objective lens 27, and objective lens 28 included in the illumination optical system 21 of the microscope are the same, so the explanation will be omitted.

The detection optical system 531 receives light generated by the sample TP as detection light. The detection optical system 531 includes, in order from the sample TP side, an objective lens 528, a second objective lens 527, a scan lens 526, a sample scanning section 525, and a dichroic mirror 524. Further, the detection optical system 531 includes, in order from the dichroic mirror 524 side, a detection filter 532, a condensing lens 533, an insertion/removal mirror 534, and a detection pinhole plate 535. Further, the detection optical system 531 includes a measurement pinhole plate 536 and a measurement relay lens 537. These functions and configurations other than the detection pinhole plate 535 are the same as those in the first embodiment, so their explanations will be omitted. In this way, the objective lens 528, the second objective lens 527, the scan lens 526, the sample scanning section 525, and the dichroic mirror 524 are included in both the illumination optical system 521 and the detection optical system 531.

The detection pinhole plate 535 is formed into a plate shape having a plurality of pinpoles (for example, three pinholes) arranged in a direction perpendicular to the optical axis AX52 of the detection optical system 531, and is optically conjugate with the sample TP. placed in position. The array type detector 541 is configured with a plurality of detectors (for example, three detectors) arranged opposite to the three pinpoles of the detection pinhole plate 535, and the detection pinhole plate 135 is A plurality of passing detection lights (for example, three detection lights) are detected. As a detector constituting the array type detector 541, for example, a photomultiplier tube, a photodiode, an avalanche photodiode, or the like is used.

The wavefront sensor 46 has the same configuration as the wavefront sensor 46 according to the first embodiment, is given the same reference numeral as in the first embodiment, and detailed description thereof will be omitted. Further, the arithmetic device 51, the storage section 52, the interface section 53, and the microscope control section 54 have the same configuration as the arithmetic device 51, the storage section 52, the interface section 53, and the microscope control section 54 according to the first embodiment. , are given the same reference numerals as in the first embodiment, and detailed description thereof will be omitted.

In the eleventh embodiment, the microscope control unit 54 transmits a control signal to the sample scanning unit 525 to control the traveling direction of the illumination light and detection light that are changed by the sample scanning unit 525. The microscope control unit 54 transmits a control signal to the objective lens holding unit 529 to control the position of the objective lens 528 held by the objective lens holding unit 529. The microscope control unit 54 transmits a control signal to the insertion/removal mirror 534 to control insertion/removal of the insertion/removal mirror 534. The microscope control unit 54 transmits a control signal to the phase modulation unit 560 to control the phase modulation of the illumination light by the phase modulation unit 560.

Illumination light (excitation light) emitted from the light source 17 of the light source unit 16 passes through the collimator lens 522 of the illumination optical system 521 and becomes substantially parallel light. The illumination light transmitted through the collimator lens 522 enters the phase modulation unit 560. When the illumination light enters the phase modulation unit 560, the phase modulation unit 560 phase-modulates the input illumination light and emits a plurality of phase-modulated illumination lights (for example, three illumination lights). Thereafter, the three illumination lights pass through a dichroic mirror 524, a sample scanning unit 525, a scan lens 526, and a second objective lens 527, and are focused on the focal plane of an objective lens 528. The sample scanning unit 525 scans multiple (for example, three) portions of the sample TP where the illumination light is focused (i.e., portions that overlap with the focal plane of the objective lens 128) in the x direction and the y direction. It is scanned two-dimensionally in the direction.

Fluorescence is emitted as detection light from fluorescent substances contained in three parts of the sample TP where illumination light (excitation light) is focused. The three detection lights (fluorescence) generated at three parts of the sample TP enter the objective lens 528 as the detection optical system 531, but then pass through the second objective lens 527, the scan lens 526, and the sample scanning unit 525. , the dichroic mirror 524, the detection filter 532, and the detection filter 532 before being transmitted through the condensing lens 533, which is the same as the first embodiment except that the detection light from three parts is transmitted, so a description thereof will be omitted.

When the removable mirror 534 is removed from the optical path between the condenser lens 533 and the detection pinhole plate 535, the three detection lights that have passed through the condenser lens 533 reach the detection pinhole plate 535. The detection light collected at a position conjugate with the focal position of the objective lens 528 passes through the detection pinhole plate 535 and enters the array type detector 541. The array type detector 541 performs photoelectric conversion of the light (detection light) that has entered each of the three detectors constituting the array type detector 541, and generates a light detection signal corresponding to the amount of light (brightness) of the light. Generate data to The array type detector 541 transmits data generated by the three detectors to the microscope control unit 54.

When the insertion/removal mirror 534 is inserted into the optical path between the condenser lens 533 and the detection pinhole plate 535, the optical axis AX52 of the detection optical system 531 of the three detection lights transmitted through the condenser lens 533 is The passing detection light is reflected by the insertion/removal mirror 534 , reaches the measurement pinhole plate 536 , passes through the measurement relay lens 537 , and enters the wavefront sensor 46 . The wavefront sensor 46 measures the aberration of the wavefront of the detection light incident on the wavefront sensor 46, and transmits data regarding the wavefront aberration as a measurement signal to the microscope control unit 54. The microscope control unit 54 acquires data regarding at least part of the aberrations occurring in the sample TP, the illumination optical system 521, and the detection optical system 531 based on the measurement signal transmitted from the wavefront sensor 46.

In the microscope 501 according to the eleventh embodiment, a beam splitter (not shown) is provided in place of the removable mirror 534, and part of the detection light incident on the beam splitter is transmitted through the beam splitter for array type detection. The configuration may be such that another part of the detection light that reaches the device 541 and enters the beam splitter is reflected by the beam splitter and reaches the wavefront sensor 46 .

[Configuration of phase modulation unit]
Next, a phase modulation unit 560 according to the eleventh embodiment will be described with reference to FIG. 19. As shown in FIG. 19, the phase modulation unit 560 according to the eleventh embodiment includes a first relay lens 561, a first mirror 562, a second relay lens 563, a first cylindrical lens 564, and a second cylindrical lens. 565, a third relay lens 566, a phase modulation scanner 567, a fourth relay lens 568, a phase modulation element 569, a second mirror 570, a fifth relay lens 571, a microlens array 572, and a third relay lens 566. 6 relay lens 573. Note that the portion surrounded by a broken line in FIG. 19 is a diagram of a part of the phase modulation unit 560 viewed from a direction perpendicular to FIG. 19.

Phase modulation unit 560 includes a first relay lens 561, a first mirror 562, a second relay lens 563, a first cylindrical lens 564, a second cylindrical lens 565, a third relay lens 566, and phase modulation. The functions and configurations of the scanner 567, the fourth relay lens 568, the phase modulation element 569, the second mirror 570, and the fifth relay lens 571 are the same as those of the phase modulation unit according to the sixth embodiment shown in FIG. 160 includes a first relay lens 161, a first mirror 162, a second relay lens 163, a first cylindrical lens 164, a second cylindrical lens 165, a third relay lens 166, and a phase modulation scanner 167. The fourth relay lens 168, the phase modulation element 169, the second mirror 170, and the fifth relay lens 171 are the same, so their explanation will be omitted. Note that the phase modulation element 569 is arranged at a position H21 that is optically conjugate with the pupil position of the objective lens 528.

FIG. 20 shows an example of arrangement of a plurality of irradiation areas. In the example of FIG. 20, three rectangular irradiation areas that can be irradiated with illumination light are provided corresponding to three phase modulation patterns displayed on the display surface of the phase modulation element 569. The three irradiation areas are provided (in order from the top in FIG. 20): a first irradiation area SC1, a second irradiation area SC2, and a third irradiation area SC3.

The first to third irradiation areas SC1 to SC3 are arranged in a direction perpendicular to the longitudinal direction of the elliptical cross section of the illumination light so as not to overlap each other. When any one of the first to third irradiation areas SC1 to SC3 is irradiated with illumination light, the phase of the illumination light changes (modulates). Specifically, the illumination light is given a phase distribution according to a phase modulation pattern set in the irradiation area irradiated with the illumination light. Further, by changing the irradiation area (that is, the phase modulation pattern) on which the illumination light is irradiated in the phase modulation element 569, it is possible to change the phase distribution imparted to the illumination light.

The microlens array 572 includes three microlenses arranged in a direction perpendicular to the optical axis AX51 of the illumination optical system 521, and divides the illumination light transmitted through the fifth relay lens 571 into three illumination lights. The sixth relay lens 573 collects the three illumination lights separated by the microlens array 572. Note that although FIG. 19 shows an example in which the number of microlenses in the microlens array 572 is three, the number is not limited to this, and these numbers are It is sufficient if the number is the same as the number of pinhole and array type detectors 541). The number of microlenses is the same in the modified example of the eleventh embodiment, and the description thereof will be omitted.

The illumination light that has passed through the collimator lens 522 of the illumination optical system 521 is irradiated onto one of the plurality of irradiation areas in the phase modulation element 569 disposed in the phase modulation unit 560. When the illumination light is irradiated onto any one of the plurality of irradiation areas (for example, the first to third irradiation areas SC1 to SC3) in the phase modulation element 569, the phase modulation occurs in the irradiation area irradiated with the illumination light. The phase of the illumination light changes (modulates) according to the pattern (phase distribution). Illumination light irradiated onto any one of the plurality of irradiation areas in the phase modulation element 569 is reflected at the irradiation area. Note that the phase modulation scanner 567 can change the irradiation area to which the illumination light is irradiated among the plurality of irradiation areas by scanning the phase modulation element 569 with the illumination light that enters the phase modulation scanner 567. It is. The illumination light reflected by the second mirror 570 passes through the fifth relay lens 571 to become substantially parallel light, and enters the microlens array 572.

The illumination light incident on the microlens array 572 passes through the microlens array 572 and is divided into three illumination lights. The three illumination lights separated in the microlens array 572 are temporarily condensed at a position B21 that is optically conjugate with the sample TP while remaining substantially parallel to each other, and then enter the sixth relay lens 573. The three illumination lights that entered the sixth relay lens 573 pass through the sixth relay lens 573 and enter the dichroic mirror 524 of the illumination optical system 521. In this way, the phase modulation unit 560 performs phase modulation on the illumination light and multi-points the illumination light. By descanning the illumination light in the phase modulation scanner 567, even though the phase modulation scanner 567 changes the traveling direction of the illumination light and changes the irradiation area, the illumination light that has passed through the fifth relay lens 171 remains approximately parallel. The light can become light and enter the microlens array 572.

In addition, for each of the three left and right observation areas of the sample TP, a phase modulation pattern is created for correcting at least part of the aberrations generated between the sample TP, the illumination optical system 521, and the detection optical system 531, that is, the sample TP and the illumination optical system. A predetermined phase distribution for correcting at least part of the aberrations caused by the detection optical system 521 and the detection optical system 531 is determined in advance. The phase modulation element 569 displays, in each irradiation area on the display surface of the phase modulation element 569, a phase modulation pattern determined in advance for the corresponding observation area. As a result, the phase modulation scanner 567 changes the irradiation area on the phase modulation element 569 for each of the three left and right observation areas, thereby adjusting the illumination light according to the phase modulation pattern (phase distribution) appropriately determined for each observation area. The phase can be changed (modulated). Therefore, it is possible to obtain a high-quality image of the sample TP in a short time, in which at least a portion of the aberrations caused by the sample TP, the illumination optical system 521, and the detection optical system 531 are corrected.

FIG. 21 shows an example of the arrangement of a plurality of observation areas in the sample TP. In the example of FIG. 21, nine observation areas in the sample TP are set within the field of view of the microscope 501. Among the nine observation areas, in the first row (from the top of FIG. 21), a first observation area KC1, a second observation area KC2, and a third observation area KC3 are set in order from the left. Among the nine observation areas, in the second row (from the top of FIG. 21), a fourth observation area KC4, a fifth observation area KC5, and a sixth observation area KC6 are set in order from the left. Among the nine observation areas, in the third row (from the top of FIG. 21), a seventh observation area KC7, an eighth observation area KC8, and a ninth observation area KC9 are set in order from the left.

In the example shown in FIGS. 20 and 21, the phase modulation element 569 displays, in the first irradiation area SC1 of the phase modulation element 569, a phase modulation pattern determined in advance for the first to third observation areas KC1 to KC3. The phase modulation element 569 displays, in the second irradiation area SC2 of the phase modulation element 569, a phase modulation pattern determined in advance for the fourth to sixth observation areas KC4 to KC6. The phase modulation element 569 displays a phase modulation pattern determined in advance for the seventh to ninth observation areas KC7 to KC9 in the third irradiation area SC3 of the phase modulation element 569.

When observing the first to ninth observation areas KC1 to KC9 in the sample TP, that is, when acquiring images of the first to ninth observation areas KC1 to KC9, first, images of the first to third observation areas KC1 to KC3 are obtained. obtain at the same time. When acquiring images of the first to third observation areas KC1 to KC3, the phase modulation scanner 567 of the phase modulation unit 560 changes the irradiation area of the illumination light onto the phase modulation element 569 to the first irradiation area SC1. The sample scanning unit 525 of the illumination optical system 521 performs raster scanning simultaneously in the first to third observation areas KC1 to KC3 using the illumination light LC1 irradiated to the first irradiation area SC1 of the phase modulation element 569.

Specifically, raster scanning is performed in the first observation area KC1 using the illumination light LC1a that is irradiated to the left side of the first irradiation area SC1 in FIG. 20 and divided. Raster scanning is performed in the second observation area KC2 using the illumination light LC1b that is irradiated to the center of the first irradiation area SC1 in FIG. 20 and divided. Raster scanning is performed in the third observation area KC3 using the illumination light LC1c that is irradiated to the right side of the first irradiation area SC1 in FIG. 20 and divided. Therefore, it becomes possible to obtain high-quality images of the first to third observation areas KC1 to KC3 in a short time, in which aberrations caused by the first to third observation areas KC1 to KC3 in the sample TP are corrected. .

After acquiring images of the first to third observation areas KC1 to KC3, images of fourth to sixth observation areas KC4 to KC6 are acquired simultaneously. When acquiring images of the fourth to sixth observation areas KC4 to KC6, the phase modulation scanner 567 of the phase modulation unit 560 changes the irradiation area of the illumination light onto the phase modulation element 569 from the first irradiation area SC1 to the second irradiation area. Change to SC2. The sample scanning unit 525 of the illumination optical system 521 simultaneously performs raster scanning in the fourth to sixth observation areas KC4 to KC6 using the illumination light LC2 irradiated to the second irradiation area SC2 of the phase modulation element 569.

Specifically, raster scanning is performed in the fourth observation area KC4 using the illumination light LC2a that is irradiated to the left side of the second irradiation area SC2 in FIG. 20 and divided. Raster scanning is performed in the fifth observation area KC5 using illumination light LC2b that is irradiated to the center of the second irradiation area SC2 in FIG. 20 and divided. A raster scan is performed in the sixth observation area KC6 using the illumination light LC2c that is irradiated to the right side of the second irradiation area SC2 in FIG. 20 and divided. Therefore, it becomes possible to obtain high-quality images of the fourth to sixth observation areas KC4 to KC6 in a short time, in which aberrations caused by the fourth to sixth observation areas KC4 to KC6 in the sample TP are corrected. .

After acquiring the images of the fourth to sixth observation areas KC4 to KC6, images of the seventh to ninth observation areas KC7 to KC9 are acquired simultaneously. When acquiring images of the seventh to ninth observation areas KC7 to KC9, the phase modulation scanner 567 of the phase modulation unit 560 changes the irradiation area of the illumination light onto the phase modulation element 569 from the second irradiation area SC2 to the third irradiation area. Change to SC3. The sample scanning unit 525 of the illumination optical system 521 simultaneously performs raster scanning in the seventh to ninth observation areas KC7 to KC9 using the illumination light LC3 irradiated to the third irradiation area SC3 of the phase modulation element 569.

Specifically, raster scanning is performed in the seventh observation area KC7 using the illumination light LC3a that is irradiated to the left side of the third irradiation area SC3 in FIG. 20 and divided. Raster scanning is performed in the eighth observation area KC8 using the illumination light LC3b that is irradiated to the center of the third irradiation area SC3 in FIG. 20 and divided. Raster scanning is performed in the ninth observation area KC9 using the illumination light LC3c that is irradiated to the right side of the third irradiation area SC3 in FIG. 20 and divided. Therefore, it becomes possible to obtain high-quality images of the seventh to ninth observation areas KC7 to KC9 in a short time, in which aberrations caused by the seventh to ninth observation areas KC7 to KC9 in the sample TP are corrected. . In this way, it becomes possible to obtain a high-quality image of the sample TP in a shorter time, in which aberrations caused by the first to ninth observation areas KC1 to KC9 in the sample TP are individually corrected.

Note that even when using the microscope 501 according to the eleventh embodiment, it is possible to acquire an image of the sample TP by the image acquisition method described in the first embodiment. According to the eleventh embodiment, the phase modulation unit 560 has a plurality of irradiation regions that can be irradiated with illumination light, and imparts a phase distribution to the illumination light according to the irradiation position of the illumination light in the plurality of irradiation regions. and a phase modulation scanner 567 that changes the irradiation area that is irradiated with illumination light. If the phase modulation pattern that changes the phase of the illumination light is changed by changing the irradiation area of the illumination light on the phase modulation element 569 using the phase modulation scanner 567, the phase modulation pattern displayed by the phase modulation element 569 can be changed. The phase modulation pattern can be changed in a shorter time than when switching the phase modulation pattern. Therefore, by changing the irradiation area of the illumination light on the phase modulation element 569 when the observation area on the sample TP is displaced, a high-quality sample can be obtained in which aberrations caused by a plurality of observation areas on the sample TP are individually corrected. It becomes possible to acquire images of TP in a short time.

Further, the phase modulation scanner 567 moves the traveling direction of the illumination light whose phase has changed by irradiating the irradiation area to a constant direction (the illumination optical system 521 (along the optical axis AX51). Thereby, only the phase of the illumination light can be changed without changing the optical path of the illumination light.

Furthermore, the plurality of irradiation areas in the phase modulation element 569 are arranged so that they do not overlap with each other. Thereby, an appropriate phase modulation pattern can be provided for each image acquisition range of the sample TP.

The phase modulation unit 560 also includes a first cylindrical lens 564 and a second cylindrical lens 565 that make the cross-sectional shape of the illumination light irradiated onto any one of the plurality of irradiation areas elliptical, and a first cylindrical lens 564 and a second cylindrical lens 565 that make the cross-sectional shape of the illumination light irradiated onto one of the plurality of irradiation areas. It has a microlens array 572 (optical element array) that divides the illumination light into a plurality of lights. Thereby, the illumination optical system 521 can illuminate the sample TP with a plurality of (for example, three) illumination lights divided by the microlens array 572, and can produce a high-quality image of the sample TP in a shorter time. It will be possible to obtain it.

[Modified example of phase modulation unit]
Next, a modification of the phase modulation unit in the eleventh embodiment will be described with reference to FIG. 22. As shown in FIG. 22, the phase modulation unit 580 according to the modification of the eleventh embodiment includes a beam splitter 591, a first relay lens 581, a mirror 582, a second relay lens 583, and a first cylindrical lens 584. , a second cylindrical lens 585, a third relay lens 586, a phase modulation scanner 587, a fourth relay lens 588, a phase modulation element 569, a microlens array 592, and a sixth relay lens 593. . Note that the portion surrounded by a broken line in FIG. 22 is a diagram of a part of the phase modulation unit 580 viewed from a direction perpendicular to FIG. 22.

A beam splitter 591, a first relay lens 581, a mirror 582, a second relay lens 583, a first cylindrical lens 584, a second cylindrical lens 585, and a third relay lens 586 included in the phase modulation unit 580. , the functions and configurations of the phase modulation scanner 587, the fourth relay lens 588, and the phase modulation element 569 are the same as those of the beam splitter 191 included in the phase modulation unit 180 according to the modification of the sixth embodiment shown in FIG. , a first relay lens 181, a mirror 182, a second relay lens 183, a first cylindrical lens 184, a second cylindrical lens 185, a third relay lens 186, a phase modulation scanner 187, and a fourth Since it is similar to the relay lens 188 and the phase modulation element 169, the explanation will be omitted. The microlens array 592 includes three microlenses arranged in a direction perpendicular to the optical axis AX51 of the illumination optical system 521, and divides the illumination light reflected by the beam splitter 591 into three illumination lights. The sixth relay lens 593 collects the three illumination lights separated by the microlens array 592.

The illumination light that has passed through the collimator lens 522 of the illumination optical system 521 is irradiated onto one of the plurality of irradiation areas in the phase modulation element 569 disposed in the phase modulation unit 580.

When the illumination light is irradiated onto any one of the plurality of irradiation areas (for example, the first to third irradiation areas SC1 to SC3) in the phase modulation element 569, the phase modulation occurs in the irradiation area irradiated with the illumination light. The phase of the illumination light changes depending on the pattern. Illumination light irradiated onto any one of the plurality of irradiation areas in the phase modulation element 569 is reflected at the irradiation area. Note that the phase modulation scanner 587 can change the irradiation area to which the illumination light is irradiated among the plurality of irradiation areas by scanning the phase modulation element 569 with the illumination light that enters the phase modulation scanner 587. It is. A part of the illumination light that enters the beam splitter 591 from the first relay lens 581 is reflected by the beam splitter 591 and enters the microlens array 592.

The illumination light incident on the microlens array 592 passes through the microlens array 592 and is divided into three illumination lights. The three illumination lights separated in the microlens array 592 are once condensed at a position B22 that is optically conjugate with the sample TP while remaining substantially parallel to each other, and then enter the sixth relay lens 593. The three illumination lights that entered the sixth relay lens 593 pass through the sixth relay lens 593 and enter the dichroic mirror 524 of the illumination optical system 521. Thereby, the phase modulation of the illumination light is performed in the same manner as in the case of the phase modulation unit 560 according to the eleventh embodiment.

In the microscope 501 according to the eleventh embodiment, the phase modulation unit 60 according to the first embodiment may be provided instead of the phase modulation unit 560. In this case, a phase pattern calculated using a computer generated hologram (CGH) is displayed on the phase modulation element 65, and the illumination light is applied to one of the plurality of irradiation areas in the phase modulation element 65. When the illumination light is irradiated, a plurality of reflected lights (diffraction lights) may be generated from the irradiation area irradiated with the illumination light due to a diffraction phenomenon. At this time, by using a computer-generated hologram (CGH), a different phase distribution can be given to each of the multiple reflected lights generated by the diffraction phenomenon, depending on the observation position of the sample illuminated with each reflected light (diffraction light). is possible. Calculations for computer-generated holograms (CGH) that give different phase distributions to multiple diffracted lights can be found in the document "Anisoplanatic adaptive optics in parallelized laser scanning microscopy, Optics EXPRESS, Vol. 28, No. 10/11 (2020)", for example. It is possible to use the methods described. Further, the phase modulation unit 160 according to the sixth embodiment may be provided instead of the phase modulation unit 560. In this case, the phase modulation element 169 is configured using a computer-generated hologram (CGH), and when illumination light is irradiated to any one of the plurality of irradiation areas in the phase modulation element 169, the illumination A plurality of reflected lights (diffraction lights) may be generated from the irradiation area where the light is irradiated. At this time, by using a computer-generated hologram (CGH), a different phase distribution can be given to each of the multiple reflected lights generated by the diffraction phenomenon, depending on the observation position of the sample illuminated with each reflected light (diffraction light). is possible. Note that the number of diffracted lights can be arbitrarily determined depending on the sample TP.

[Twelfth embodiment]
Next, a microscope according to a twelfth embodiment will be described with reference to FIG. 23. The microscope according to the twelfth embodiment has the same configuration as the microscope 1 according to the first embodiment, except that a phase modulation unit 610 according to the twelfth embodiment is provided instead of the phase modulation unit 60, and Common parts other than the unit 610 are given the same reference numerals as in the first embodiment, and detailed illustrations and explanations are omitted. In the microscope according to the twelfth embodiment, the phase modulation unit 610 performs phase modulation on the illumination light that has passed through the collimator lens 22, and corrects at least part of the aberrations generated between the sample TP and the illumination optical system 21. . As shown in FIG. 23, a phase modulation unit 610 according to the twelfth embodiment includes a first microlens array 611, a first mirror 612, a first relay lens 613, a first phase modulation scanner 614, and a first phase modulation scanner 614. It has two relay lenses 615, a phase modulation element 616, a second phase modulation scanner 617, a third relay lens 618, a second mirror 619, and a second microlens array 620. Note that instead of the first phase modulation scanner 614 and the second phase modulation scanner 617, a single phase modulation scanner may be used.

The first microlens array 611 includes a plurality of microlenses (for example, 12 microlenses) arranged radially in a direction perpendicular to the optical axis AX1 of the illumination optical system 21. The first microlens array 611 divides the illumination light that has passed through the collimator lens 22 of the illumination optical system 21 and become substantially parallel light into a plurality of illumination lights (for example, 12 illumination lights) and focuses them at a plurality of points. Shine. The first mirror 612 reflects the plurality of illumination lights separated by the first microlens array 611 toward the first relay lens 613 . The first relay lens 613 makes the plurality of illumination lights reflected by the first mirror 612 substantially parallel.

The first phase modulation scanner 614 is arranged at a position where a plurality of illumination lights separated by the first microlens array 611 overlap (near the focal position of the first relay lens 113). The first phase modulation scanner 614 uses a mirror (for example, a galvanometer mirror, a resonant mirror, a MEMS mirror, etc.) held so that the direction (azimuth angle) of the reflecting surface changes by one-axis rotation or two-axis rotation. configured. The first phase modulation scanner 614 changes the traveling direction of the plurality of illumination lights separated by the first microlens array 611. Thereby, the first phase modulation scanner 614 scans the phase modulation element 616 with a plurality of illumination lights separated by the first microlens array 611, as shown in FIG.

The second relay lens 615 is arranged facing the phase modulation element 616. The second relay lens 615 focuses the plurality of illumination lights that have passed through the first phase modulation scanner 614 onto a plurality of points. The second relay lens 615 makes the plurality of illumination lights reflected by the phase modulation element 616 substantially parallel.

The phase modulation element 616 is arranged at a position where the illumination lights separated by the first microlens array 611 are focused (a position conjugate with the position where the illumination lights separated by the first microlens array are focused). The phase modulation element 616 is configured using, for example, a reflective liquid crystal element or a transmissive liquid crystal element, and divides and displays a plurality of phase modulation patterns on the display surface. Note that the display surface of the phase modulation element 616 is provided with a plurality of irradiation areas (for example, 36 sets of irradiation areas) that can be irradiated with illumination light. Each irradiation area is divided into a plurality of partial areas (for example, 12 partial areas) separated from each other. The partial areas constituting each irradiation area on the display surface of the phase modulation element 616 are arranged vertically and horizontally so as not to overlap each other, corresponding to the arrangement of the observation areas. The phase modulation element 616 divides and displays a plurality of phase modulation patterns into a plurality of partial areas in a plurality of irradiation areas.

FIG. 24 shows an example of the arrangement of a plurality of irradiation areas and a plurality of partial areas forming each irradiation area. In the example of FIG. 24, the display surface of the phase modulation element 616 is provided with 36 sets of irradiation areas divided into 12 partial areas (A to L). For example, in the first irradiation area SD1, in the first stage of the 12 partial areas (from the top of FIG. 24), a first partial area SD1A and a second partial area SD1B are separated in order from the left side. Placed. In the first irradiation area SD1, in the second stage (from the top of FIG. 24) among the 12 partial areas, from the left side, there are a third partial area SD1C, a fourth partial area SD1D, and a fifth partial area SD1E. and the sixth partial region SD1F are arranged separately. In the first irradiation area SD1, in the third row (from the top of FIG. 24) among the 12 partial areas, from the left side, there are a seventh partial area SD1G, an eighth partial area SD1H, and a ninth partial area SD1I. and the tenth partial region SD1J are arranged separately. In the first irradiation area SD1, in the fourth stage (from the top of FIG. 24) among the 12 partial areas, an 11th partial area SD1K and a 12th partial area SD1L are separately arranged in order from the left side. Ru.

Further, as shown in FIGS. 24 and 25, the first partial area SD1A of the first irradiation area SD1
First partial areas SD1A to SD36A of the first to 36th irradiation areas are arranged in the 6×6 36 partial areas including the first to 36th irradiation areas. Specifically, in the first row (from the top of FIG. 25) among the 36 partial areas, in order from the left side, the first partial area SD1A of the first irradiation area SD1 and the first part of the second irradiation area area SD2A, first partial area SD3A of the third irradiation area, first partial area SD4A of the fourth irradiation area, first partial area SD5A of the fifth irradiation area, and first partial area SD6A of the sixth irradiation area. and are placed. Among the 36 partial areas, in the second row (from the top of FIG. 25), from the left side, the first partial area SD7A of the seventh irradiation area, the first partial area SD8A of the eighth irradiation area, and the ninth partial area A first partial area SD9A of the irradiation area, a first partial area SD10A of the tenth irradiation area, a first partial area SD11A of the eleventh irradiation area, and a first partial area SD12A of the twelfth irradiation area are arranged. Among the 36 partial areas, in the third row (from the top of FIG. 25), from the left side, the first partial area SD13A of the 13th irradiation area, the first partial area SD14A of the 14th irradiation area, and the 15th partial area SD13A of the 14th irradiation area, A first partial area SD15A of the irradiation area, a first partial area SD16A of the 16th irradiation area, a first partial area SD17A of the 17th irradiation area, and a first partial area SD18A of the 18th irradiation area are arranged.

Among the 36 partial areas, the fourth row (from the top of FIG. 25) includes, in order from the left, the first partial area SD19A of the 19th irradiation area, the first partial area SD20A of the 20th irradiation area, and the 21st partial area SD19A of the 19th irradiation area. A first partial area SD21A of the irradiation area, a first partial area SD22A of the 22nd irradiation area, a first partial area SD23A of the 23rd irradiation area, and a first partial area SD24A of the 24th irradiation area are arranged. Among the 36 partial areas, in the fifth row (from the top of FIG. 25), from the left side, the first partial area SD25A of the 25th irradiation area, the first partial area SD26A of the 26th irradiation area, and the 27th partial area A first partial area SD27A of the irradiation area, a first partial area SD28A of the 28th irradiation area, a first partial area SD29A of the 29th irradiation area, and a first partial area SD30A of the 30th irradiation area are arranged. Among the 36 partial areas, the 6th row (from the top of FIG. 25) includes, in order from the left, the first partial area SD31A of the 31st irradiation area, the first partial area SD32A of the 32nd irradiation area, and the 33rd partial area SD31A of the 32nd irradiation area. A first partial area SD33A of the irradiation area, a first partial area SD34A of the 34th irradiation area, a first partial area SD35A of the 35th irradiation area, and a first partial area SD36A of the 36th irradiation area are arranged.

Similarly to the first partial areas SD1A to SD36A of the first to 36th irradiation areas, the 6×6 36 partial areas including the second partial area SD1B of the first irradiation area SD1 include the first to 36th partial areas SD1A to SD36A of the first to 36th irradiation areas. A second partial area (not shown) of the irradiation area is arranged. The third partial areas (not shown) of the first to 36th irradiation areas are arranged in the 6×6 36 partial areas including the third partial area SD1C of the first irradiation area SD1. The fourth partial areas (not shown) of the first to 36th irradiation areas are arranged in the 6×6 36 partial areas including the fourth partial area SD1D of the first irradiation area SD1. The fifth partial areas (not shown) of the first to 36th irradiation areas are arranged in the 6×6 36 partial areas including the fifth partial area SD1E of the first irradiation area SD1. Sixth partial areas (not shown) of the first to 36th irradiation areas are arranged in 6×6 36 partial areas including the sixth partial area SD1F of the first irradiation area SD1.

The seventh partial areas (not shown) of the first to 36th irradiation areas are arranged in the 6×6 36 partial areas including the seventh partial area SD1G of the first irradiation area SD1. Eighth partial areas (not shown) of the first to 36th irradiation areas are arranged in the 6×6 36 partial areas including the eighth partial area SD1H of the first irradiation area SD1. The ninth partial areas (not shown) of the first to 36th irradiation areas are arranged in the 6×6 36 partial areas including the ninth partial area SD1I of the first irradiation area SD1. The 10th partial areas (not shown) of the first to 36th irradiation areas are arranged in the 6×6 36 partial areas including the 10th partial area SD1J of the first irradiation area SD1. The 11th partial areas (not shown) of the first to 36th irradiation areas are arranged in the 6×6 36 partial areas including the 11th partial area SD1K of the first irradiation area SD1. The 12th partial areas (not shown) of the first to 36th irradiation areas are arranged in the 6×6 36 partial areas including the 12th partial area SD1L of the first irradiation area SD1.

As shown in FIG. 24, the phase modulation element 616 divides and displays the phase modulation pattern of the first irradiation area SD1 into first to twelfth partial areas SD1A to SD1L in the first irradiation area SD1. The phase modulation element 616 divides and displays the phase modulation pattern of the second irradiation area SD2 into first to twelfth partial areas SD2A to SD2L in the second irradiation area SD2. In this way, the phase modulation element 616 divides and displays the phase modulation pattern of the n-th irradiation area SDn (n=1 to 36) into the first to twelfth partial areas SDnA to SDnL in the n-th irradiation area SDn. . In the example shown in FIG. 24, the 12 illumination lights divided by the first microlens array 611 are used for the first to twelfth partial areas SDnA to SDnL in the n-th irradiation area SDn, that is, the first to 36th irradiation areas. The first to twelfth partial areas in any one of the irradiation areas are irradiated. Note that the arrangement of the plurality of irradiation areas set on the phase modulation element 616 is not necessarily limited to the grid-like arrangement as shown in the figure, but may be any trajectory that the first phase modulation scanner 614 scans over the phase modulation element 616. may be placed on top.

When the first to twelfth partial regions in any one of the first to 36th irradiation regions are irradiated with the 12 divided illumination lights, the phase of each illumination light changes (modulates). Specifically, a phase is given to each illumination light according to a phase value in a partial region irradiated with the illumination light. As a result, as shown in FIG. 26, the illumination light that is combined by the microlens array 620 and emitted from the phase modulation unit 610 (described later) is set in each of the first to twelfth partial regions constituting the irradiation region. A phase distribution is given according to the phase value. In addition, by changing the irradiation area (that is, the set of the first to twelfth partial areas) on which the illumination light is irradiated in the phase modulation element 616, the phase distribution (detection light When the detection light is irradiated onto the irradiation area, it is possible to change the phase distribution imparted to the detection light.

The second phase modulation scanner 617 is arranged at a position where a plurality of illumination lights separated by the first microlens array 611 overlap (near the focal position of the second relay lens 615). The second phase modulation scanner 617 is configured similarly to the first phase modulation scanner 614. The second phase modulation scanner 617 is arranged so that the plurality of illumination lights transmitted or reflected through the phase modulation element and made substantially parallel by the second relay lens 615 travel toward the third relay lens 618, that is, the phase modulation unit 610. The traveling direction of the illumination light is changed so that the illumination light emitted from the illumination optical system 21 travels along the optical axis AX1 of the illumination optical system 21. The third relay lens 618 focuses the plurality of illumination lights that have passed through the second phase modulation scanner 617 onto a plurality of points. The second mirror 619 reflects the plurality of illumination lights that have passed through the third relay lens 618 toward the second microlens array 620 . The second microlens array 620 is configured similarly to the first microlens array 611. The second microlens array 620 combines the plurality of illumination lights reflected by the second mirror 619 into substantially parallel light. In this way, the phase modulation unit 610 performs phase modulation on the illumination light.

Note that when a plurality of illumination lights reflected by a plurality of partial areas in any one of the plurality of irradiation areas passes through the second relay lens 615 and passes through the second phase modulation scanner 617, phase modulation occurs. The illumination light emitted from the unit 610 travels along the optical axis AX1 of the illumination optical system 21. Due to the descanning of the illumination light by the second phase modulation scanner 617, a plurality of light beams transmitted through the second microlens array 620, even though the first phase modulation scanner 614 changes the traveling direction of the illumination light and changes the irradiation area. The illumination light can be made into one substantially parallel illumination light and can be incident on the dichroic mirror 24 of the illumination optical system 21.

As described above, the first phase modulation scanner 614 scans the phase modulation element 616 with a plurality of illumination lights incident on the first phase modulation scanner 614, thereby detecting the inside of the plurality of irradiation areas in the phase modulation element 616. It is possible to change the irradiation area to which the illumination light is irradiated.
In the twelfth embodiment, the sample scanning section 25 of the illumination optical system 21 performs scanning across a plurality of observation areas on the sample TP using the illumination light emitted from the phase modulation unit 610. The first phase modulation scanner 614 sequentially changes the irradiation area to which the illumination light is irradiated in the phase modulation element 616 in synchronization with scanning across a plurality of observation areas. Note that, for each of the plurality of observation areas on the sample TP, a phase modulation pattern for correcting at least part of the aberrations generated between the sample TP and the illumination optical system 21, that is, a phase modulation pattern for correcting at least part of the aberrations generated between the sample TP and the illumination optical system 21, is used. , a predetermined phase distribution for correcting at least a portion thereof is determined in advance. The phase modulation element 616 divides and displays a phase modulation pattern determined in advance for the corresponding observation area in a plurality of partial areas in each irradiation area of the phase modulation element 616. A case in which the irradiation area is changed each time one observation area is scanned by sequentially changing the irradiation area that is irradiated with illumination light in the phase modulation element 616 in synchronization with scanning across multiple observation areas. It becomes possible to obtain an image of the sample TP in which aberrations are corrected in a shorter time than in the case of FIG.

FIG. 27 shows an example of the arrangement of a plurality of observation areas in the sample TP. In the example of FIG. 27, 36 observation areas in the sample TP are set within the field of view of the microscope 1. Among the 36 observation areas, in the first row (from the top of FIG. 27), from the left side, there are a first observation area KD1, a second observation area KD2, a third observation area KD3, and a fourth observation area KD4. , a fifth observation area KD5, and a sixth observation area KD6 are set. Among the 36 observation areas, in the second row (from the top of FIG. 27), from the left side, there are a seventh observation area KD7, an eighth observation area KD8, a ninth observation area KD9, and a tenth observation area KD10. , an eleventh observation area KD11, and a twelfth observation area KD12 are set. Among the 36 observation areas, in the third row (from the top of FIG. 27), from the left side, there are a 13th observation area KD13, a 14th observation area KD14, a 15th observation area KD15, and a 16th observation area KD16. , a 17th observation area KD17, and an 18th observation area KD18 are set.

Among the 36 observation areas, in the fourth row (from the top of FIG. 27), from the left side, there are a 19th observation area KD19, a 20th observation area KD20, a 21st observation area KD21, and a 22nd observation area KD22. , a 23rd observation area KD23, and a 24th observation area KD24 are set. Among the 36 observation areas, in the fifth row (from the top of FIG. 27), from the left side, there are a 25th observation area KD25, a 26th observation area KD2, a 27th observation area KD27, and a 28th observation area KD28. , a 29th observation area KD29, and a 30th observation area KD30 are set. Among the 36 observation areas, in the 6th row (from the top of FIG. 27), from the left side, there are a 31st observation area KD31, a 32nd observation area KD32, a 33rd observation area KD33, and a 34th observation area KD34. , a 35th observation area KD35, and a 36th observation area KD36 are set. Note that the area of each of the observation regions KD1 to KD36 may be larger than the cross-sectional area of the illumination light LD1, or may be approximately the same as the cross-sectional area of the illumination light LD1.

In the examples shown in FIGS. 24 to 27, the phase modulation element 616 applies the phase modulation pattern determined in advance for the first observation area KD1 to the first to twelfth partial areas SD1A in the first irradiation area SD1 of the phase modulation element 616. - Divide into SD1L and display. The phase modulation element 616 divides and displays the phase modulation pattern previously determined for the second observation area KA2 into first to twelfth partial areas SD2A to SD2L in the second irradiation area SD2 of the phase modulation element 616. In this way, the phase modulation element 616 applies the phase modulation pattern determined in advance for the n-th observation area KDn (n=1 to 36) to the first to twelfth portions in the n-th irradiation area SDn of the phase modulation element 616. It is divided into areas SDnA to SDnL and displayed.

When observing the first to 36th observation areas KD1 to KD36 in the sample TP, that is, when acquiring images of the first to 36th observation areas KD1 to KD36, the sample scanning section 25 of the illumination optical system 21 uses a phase modulation unit. The illumination light emitted from 610 scans across the first to 36th observation areas KD1 to KD36 on the sample TP. For example, in synchronization with the timing of linearly scanning the first to sixth observation areas KD1 to KD6, the first phase modulation scanner 614 changes the irradiation area of the illumination light onto the phase modulation element 616 to the first to sixth observation areas KD1 to KD6. Change in order up to the irradiation area. At this time, each partial area irradiated with the divided illumination light changes continuously from each partial area in the first irradiation area to each partial area in the sixth irradiation area. For example, as shown in FIG. 25, the first partial area irradiated with the divided illumination light LD1 changes continuously from the first partial area SD1A in the first irradiation area to the first partial area SD6A in the sixth irradiation area. .

For example, in synchronization with the timing of linearly scanning the seventh to twelfth observation areas KD7 to KD12, the first phase modulation scanner 614 changes the irradiation area of the illumination light onto the phase modulation element 616 to the seventh to twelfth observation areas KD7 to KD12. Change sequentially up to 12 irradiation areas. At this time, each partial area irradiated with the divided illumination light changes continuously from each partial area in the seventh irradiation area to each partial area in the twelfth irradiation area. For example, as shown in FIG. 25, the first partial area irradiated with the divided illumination light LD1 changes continuously from the first partial area SD7A in the seventh irradiation area to the first partial area SD12A in the twelfth irradiation area. .

Similarly, in synchronization with the timing of linearly scanning the 13th to 18th observation areas KD13 to KD18, the first phase modulation scanner 614 shifts the irradiation area of the illumination light onto the phase modulation element 616 to the 13th to 18th observation areas KD13 to KD18. to the 18th irradiation area in order. In synchronization with the timing of linearly scanning the 19th to 24th observation areas KD19 to KD24, the first phase modulation scanner 614 changes the irradiation area of the illumination light onto the phase modulation element 616 to the 19th to 24th irradiation areas. Change in order up to In synchronization with the timing of linearly scanning the 25th to 30th observation areas KD25 to KD30, the first phase modulation scanner 614 changes the irradiation area of the illumination light onto the phase modulation element 616 to the 25th to 30th irradiation areas. Change in order up to In synchronization with the timing of linearly scanning the 31st to 36th observation areas KD31 to KD36, the first phase modulation scanner 614 changes the irradiation area of the illumination light onto the phase modulation element 616 to the 31st to 36th irradiation areas. Change in order up to

This allows the aberrations caused by the first to 36th observation areas KD1 to KD36 in the sample TP to be individually corrected, compared to the case where the irradiation area is changed each time one observation area is scanned. It becomes possible to obtain a high-quality image of the sample TP in a shorter time. The second phase modulation scanner 617 operates in synchronization with the first phase modulation scanner 614 so that the illumination light emitted from the phase modulation unit 610 travels along the optical axis AX1 of the illumination optical system 21. , the traveling direction of the 12 illumination lights that are irradiated and reflected from the first to twelfth partial areas in each irradiation area is changed.

Note that even when using the microscope according to the twelfth embodiment, it is possible to acquire an image of the sample TP by the image acquisition method described in the first embodiment. According to the twelfth embodiment, the phase modulation unit 610 has a plurality of irradiation regions that can be irradiated with illumination light, and imparts a phase distribution to the illumination light according to the irradiation position of the illumination light in the plurality of irradiation regions. and a first phase modulation scanner 614 that changes the irradiation area that is irradiated with illumination light. If the first phase modulation scanner 614 changes the irradiation area of the illumination light onto the phase modulation element 616 to change the phase modulation pattern that changes the phase of the illumination light, the phase displayed by the phase modulation element 616 can be changed. The phase modulation pattern can be changed in a shorter time than when changing the modulation pattern.

Further, the phase modulation unit 610 includes a first microlens array 611 (optical element array) that divides the illumination light into a plurality of illumination lights and irradiates the plurality of partial regions in each irradiation region. Therefore, the first phase modulation scanner 614 performs irradiation divided into a plurality of partial areas to which a plurality of illumination lights are irradiated in synchronization with the sample scanning section 25 of the illumination optical system 21 scanning the sample TP. By changing the area, aberrations caused by multiple observation areas in the sample TP are individually corrected, compared to changing the irradiation area each time one observation area is scanned, resulting in a high-quality sample. It becomes possible to acquire a TP image in a shorter time.

Further, the phase modulation unit 610 keeps the traveling direction of the illumination light whose phase has changed by being irradiated onto the irradiation area constant, even though the first phase modulation scanner 614 changes the irradiation area of the illumination light on the phase modulation element 616. (direction along the optical axis AX1 of the illumination optical system 21). Thereby, only the phase of the illumination light can be changed without changing the optical path of the illumination light.

[First modification of phase modulation unit]
Next, a first modification of the phase modulation unit in the twelfth embodiment will be described with reference to FIG. 28. As shown in FIG. 28, a phase modulation unit 630 according to the first modification of the twelfth embodiment includes a beam splitter 637, a microlens array 631, a mirror 632, a first relay lens 633, and a phase modulation scanner. 634, a second relay lens 635, and a phase modulation element 616, except for the beam splitter 637, which includes a first microlens array 611, a first mirror 612, and a first relay lens included in the phase modulation unit 610. 613, a first phase modulation scanner 614, a second relay lens 615, a phase modulation element 616, a second phase modulation scanner 617, a third relay lens 618, a second mirror 619, and a second micro Since each function and each configuration of the lens array 620 are the same, explanations thereof will be omitted. The ratio of transmittance and reflectance of the beam splitter 637 is set to, for example, 1:1. A part of the illumination light that has entered the beam splitter 637 from the collimator lens 22 of the illumination optical system 21 passes through the beam splitter 637 and enters the microlens array 631. A part of the illumination light that has entered the beam splitter 637 from the microlens array 631 is reflected by the beam splitter 637 and enters the dichroic mirror 24 of the illumination optical system 21 .

[Second modification of phase modulation unit]
Next, a second modification of the phase modulation unit in the twelfth embodiment will be described with reference to FIG. 29. As shown in FIG. 29, a phase modulation unit 640 according to the second modification of the twelfth embodiment includes a first microlens array disk 641, a motor 645, a first mirror 642, a first relay lens 643, It has a second relay lens 644, a phase modulation element 616, a third relay lens 647, a second mirror 648, and a second microlens array disk 649. Except for the two microlens array disks 649, the first mirror 612, the first relay lens 613, the second relay lens 615, the phase modulation element 616, the third relay lens 618, and the third relay lens included in the phase modulation unit 610 are Since the mechanisms and configurations of the two mirrors 619 are the same, their explanations will be omitted. The phase modulation unit 640 controls the functions and configurations of the first microlens array 611, first phase modulation scanner 614, second phase modulation scanner 617, and second microlens array 620 included in the phase modulation unit 610. This corresponds to a lens array disk 641, a motor 645, and a second microlens array disk 649.

The first microlens array disk 641 is formed into a disk shape and includes a plurality of microlenses (for example, 12 microlenses) arranged radially in a direction perpendicular to the optical axis AX1 of the illumination optical system 21. The first microlens array disk 641 divides the illumination light that has passed through the collimator lens 22 and turned into substantially parallel light into a plurality of illumination lights (for example, 12 illumination lights). The first microlens array disk 641 is rotatably supported by a motor 645 via a first rotating shaft 646a. The motor 645 rotates the first microlens array disk 641 to change the traveling direction of the plurality of illumination lights separated by the first microlens array disk 641. Thereby, the motor 645 scans the phase modulation element 616 with a plurality of illumination lights separated by the first microlens array disk 641.

The second microlens array disk 649 is configured similarly to the first microlens array disk 641. The second microlens array disk 649 combines the plurality of illumination lights incident on the second microlens array disk 649 into substantially parallel light. The second microlens array disk 649 is rotatably supported by the motor 645 via a second rotation shaft 646b. The motor 645 rotates and moves the second microlens array disk 649 in synchronization with the first microlens array disk 641, so that the illumination light transmitted through the second microlens array disk 649 is aligned with the optical axis AX1 of the illumination optical system 21. Make sure to follow the directions.

A plurality of illumination lights separated by the first microlens array disk 641 are applied to a plurality of partial areas in any one of the plurality of irradiation areas (for example, one of the irradiation areas among the first to 36th irradiation areas). When the illumination light beams are irradiated onto the first to twelfth partial regions), the phase of each illumination light beam changes according to the phase modulation pattern in the partial region irradiated with the illumination light beams. A plurality of illumination lights irradiated onto a plurality of partial regions in any one of the plurality of irradiation regions is reflected at each partial region. Note that the motor 645 rotates the first microlens array disk 641 and scans the phase modulation element 616 with a plurality of illumination lights separated by the first microlens array disk 641. It is possible to change the irradiation area (that is, the set of the first to twelfth partial areas) to which the illumination light is irradiated within the irradiation area. On the phase modulation element 616, a plurality of irradiation areas and each irradiation area is divided apart from each other at positions along the positions and trajectories of the plurality of illumination lights scanned by the rotation of the first microlens array disk 641. A partial area is set.

[Third modification of phase modulation unit]
Next, a third modification of the phase modulation unit in the twelfth embodiment will be described with reference to FIG. 30. As shown in FIG. 30, a phase modulation unit 650 according to the third modification of the twelfth embodiment includes a first relay lens 651, a first mirror 652, a second relay lens 653, and a microlens array disk 654. The phase modulation unit 60 shown in FIG. Since the first relay lens 61, the first mirror 62, the second relay lens 64, the second mirror 67, and the third relay lens 68 included in the above are the same, the description thereof will be omitted. The first mirror 652 is arranged near a position B34 that is optically conjugate with the sample TP. The first mirror 652 reflects the illumination light that has passed through the first relay lens 651 toward the second relay lens 653 . The second relay lens 653 converts the illumination light reflected by the first mirror 652 into substantially parallel light. Further, the second relay lens 653 collects the illumination light emitted from the microlens array disk 654.

The microlens array disk 654 is formed into a disk shape and includes a plurality of microlenses (for example, 12 microlenses) arranged radially in a direction perpendicular to the optical axis AX1 of the illumination optical system 21. The microlens array disk 654 divides the illumination light that has passed through the second relay lens 653 into a plurality of illumination lights (for example, 12 illumination lights) and collects the illumination light. Further, the microlens array disk 654 combines a plurality of illumination lights reflected by the phase modulation element 616 into substantially parallel light. The microlens array disk 654 is rotatably supported by a motor 655 via a rotation shaft 656. The motor 655 rotates the microlens array disk 654 to change the traveling direction of the plurality of illumination lights separated by the microlens array disk 654. Thereby, the motor 655 scans the phase modulation element 616 with a plurality of illumination lights separated by the microlens array disk 654.

The phase modulation element 616 has the same configuration as the phase modulation element 616 according to the twelfth embodiment, is given the same reference numeral as in the twelfth embodiment, and detailed description thereof will be omitted. The second mirror 657 is arranged near a position B34 that is optically conjugate with the sample TP. The second mirror 657 reflects the illumination light that has passed through the second relay lens 653 toward the third relay lens 658 . The third relay lens 658 converts the illumination light reflected by the second mirror 657 into substantially parallel light.

A plurality of illumination lights separated by the microlens array disk 654 are applied to a plurality of partial areas in any one of the plurality of irradiation areas (for example, a plurality of partial areas in any one of the first to 36th irradiation areas). When the first to twelfth partial regions) are irradiated, the phase of each illumination light changes according to the phase modulation pattern in the partial region irradiated with the illumination light. A plurality of illumination lights irradiated onto a plurality of partial regions in any one of the plurality of irradiation regions is reflected at each partial region. The motor 655 rotates the microlens array disk 654 and scans the phase modulation element 616 with a plurality of illumination lights separated by the microlens array disk 654, thereby scanning the plurality of irradiation areas on the phase modulation element 616. It is possible to change the irradiation area (ie, the set of the first to twelfth partial areas) to which the illumination light is irradiated. On the phase modulation element 616, a plurality of irradiation areas and partial areas in which each irradiation area is separated from each other are formed at positions along the positions and trajectories of the plurality of illumination lights scanned by the rotation of the microlens array disk 654. is set.

A plurality of illumination lights that are irradiated and reflected by a plurality of partial areas in one of the plurality of irradiation areas enter the microlens array disk 654. The plurality of illumination lights incident on the microlens array disk 654 are transmitted through the microlens array disk 654 and combined into one substantially parallel illumination light, which is then incident on the second relay lens 653. The illumination light incident on the second relay lens 653 is transmitted through the second relay lens 653 and reflected by the second mirror 657. The illumination light reflected by the second mirror 657 passes through the third relay lens 658 to become substantially parallel light, and enters the dichroic mirror 24 of the illumination optical system 21 . Thereby, the phase modulation of the illumination light is performed in the same manner as in the case of the phase modulation unit 610 according to the twelfth embodiment.

[Fourth modification of phase modulation unit]
Next, a fourth modification of the phase modulation unit in the twelfth embodiment will be described with reference to FIG. 31. As shown in FIG. 31, a phase modulation unit 660 according to the fourth modification of the twelfth embodiment includes a beam splitter 661, a first relay lens 662, a second relay lens 663, a microlens array disk 664, It has a phase modulation element 616. The ratio of transmittance and reflectance of the beam splitter 661 is set to, for example, 1:1. A part of the illumination light that has entered the beam splitter 661 from the collimator lens 22 of the illumination optical system 21 passes through the beam splitter 661 and enters the first relay lens 662 . A part of the illumination light that has entered the beam splitter 661 from the first relay lens 662 is reflected by the beam splitter 661 and enters the dichroic mirror 24 of the illumination optical system 21 .

The first relay lens 662 collects the illumination light that has passed through the beam splitter 661. The second relay lens 663 converts the illumination light transmitted through the first relay lens 662 into substantially parallel light. Further, the second relay lens 663 collects the illumination light from the microlens array disk 664. The phase modulation element 616 has the same configuration as the phase modulation element 616 according to the twelfth embodiment, is given the same reference numeral as in the twelfth embodiment, and detailed description thereof will be omitted.

The microlens array disk 664 has the same function and configuration as the microlens array disk 654 included in the phase modulation unit 650 shown in FIG. 30, so a description thereof will be omitted.

The illumination light transmitted through the collimator lens 22 of the illumination optical system 21 enters the beam splitter 661 of the phase modulation unit 660. A part of the illumination light that has entered the beam splitter 661 from the collimator lens 22 passes through the beam splitter 661 and then passes through the first relay lens 662 . The illumination light that has passed through the first relay lens 662 is once condensed at a position B35 that is optically conjugate with the sample TP, and then enters the second relay lens 663. The illumination light that has entered the second relay lens 663 passes through the second relay lens 663 and enters the microlens array disk 664 . The illumination light incident on the microlens array disk 664 passes through the microlens array disk 664 and is divided into a plurality of illumination lights (for example, 12 illumination lights). The plurality of illumination lights separated in the microlens array disk 664 are irradiated to a plurality of partial areas in one of the plurality of irradiation areas of the phase modulation element 616, but after that, the illumination lights of the third modification are applied. Since it is the same as described above, the explanation will be omitted.

[13th embodiment]
Next, a microscope according to a thirteenth embodiment will be described. The microscope according to the thirteenth embodiment has the same configuration as the microscope 101 according to the second embodiment, except that the phase modulation unit 610 according to the twelfth embodiment is provided instead of the phase modulation unit 60, and Common parts other than the unit 610 are given the same reference numerals as in the second embodiment, and detailed illustrations and explanations are omitted. In the microscope according to the thirteenth embodiment, a phase modulation unit 610 performs phase modulation on the illumination light that has passed through the dichroic mirror 123 of the illumination optical system 121, and also performs phase modulation on the detection light that has passed through the sample scanning section 125. Modulation is performed to correct at least a portion of aberrations occurring in the sample TP, the illumination optical system 121, and the detection optical system 131.

The illumination light that has passed through the dichroic mirror 123 of the illumination optical system 121 enters the phase modulation unit 610, but the subsequent steps are the same as in the twelfth embodiment, so the explanation will be omitted. The illumination light emitted from the phase modulation unit 610 enters the sample scanning section 125 of the illumination optical system 121.

In addition, the detection light that has passed through the sample scanning unit 125 as the detection optical system 131 enters the phase modulation unit 610, then follows a path in the opposite direction to the illumination light described above, and undergoes phase modulation similar to the illumination light. The light is emitted from the phase modulation unit 610 toward the dichroic mirror 123 in a substantially parallel manner. As in the twelfth embodiment, by sequentially changing the irradiation area to which the illumination light and detection light are irradiated in the phase modulation element 616 in synchronization with the scanning across a plurality of observation areas, one observation area can be Compared to the case where the irradiation area is changed each time a scan is performed, a high-quality image of the sample TP can be obtained in a shorter time.

Further, even when using the microscope according to the thirteenth embodiment, it is possible to acquire an image of the sample TP by the image acquisition method described in the first embodiment, similarly to the microscope 101 according to the second embodiment. Therefore, according to the thirteenth embodiment, effects similar to those of the twelfth embodiment and the second embodiment can be obtained. Further, according to the thirteenth embodiment, the phase modulation unit 610 performs phase modulation on the detection light in addition to the phase modulation on the illumination light, so that it is possible to obtain a higher quality image of the sample TP in a short time. It becomes possible.

In the microscope according to the thirteenth embodiment, the phase modulation unit 610 may be replaced with a phase modulation unit 630 according to the first modification of the twelfth embodiment. As in the thirteenth embodiment described above, the phase modulation unit 630 according to the first modification of the twelfth embodiment can perform phase modulation on the illumination light and the detection light. Furthermore, instead of the phase modulation unit 610, a phase modulation unit 640 according to a second modification of the twelfth embodiment, a phase modulation unit 650 according to a third modification, or a phase modulation unit 660 according to a fourth modification is provided. It may be possible to do so.

[Fourteenth embodiment]
Next, a microscope according to a fourteenth embodiment will be described. The microscope according to the fourteenth embodiment has the same configuration as the microscope 201 according to the third embodiment, except that the phase modulation unit 610 according to the twelfth embodiment is provided instead of the phase modulation unit 60, and Common parts other than the unit 610 are given the same reference numerals as in the third embodiment, and detailed illustrations and explanations are omitted.

In the microscope according to the fourteenth embodiment, the phase modulation unit 610 performs phase modulation on the illumination light that has passed through the collimator lens 222 of the illumination optical system 221, so that at least Correct some parts. At this time, the phase modulation unit 610 performs phase modulation on the illumination light in the same manner as in the twelfth embodiment. Further, even when using the microscope according to the fourteenth embodiment, it is possible to acquire an image of the sample TP by the image acquisition method described in the first embodiment, similarly to the microscope 201 according to the third embodiment. Therefore, according to the fourteenth embodiment, the same effects as the twelfth and third embodiments can be obtained.

In the microscope according to the fourteenth embodiment, a phase modulation unit 630 according to the first modification of the twelfth embodiment may be provided instead of the phase modulation unit 610. Similarly to the first modification of the twelfth embodiment described above, the phase modulation unit 630 according to the first modification of the twelfth embodiment can perform phase modulation on illumination light. Furthermore, instead of the phase modulation unit 610, a phase modulation unit 640 according to a second modification of the twelfth embodiment, a phase modulation unit 650 according to a third modification, or a phase modulation unit 660 according to a fourth modification is provided. It may be possible to do so.

[15th embodiment]
Next, a microscope according to a fifteenth embodiment will be described. The microscope according to the fifteenth embodiment has the same configuration as the microscope 301 according to the fourth embodiment, except that the phase modulation unit 610 according to the twelfth embodiment is provided instead of the phase modulation unit 60, and Common parts other than the unit 610 are given the same reference numerals as in the fourth embodiment, and detailed illustrations and explanations are omitted.

In the microscope according to the fifteenth embodiment, the phase modulation unit 610 performs phase modulation on the illumination light that has passed through the collimator lens 322 of the illumination optical system 321, and the phase modulation that occurs between the sample TP, the illumination optical system 321, and the detection optical system 331 At least some of the aberrations are corrected. At this time, the phase modulation unit 610 performs phase modulation on the illumination light in the same manner as in the twelfth embodiment. Further, even when using the microscope according to the fifteenth embodiment, it is possible to acquire an image of the sample TP by the image acquisition method described in the first embodiment, similarly to the microscope 301 according to the fourth embodiment. Therefore, according to the fifteenth embodiment, the same effects as those of the twelfth embodiment and the fourth embodiment can be obtained.

In the microscope according to the fifteenth embodiment, the phase modulation unit 610 may be replaced with a phase modulation unit 630 according to the first modification of the twelfth embodiment. Similarly to the first modification of the twelfth embodiment described above, the phase modulation unit 630 according to the first modification of the twelfth embodiment can perform phase modulation on illumination light. Furthermore, instead of the phase modulation unit 610, a phase modulation unit 640 according to a second modification of the twelfth embodiment, a phase modulation unit 650 according to a third modification, or a phase modulation unit 660 according to a fourth modification is provided. It may be possible to do so.

[Sixteenth embodiment]
Next, a microscope according to the sixteenth embodiment will be described with reference to FIG. 32. The microscope according to the 16th embodiment has the same configuration as the microscope 1 according to the 1st embodiment, except that a phase modulation unit 710 according to the 16th embodiment is provided instead of the phase modulation unit 60. Common parts other than the unit 710 are given the same reference numerals as in the first embodiment, and detailed illustrations and explanations are omitted. In the microscope according to the sixteenth embodiment, the phase modulation unit 710 performs phase modulation on the illumination light that has passed through the collimator lens 22, and corrects at least part of the aberrations that occur between the sample TP and the illumination optical system 21. . As shown in FIG. 32, the phase modulation unit 710 according to the sixteenth embodiment includes a first relay lens 711, a first mirror 712, a second relay lens 713, a cylindrical lens array 714, and a third relay lens 715. , a phase modulation scanner 716, a fourth relay lens 717, a phase modulation element 718, a second mirror 719, and a fifth relay lens 720, except for the cylindrical lens array 714 and the phase modulation element 718. A first relay lens 161, a first mirror 162, a second relay lens 163, a second cylindrical lens 165, a third relay lens 166, and a phase modulation scanner 167 included in the phase modulation unit 160 shown in FIG. Since the functions and configurations of the fourth relay lens 168, second mirror 170, and fifth relay lens 171 are the same, their explanations will be omitted. Note that the portion surrounded by a broken line in FIG. 32 is a diagram of a part of the phase modulation unit 710 viewed from a direction perpendicular to FIG. 32.

The cylindrical lens array 714 includes a plurality of cylindrical lenses (for example, four cylindrical lenses) arranged in a row in a direction perpendicular to the optical axis AX1 of the illumination optical system 21. The cylindrical lens array 714 divides the illumination light from the second relay lens 713 into a plurality of illumination lights (for example, four illumination lights) and focuses them into a line. The third relay lens 715 overlaps the plurality of illumination lights from the cylindrical lens array 714.

The phase modulation scanner 716 is arranged at a position B41 that is optically conjugate with the sample TP. The phase modulation scanner 716 changes the traveling direction of a plurality of illumination lights separated by the cylindrical lens array 714. Thereby, the phase modulation scanner 716 scans the phase modulation element 718 with a plurality of illumination lights separated by the cylindrical lens array 714, as shown in FIG.

Further, the phase modulation scanner 716 is configured such that the illumination light that has entered the phase modulation scanner 716 from the fourth relay lens 717 travels toward the third relay lens 715, that is, the illumination light emitted from the phase modulation unit 710 is The traveling direction of the illumination light is changed so that it travels along the optical axis AX1 of the illumination optical system 21.

The phase modulation element 718 is arranged at a position H41 that is conjugate with the position where the illumination light is focused into a plurality of lines by the cylindrical lens array 714. The phase modulation element 718 is configured using, for example, a reflective liquid crystal element or a transmissive liquid crystal element, and divides and displays a plurality of phase modulation patterns on the display surface. The display surface of the phase modulation element 718 is provided with a plurality of irradiation areas (for example, six sets of irradiation areas) that can be irradiated with illumination light. Each irradiation area is divided into a plurality of rectangular partial areas (for example, four partial areas) separated from each other. The partial regions constituting each irradiation region on the display surface of the phase modulation element 718 are arranged in a direction perpendicular to the longitudinal direction of the elliptical cross section of the illumination light. The phase modulation element 718 displays a plurality of phase modulation patterns by dividing them into a plurality of partial areas in a plurality of irradiation areas.

FIG. 33 shows an arrangement example of a plurality of irradiation areas and a plurality of partial areas forming each irradiation area. In the example of FIG. 33, the display surface of the phase modulation element 718 is provided with six sets of irradiation areas divided into four rectangular partial areas. For example, as the four partial areas constituting the first irradiation area SE1, (in order from the top of FIG. 33) the first partial area SE1A and the second partial area S
E1B, third partial area SE1C, and fourth partial area SE1D are arranged separately.

Also, along with the first partial area SE1A of the first irradiation area SE1, a first partial area SE2A of the second irradiation area, a first partial area SE3A of the third irradiation area, and a first partial area of the fourth irradiation area. SE4A, the first partial area SE5A of the fifth irradiation area, and the first partial area SE6A of the sixth irradiation area are arranged. Along with the second partial area SE1B of the first irradiation area SE1, a second partial area SE2B of the second irradiation area, a second partial area SE3B of the third irradiation area, and a second partial area SE4B of the fourth irradiation area. , a second partial area SE5B of the fifth irradiation area, and a second partial area SE6B of the sixth irradiation area are arranged.

Along with the third partial area SE1C of the first irradiation area SE1, a third partial area SE2C of the second irradiation area, a third partial area SE3C of the third irradiation area, and a third partial area SE4C of the fourth irradiation area. , a third partial area SE5C of the fifth irradiation area, and a third partial area SE6C of the sixth irradiation area are arranged. Along with the fourth partial area SE1D of the first irradiation area SE1, a fourth partial area SE2D of the second irradiation area, a fourth partial area SE3D of the third irradiation area, and a fourth partial area SE4D of the fourth irradiation area. , a fourth partial area SE5D of the fifth irradiation area, and a fourth partial area SE6D of the sixth irradiation area are arranged.

As shown in FIG. 33, the phase modulation element 718 displays the phase modulation pattern of the first irradiation area SE1 by dividing it into first to fourth partial areas SE1A to SE1D in the first irradiation area SE1. The phase modulation element 718 displays the phase modulation pattern of the second irradiation area SE2 by dividing it into first to fourth partial areas SE2A to SE2D in the second irradiation area SE2. In this way, the phase modulation element 718 divides and displays the phase modulation pattern of the n-th irradiation area SEn (n=1 to 6) into the first to fourth partial areas SEnA to SEnD in the n-th irradiation area SEn. . A horizontal one-dimensional phase pattern in FIG. 33 is displayed in each partial area in each irradiation area set on the phase modulation element 718. In the example shown in FIG. 33, the four illumination lights divided by the cylindrical lens array 714 are directed to the first to fourth partial areas SEnA to SEnD in the n-th irradiation area SEn, that is, to any one of the first to sixth irradiation areas. The first to fourth partial areas in the irradiation area are irradiated.

When the first to fourth partial regions in any one of the first to sixth irradiation regions are irradiated with four separate illumination lights, the phase of each illumination light changes (modulates). Specifically, each illumination light is given a phase distribution according to a one-dimensional phase modulation pattern in a partial region irradiated with the illumination light. As a result, as shown in FIG. 34, the illumination light combined by the cylindrical lens array 714 (described later) and emitted from the phase modulation unit 710 has a phase modulation set in the irradiation area consisting of the first to fourth partial areas. A phase distribution corresponding to the pattern is given. In addition, by changing the irradiation region (that is, the set of the first to fourth partial regions) to which the illumination light is irradiated in the phase modulation element 718, the phase distribution imparted to the illumination light (the detection light is irradiated to the irradiation region In this case, it is possible to change the phase distribution imparted to the detection light.

A plurality of observation areas in the sample TP can be set in the same manner as the observation areas illustrated in the twelfth embodiment. Specifically, as shown in FIG. 27, for example, 36 observation areas KD1 to KD36 in the sample TP are set within the field of view of the microscope. In the examples shown in FIGS. 33 and 27, a method for acquiring images of six observation areas KD1 to KD6 will be described here. The phase modulation element 718 divides and displays the phase modulation pattern previously determined for the first observation area KD1 into first to fourth partial areas SE1A to SE1D in the first irradiation area SE1 of the phase modulation element 718. The phase modulation element 718 divides and displays the phase modulation pattern previously determined for the second observation area KD2 into first to fourth partial areas SE2A to SE2D in the second irradiation area SE2 of the phase modulation element 718. The phase modulation element 718 divides and displays the phase modulation pattern previously determined for the third observation area KD3 into first to fourth partial areas SE3A to SE3D in the third irradiation area of the phase modulation element 718.

The phase modulation element 718 divides and displays the phase modulation pattern previously determined for the fourth observation area KD4 into first to fourth partial areas SE4A to SE4D in the fourth irradiation area of the phase modulation element 718. The phase modulation element 718 divides and displays the phase modulation pattern previously determined for the fifth observation area KD5 into first to fourth partial areas SE5A to SE5D in the fifth irradiation area of the phase modulation element 718. The phase modulation element 718 divides and displays the phase modulation pattern previously determined for the sixth observation area KD6 into first to fourth partial areas SE6A to SE6D in the sixth irradiation area of the phase modulation element 718.

When observing the first to sixth observation areas KD1 to KD6 in the sample TP, that is, when acquiring images of the first to sixth observation areas KD1 to KD6, the sample scanning section 25 of the illumination optical system 21 uses a phase modulation unit. The illumination light emitted from 710 scans across the first to sixth observation areas KD1 to KD6 on the sample TP. For example, in synchronization with the timing of linearly scanning the first observation area KD1, the phase modulation scanner 716 changes the irradiation area of the illumination light onto the phase modulation element 718 to the first irradiation area SE1. In synchronization with the timing of linearly scanning the second observation area KD2, the phase modulation scanner 716 changes the irradiation area of the illumination light onto the phase modulation element 718 to the second irradiation area SE2.

In synchronization with the timing of linearly scanning the third observation area KD3, the phase modulation scanner 716 changes the irradiation area of the illumination light onto the phase modulation element 718 to the third irradiation area. In synchronization with the timing of linearly scanning the fourth observation area KD4, the phase modulation scanner 716 changes the irradiation area of the illumination light onto the phase modulation element 718 to the fourth irradiation area. In synchronization with the timing of linearly scanning the fifth observation area KD5, the phase modulation scanner 716 changes the irradiation area of the illumination light onto the phase modulation element 718 to the fifth irradiation area. In synchronization with the timing of linearly scanning the sixth observation area KD6, the phase modulation scanner 716 changes the irradiation area of the illumination light onto the phase modulation element 718 to the sixth irradiation area.

At this time, each partial area irradiated with the divided illumination light sequentially changes from each partial area in the first irradiation area to each partial area in the sixth irradiation area. For example, as shown in FIG. 33, the first partial area irradiated with the divided illumination light LE1 changes sequentially from the first partial area SE1A in the first irradiation area to the first partial area SE6A in the sixth irradiation area. The second partial area irradiated with the divided illumination light LE1 changes sequentially from the second partial area SE1B in the first irradiation area to the second partial area SE6B in the sixth irradiation area. The third partial area irradiated with the divided illumination light LE1 changes sequentially from the third partial area SE1C in the first irradiation area to the third partial area SE6C in the sixth irradiation area. The fourth partial area irradiated with the divided illumination light LE1 changes sequentially from the fourth partial area SE1D in the first irradiation area to the fourth partial area SE6D in the sixth irradiation area.

When acquiring images of the seventh to twelfth observation areas KD7 to KD12, the phase modulation patterns displayed in the first to sixth irradiation areas SE1 to SE6 of the phase modulation element 718 are The phase modulation patterns are changed to those obtained in advance for the regions KD7 to KD12, and images are acquired in the same manner as for the first to sixth observation regions KD1 to KD6. Similarly, images of the 13th to 18th observation areas KD13 to KD18, the 19th to 24th observation areas KD19 to KD24, the 25th to 30th observation areas KD25 to KD30, and the 31st to 36th observation areas KD31 to KD36 are When acquiring each image, the phase modulation pattern displayed in each irradiation area of the phase modulation element 718 is sequentially changed to a phase modulation pattern determined in advance for the corresponding observation area, and the above-described image acquisition is performed.

[First modification of phase modulation unit]
Next, a first modification of the phase modulation unit in the sixteenth embodiment will be described with reference to FIG. 35. As shown in FIG. 35, a phase modulation unit 730 according to the first modification of the sixteenth embodiment includes a first relay lens 731, a first mirror 732, a second relay lens 733, a cylindrical lens array 734, It has a lens drive section 735, a phase modulation element 718, a second mirror 739, and a third relay lens 740, but the components other than the cylindrical lens array 734, the lens drive section 735, and the phase modulation element 718 have the phase shown in FIG. The functions and configurations of the first relay lens 61, first mirror 62, second relay lens 64, second mirror 67, and third relay lens 68 included in the modulation unit 60 are the same, and The functions and configurations of the first relay lens 651, first mirror 652, second relay lens 653, second mirror 657, and third relay lens 658 included in the phase modulation unit 650 shown in FIG. 30 are the same. Therefore, the explanation will be omitted. The phase modulation unit 730 corresponds to the microlens array disk 654, motor 655, and phase modulation element 616 included in the phase modulation unit 650, which are replaced by a cylindrical lens array 734, a lens driving section 735, and a phase modulation element 718. Note that the portion surrounded by a broken line in FIG. 35 is a diagram of a part of the phase modulation unit 730 viewed from a direction perpendicular to FIG. 35.

The first relay lens 731 is arranged near a position B42 that is optically conjugate with the sample TP. The first mirror 732 is arranged near a position B42 that is optically conjugate with the sample TP.

The cylindrical lens array 734 includes a plurality of cylindrical lenses (for example, four cylindrical lenses) arranged in a row in a direction perpendicular to the optical axis AX1 of the illumination optical system 21. The cylindrical lens array 734 divides the illumination light from the second relay lens 733 into a plurality of line-shaped illumination lights (for example, four illumination lights). Further, the cylindrical lens array 734 combines a plurality of illumination lights reflected by the phase modulation element 718 into substantially parallel light. The cylindrical lens array 734 is supported by a lens drive unit 735 so as to be movable in the direction in which the cylindrical lenses are arranged. The lens driving section 735 is configured using a voice coil motor, a piezo element, or the like. The lens drive unit 735 linearly moves the cylindrical lens array 734 in the direction in which the cylindrical lenses are arranged.

The phase modulation element 718 is arranged at a position H42 that is optically conjugate with the pupil position of the objective lens 28. The phase modulation element 718 has the same configuration as the phase modulation element 718 according to the sixteenth embodiment, is given the same reference numeral as in the sixteenth embodiment, and detailed description thereof will be omitted. The second mirror 739 is arranged near a position B42 that is optically conjugate with the sample TP. The second mirror 739 reflects the illumination light that has passed through the second relay lens 733 and entered the second mirror 739 toward the third relay lens 740 . The third relay lens 740 converts the illumination light reflected by the second mirror 739 into substantially parallel light.

A plurality of illumination lights separated by the cylindrical lens array 734 are applied to a plurality of partial areas in any one of the plurality of irradiation areas (for example, the first to sixth irradiation areas in any one of the first to sixth irradiation areas). to the fourth partial region), the phase distribution of each illumination light changes according to the phase modulation pattern in the longitudinal direction in the partial region irradiated with the illumination light. A plurality of illumination lights irradiated onto a plurality of partial regions in any one of the plurality of irradiation regions is reflected at each partial region. Note that the lens driving unit 735 moves the cylindrical lens array 734 in the direction in which the cylindrical lenses are lined up, and scans the phase modulation element 718 with a plurality of illumination lights separated in the cylindrical lens array 734. It is possible to change the irradiation area (ie, the set of the first to fourth partial areas) to which the illumination light is irradiated among the plurality of irradiation areas.

[Second modification of phase modulation unit]
Next, a second modification of the phase modulation unit in the sixteenth embodiment will be described with reference to FIG. 36. As shown in FIG. 36, a phase modulation unit 750 according to the second modification of the sixteenth embodiment includes a beam splitter 751, a first relay lens 752, a second relay lens 753, a cylindrical lens array 754, and a lens Although it has a driving section 755 and a phase modulation element 718, except for the cylindrical lens array 754, the lens driving section 755, and the phase modulation element 718, the beam splitter 661 and the first relay included in the phase modulation unit 660 shown in FIG. Since the functions and configurations of the lens 662 and the second relay lens 663 are the same, their explanations will be omitted. Note that the portion surrounded by a broken line in FIG. 36 is a diagram of a part of the phase modulation unit 750 viewed from a direction perpendicular to FIG. 36.

The phase modulation element 718 is arranged at a position H43 that is optically conjugate with the pupil position of the objective lens 28. The phase modulation element 718 has the same configuration as the phase modulation element 718 according to the sixteenth embodiment, is given the same reference numeral as in the sixteenth embodiment, and detailed description thereof will be omitted. The cylindrical lens array 754 is the same as the cylindrical lens array 734 included in the phase modulation unit 730 shown in FIG. 35, so a description thereof will be omitted. Note that the illumination light that enters the beam splitter 751 from the collimator lens 22 of the illumination optical system 21 and passes through the beam splitter 751 and the first relay lens 752 is once focused at a position B43 that is optically conjugate with the sample TP. , passes through the second relay lens 753 and enters the cylindrical lens array 754.

[Seventeenth embodiment]
Next, a microscope according to a seventeenth embodiment will be described. The microscope according to the seventeenth embodiment has the same configuration as the microscope 101 according to the second embodiment, except that the phase modulation unit 710 according to the sixteenth embodiment is provided instead of the phase modulation unit 60, and Common parts other than the unit 710 are given the same reference numerals as in the second embodiment, and detailed illustrations and explanations are omitted. In the microscope according to the seventeenth embodiment, a phase modulation unit 710 performs phase modulation on the illumination light that has passed through the dichroic mirror 123 of the illumination optical system 121, and also performs phase modulation on the detection light that has passed through the sample scanning unit 125. Modulation is performed to correct at least a portion of aberrations occurring in the sample TP, the illumination optical system 121, and the detection optical system 131.

The illumination light that has passed through the dichroic mirror 123 of the illumination optical system 121 is incident on the first relay lens 711 of the phase modulation unit 710, but the subsequent steps are the same as in the sixteenth embodiment, so a description thereof will be omitted.

In addition, the detection light that has passed through the sample scanning unit 125 as the detection optical system 131 enters the phase modulation unit 710, then follows a path in the opposite direction to that of the illumination light described above, and is subjected to phase modulation similar to that of the illumination light. The detection light is emitted from the phase modulation unit 710 toward the dichroic mirror 123 in a substantially parallel manner. As in the 16th embodiment, by sequentially changing the irradiation area to which illumination light and detection light are irradiated in the phase modulation element 616 in synchronization with scanning across multiple observation areas, one observation area can be Compared to the case where the irradiation area is changed each time a scan is performed, a high-quality image of the sample TP can be obtained in a shorter time.

Further, even when using the microscope according to the seventeenth embodiment, it is possible to acquire an image of the sample TP by the image acquisition method described in the first embodiment, as with the microscope 101 according to the second embodiment. Therefore, according to the seventeenth embodiment, the same effects as the sixteenth embodiment and the second embodiment can be obtained. Further, according to the seventeenth embodiment, the phase modulation unit 710 performs phase modulation on the detection light in addition to the phase modulation on the illumination light, so that it is possible to obtain a higher quality image of the sample TP in a short time. It becomes possible.

In the microscope according to the seventeenth embodiment, a phase modulation unit 730 according to the first modification of the sixteenth embodiment may be provided instead of the phase modulation unit 710. Similarly to the seventeenth embodiment described above, the phase modulation unit 730 according to the first modification of the sixteenth embodiment can perform phase modulation on the illumination light and the detection light. Furthermore, instead of the phase modulation unit 710, a phase modulation unit 750 according to a second modification of the sixteenth embodiment may be provided.

[18th embodiment]
Next, a microscope according to the eighteenth embodiment will be described. The microscope according to the 18th embodiment has the same configuration as the microscope 201 according to the 3rd embodiment, except that the phase modulation unit 710 according to the 16th embodiment is provided instead of the phase modulation unit 60. Common parts other than the unit 710 are given the same reference numerals as in the third embodiment, and detailed illustrations and explanations are omitted.

In the microscope according to the 18th embodiment, the phase modulation unit 710 performs phase modulation on the illumination light transmitted through the collimator lens 222 of the illumination optical system 221, and eliminates at least aberrations occurring between the sample TP and the illumination optical system 221. Correct some parts. At this time, the phase modulation unit 710 performs phase modulation on the illumination light in the same manner as in the sixteenth embodiment. Further, even when using the microscope according to the eighteenth embodiment, it is possible to acquire an image of the sample TP by the image acquisition method described in the first embodiment, similarly to the microscope 201 according to the third embodiment. Therefore, according to the 18th embodiment, the same effects as the 16th embodiment and the 3rd embodiment can be obtained.

In the microscope according to the eighteenth embodiment, the phase modulation unit 710 may be replaced with a phase modulation unit 730 according to the first modification of the sixteenth embodiment. Similarly to the first modification of the sixteenth embodiment described above, the phase modulation unit 730 according to the first modification of the sixteenth embodiment can perform phase modulation on illumination light. Furthermore, instead of the phase modulation unit 710, a phase modulation unit 750 according to a second modification of the sixteenth embodiment may be provided.

[19th embodiment]
Next, a microscope according to a nineteenth embodiment will be described. The microscope according to the nineteenth embodiment has the same configuration as the microscope 301 according to the fourth embodiment, except that the phase modulation unit 710 according to the sixteenth embodiment is provided instead of the phase modulation unit 60, and Common parts other than the unit 710 are given the same reference numerals as in the fourth embodiment, and detailed illustrations and explanations are omitted.

In the microscope according to the nineteenth embodiment, the phase modulation unit 710 performs phase modulation on the illumination light that has passed through the collimator lens 322 of the illumination optical system 321, and the phase modulation that occurs between the sample TP, the illumination optical system 321, and the detection optical system 331 At least some of the aberrations are corrected. At this time, the phase modulation unit 710 performs phase modulation on the illumination light in the same manner as in the sixteenth embodiment. Further, even when using the microscope according to the nineteenth embodiment, it is possible to acquire an image of the sample TP by the image acquisition method described in the first embodiment, similarly to the microscope 301 according to the fourth embodiment. Therefore, according to the nineteenth embodiment, the same effects as the sixteenth embodiment and the fourth embodiment can be obtained.

In the microscope according to the nineteenth embodiment, a phase modulation unit 730 according to the first modification of the sixteenth embodiment may be provided instead of the phase modulation unit 710. Similarly to the first modification of the sixteenth embodiment described above, the phase modulation unit 730 according to the first modification of the sixteenth embodiment can perform phase modulation on illumination light. Furthermore, instead of the phase modulation unit 710, a phase modulation unit 750 according to a second modification of the sixteenth embodiment may be provided.

[Twentieth embodiment]
Next, a microscope according to the 20th embodiment will be described with reference to FIG. 37. The microscope according to the 20th embodiment has the same configuration as the microscope 501 according to the 11th embodiment, except that the phase modulation unit 810 according to the 20th embodiment is provided instead of the phase modulation unit 60, and Common parts other than the unit 810 are given the same reference numerals as in the eleventh embodiment, and detailed illustrations and explanations are omitted. In the microscope according to the twentieth embodiment, the phase modulation unit 810 performs phase modulation on the illumination light transmitted through the collimator lens 522 of the illumination optical system 521, and eliminates at least aberrations occurring between the sample TP and the illumination optical system 521. Correct some parts. As shown in FIG. 37, the phase modulation unit 810 according to the twentieth embodiment includes a first relay lens 811, a first mirror 812, a second relay lens 813, a cylindrical lens array 814, and a third relay lens 815. , a phase modulation scanner 816, a fourth relay lens 817, a phase modulation element 818, a second mirror 819, and a fifth relay lens 820, but each function and configuration other than the phase modulation element 818 are , a first relay lens 711, a first mirror 712, a second relay lens 713, a cylindrical lens array 714, a third relay lens 715, and a phase modulation scanner 716 included in the phase modulation unit 710 shown in FIG. The fourth relay lens 717, the second mirror 719, and the fifth relay lens 720 are the same, so their explanation will be omitted. Note that the part surrounded by a broken line in FIG. 37 is a diagram of a part of the phase modulation unit 810 viewed from a direction perpendicular to FIG. 37.

The cylindrical lens array 814 includes a plurality of cylindrical lenses (for example, four cylindrical lenses) arranged in a row in a direction perpendicular to the optical axis AX51 of the illumination optical system 521. Further, the phase modulation scanner 816 is arranged at a position B51 that is optically conjugate with the sample TP. The cylindrical lens array 814 and the phase modulation scanner 816 are the same as the cylindrical lens array 714 and the phase modulation scanner 716 included in the phase modulation unit 710 shown in FIG. 32, so a description thereof will be omitted.

The phase modulation element 818 is arranged at a position H51 that is optically conjugate with the pupil position of the objective lens 528. The one-dimensional phase modulation pattern in the longitudinal direction of each partial region displayed by the phase modulation element 818 is constructed using a computer-generated hologram (CGH), and any one of the plurality of irradiation areas in the phase modulation element 65 is illuminated. When light is irradiated, a plurality of reflected lights (diffraction lights) are generated from the irradiation area irradiated with the illumination light due to a diffraction phenomenon (FIG. 37 shows an example in which two diffraction lights are generated). At this time, by using a computer-generated hologram (CGH), a different phase distribution can be given to each of the multiple reflected lights generated by the diffraction phenomenon, depending on the observation position of the sample illuminated with each reflected light (diffraction light). is possible. Note that the computer-generated hologram (CGH) can be calculated using the same method as in the eleventh embodiment.

The arrangement of the plurality of irradiation regions and the plurality of partial regions constituting each irradiation region is set in the same manner as in the case of the phase modulation element 718 according to the sixteenth embodiment. The phase modulation element 818 according to the 20th embodiment divides the phase modulation pattern of the n-th irradiation area SEn (n=1 to 6) into the first to fourth partial areas SEnA to SEnD in the n-th irradiation area SEn. indicate. The four illumination lights divided by the cylindrical lens array 814 are transmitted to the first to fourth partial areas SEnA to SEnD in the n-th irradiation area SEn, that is, to the first to fourth partial areas SEnA to SEnD in the n-th irradiation area SEn, that is, to the ~4th partial region is irradiated. Note that the phase modulation element 818 according to the twentieth embodiment displays a phase modulation pattern that can generate diffracted light.

When the first to fourth partial areas set in any one of the first to sixth irradiation areas are irradiated with four separate illumination lights, the phase distribution of each illumination light changes (modulation). )do. Specifically, according to the phase modulation pattern (phase distribution) set in the partial area irradiated with the illumination light,
Due to the diffraction phenomenon, reflected light (diffraction light) is generated in a plurality of directions (for example, two directions). A different phase distribution is given to each of the plurality of diffracted lights. The diffracted lights diffracted in the same direction in each of the first to fourth partial regions are combined into one illumination light by the cylindrical lens array 814 (described later), and the illumination light emitted from the phase modulation unit 810 is combined with the first A phase distribution according to the phase modulation pattern set in the irradiation area consisting of the ~4th partial area is imparted. Further, a plurality of illumination lights (for example, two illumination lights) are emitted from the phase modulation unit 810 by the number of diffracted lights generated in the partial region of the phase modulation element 818. Each of the plurality of illumination lights emitted from the phase modulation unit 810 is given a different phase distribution depending on the observation position of the sample to be illuminated. The phase distribution imparted to each of the plurality of illumination lights emitted from the phase modulation unit 810 can be changed by changing the irradiation area (that is, the set of the first to fourth partial areas) on which the illumination light is irradiated in the phase modulation element 818. It is possible to change.

Note that the fourth relay lens 817 overlaps the plurality of diffracted lights reflected by the phase modulation element 818. The phase modulation scanner 816 is configured such that the diffracted light incident on the phase modulation scanner 816 from the fourth relay lens 817 travels toward the third relay lens 815, that is, the illumination light emitted from the phase modulation unit 810 is configured as an illumination optical system. The traveling directions of the illumination light and the diffracted light are changed so that they travel along the optical axis AX51 of the system 521. The plurality of diffracted lights that entered the third relay lens 815 pass through the third relay lens 815 and enter the cylindrical lens array 814 . Among the plurality of diffracted lights that have entered the cylindrical lens array 814, the diffracted lights that travel in the same direction are transmitted through the cylindrical lens array 814 and combined into one substantially parallel illumination light beam. On the other hand, since the diffracted lights traveling in different directions are not combined by the cylindrical lens array 814, substantially parallel illumination lights are emitted equal to the number of traveling directions of the diffracted lights incident on the lindrical lens array 814. The second relay lens 813 focuses the plurality of illumination lights emitted from the cylindrical lens array 814 onto a plurality of positions. The second mirror 819 reflects the plurality of illumination lights that have passed through the second relay lens 813 and entered the second mirror 819 toward the fifth relay lens 820 . The fifth relay lens 820 converts the illumination light reflected by the second mirror 819 into substantially parallel light. The two illumination lights emitted from the phase modulation unit 810 enter the dichroic mirror 524 of the illumination optical system 521. In this manner, the illumination light is divided and phase modulated by the phase modulation unit 810.

Note that even when using the microscope according to the twentieth embodiment, it is possible to acquire an image of the sample TP by the image acquisition method described in the first embodiment. According to the 20th embodiment, the phase modulation unit 810 has a plurality of irradiation areas that can be irradiated with illumination light, and imparts a phase distribution to the illumination light according to the irradiation position of the illumination light in the plurality of irradiation areas. and a phase modulation scanner 816 that changes the irradiation area that is irradiated with illumination light. If the phase modulation pattern that changes the phase of the illumination light is changed by changing the irradiation area of the illumination light on the phase modulation element 818 using the phase modulation scanner 816, the phase modulation pattern displayed by the phase modulation element 818 can be changed. The phase modulation pattern can be changed in a shorter time than when switching the phase modulation pattern.

Further, the phase modulation unit 810 includes a cylindrical lens array 814 (optical element array) that divides the illumination light into a plurality of line-shaped illumination lights and irradiates the plurality of partial regions in each irradiation region. Therefore, as the sample scanning unit 525 of the illumination optical system 521 scans the sample TP, the phase modulation scanner 816 changes the irradiation area that is divided into a plurality of partial areas that are irradiated with a plurality of illumination lights. This makes it possible to obtain a high-quality image of the sample TP in a shorter time, in which aberrations caused by a plurality of observation areas on the sample TP are individually corrected. Further, it is possible to provide a phase modulation pattern having a smooth phase distribution in the longitudinal direction of each partial region in the irradiation region of the phase modulation element 818.

Further, the phase modulation scanner 816 moves the traveling direction of the illumination light whose phase has changed by irradiating the irradiation area to a constant direction (the illumination optical system 521 (along the optical axis AX51). Thereby, only the phase of the illumination light can be changed without changing the optical path of the illumination light.

Furthermore, the phase modulation pattern displayed by the phase modulation element 818 is constructed using a computer-generated hologram (CGH). Thereby, the illumination optical system 521 can illuminate the sample TP with a plurality of (for example, two) illumination lights separated by the diffraction of the phase modulation element 818, and can produce a higher quality image of the sample TP. It can be obtained in a short time.

In each of the embodiments described above, an inverted microscope is exemplified as the microscope, but the present invention is not limited to this, and the microscope according to each embodiment may be an upright microscope.

1 Microscope (first embodiment)
21 Illumination optical system 31 Detection optical system 60 Phase modulation unit (first embodiment)
63 First phase modulation scanner 65 Phase modulation element 66 Second phase modulation scanner 80 Phase modulation unit (modification of the first embodiment)
83 Phase modulation scanner 101 Microscope (second embodiment)
121 Illumination optical system 131 Detection optical system 201 Microscope (third embodiment)
221 Illumination optical system 231 Detection optical system 301 Microscope (4th embodiment)
321 Illumination optical system 331 Detection optical system 401 Microscope (fifth embodiment)
421 Illumination optical system 431 Detection optical system 160 Phase modulation unit (6th embodiment)
164 First cylindrical lens 165 Second cylindrical lens 167 Phase modulation scanner 169 Phase modulation element 180 Phase modulation unit (modification of the sixth embodiment)
184 First cylindrical lens 185 Second cylindrical lens 187 Phase modulation scanner 501 Microscope (11th embodiment)
521 Illumination optical system 531 Detection optical system 560 Phase modulation unit (11th embodiment)
564 First cylindrical lens 565 Second cylindrical lens 567 Phase modulation scanner 569 Phase modulation element 572 Microlens array 580 Phase modulation unit (modification of the 11th embodiment)
584 First cylindrical lens 585 Second cylindrical lens 587 Phase modulation scanner 592 Microlens array 610 Phase modulation unit (12th embodiment)
611 First microlens array 614 First phase modulation scanner 616 Phase modulation element 617 Second phase modulation scanner 620 Second microlens array 630 Phase modulation unit (first modification of the twelfth embodiment)
631 Microlens array 634 Phase modulation scanner 640 Phase modulation unit (second modification of the twelfth embodiment)
641 First microlens array disk 649 Second microlens array disk 650 Phase modulation unit (third modification of the twelfth embodiment)
654 Microlens array disk 660 Phase modulation unit (4th modification of 12th embodiment)
664 Microlens array disk 710 Phase modulation unit (16th embodiment)
714 Cylindrical lens array 716 Phase modulation scanner 718 Phase modulation element 730 Phase modulation unit (first modification of the twelfth embodiment)
734 Cylindrical lens array 735 Linear motor 750 Phase modulation unit (second modification of the twelfth embodiment)
754 Cylindrical lens array 755 Linear motor 810 Phase modulation unit (20th embodiment)
814 Cylindrical lens array 816 Phase modulation scanner 818 Phase modulation element

Claims (10)

an illumination optical system that irradiates the sample with illumination light from a light source;
a detection optical system that receives detection light from the sample;
a phase modulation unit disposed in at least one of the illumination optical system and the detection optical system,
The phase modulation unit is
a plurality of irradiation areas that can be irradiated with at least one of the illumination light and the detection light; a phase modulation element that imparts a predetermined phase distribution;
A microscope including a first scanning unit that relatively moves the irradiation position and the plurality of irradiation areas. 2. The microscope according to claim 1, wherein the phase modulation unit includes a second scanning section that makes the traveling direction of the at least one light beam imparted with the predetermined phase distribution constant. The microscope according to claim 1 or 2, wherein the plurality of irradiation areas do not overlap with each other. 4. The first scanning unit changes the irradiation position of the at least one light in the plurality of irradiation areas when the observation area in which the sample is subjected to microscopic observation is displaced. The microscope described. The microscope according to any one of claims 1 to 4, wherein the phase modulation unit includes a cylindrical lens that makes the cross-sectional shape of the at least one light beam elliptical. the phase modulation unit is arranged in the illumination optical system,
the at least one light is the illumination light,
The phase modulation unit has an optical element array that divides the illumination light into a plurality of lights,
The microscope according to claim 5, wherein the illumination optical system irradiates the sample with the plurality of lights. The irradiation area is divided into a plurality of partial areas separated from each other,
5. The phase modulation unit includes an optical element array that divides the at least one light into a plurality of lights and irradiates the plurality of partial regions in the irradiation region. microscope. The illumination optical system includes a third scanning unit that scans the sample with the illumination light,
8. The microscope according to claim 7, wherein the first scanning section changes the irradiation area to which the plurality of lights are irradiated in synchronization with the third scanning section scanning the sample with the illumination light. The microscope according to claim 7 or 8, wherein the plurality of lights separated by the optical element array form a line shape on each of the plurality of partial regions. the phase modulation unit is arranged in the illumination optical system,
the at least one light is the illumination light,
The illumination light is divided by the phase modulation element into a plurality of diffracted lights each having a mutually different phase distribution,
10. The microscope according to claim 9, wherein the illumination optical system irradiates the sample with the plurality of diffracted lights.

Images