JP2013045014A - Microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an image having high resolution in an optical axis direction by a simple optical adjustment.SOLUTION: A microscope device 1 of the present invention includes a light source 4 oscillating laser light L with which a sample S is irradiated, a first objective lens 21 focusing the laser light L on the sample S, a corner cube 24 retro-reflecting the laser light L after passing through the sample S, a second objective lens 23 focusing the laser light L retro-reflected by the corner cube 24 on the sample S, a camera 16 detecting return light R produced by the fluorescence of the sample S by the laser light L, a dish drive mechanism 25 relatively moving the sample S and a focal point in the optical axis direction of the laser light L, and a control part 17 performing calculation for deconvolution processing on the basis of the cross section information of the sample S obtained by the relative movement by the dish drive mechanism 25.

Description

  The present invention relates to a microscope apparatus that irradiates a sample with illumination light and observes an image of the sample.

  The microscope apparatus is used for irradiating a sample with illumination light and observing an image thereof. As a microscope apparatus, a confocal microscope having a high resolution in the optical axis direction is often used, but a 4Pi confocal microscope having a higher resolution is used.

  In the 4Pi confocal microscope, two objective lenses are arranged to face each other so that the focal position overlaps with the sample. The resolution is improved in the optical axis direction by causing the excitation light to interfere from the two objective lenses. In addition, the return light that is fluorescent from the focal point narrowed by the interference is collected from the two objective lenses, and the return light is made to interfere to further improve the resolution in the optical axis direction.

  A microscope similar to a 4Pi confocal microscope is disclosed in Patent Document 1. The microscope of Patent Document 1 includes an objective lens and an infinity correction objective lens with a sample interposed therebetween, and reflects light transmitted through the infinity correction objective lens. Thereby, light is passed through the sample twice to enhance the contrast of the image.

JP-A-4-27909

  In the 4Pi confocal microscope, the optical adjustment must be performed so that the focal positions of the two objective lenses coincide strictly with the optical axis direction and the direction orthogonal to the optical axis direction. In addition, the optical path for collecting the fluorescence needs to be accurately superimposed again. This has caused a problem that optical adjustment becomes very difficult.

  Moreover, in the microscope of Patent Document 1, although the contrast of an image can be enhanced by passing light through the sample twice, the light is simply passed through the sample twice. Therefore, an image with high resolution in the optical axis direction is not obtained. In recent years, it has become important to obtain a clear image with sufficiently high resolution in the optical axis direction.

  Accordingly, an object of the present invention is to obtain a clear image with high resolution in the optical axis direction by simple optical adjustment.

  In order to solve the above problems, a microscope apparatus according to the present invention includes a light source that oscillates illumination light that irradiates a sample, a first objective lens that focuses the illumination light on the sample, and the sample that has passed through the sample. A retroreflective optical system that retroreflects illumination light, a second objective lens that connects the focal point of the illumination light retroreflected by the retroreflective optical system to the sample, and return light that the sample is fluorescent by the illumination light. Based on a detection unit for detection, a relative movement mechanism for relatively moving the sample and the focal point in the optical axis direction of the illumination light, and cross-sectional information of the sample obtained by relative movement by the relative movement mechanism An arithmetic unit that performs a deconvolution process.

  According to this microscope apparatus, the illumination light focused by the first objective lens interferes with the illumination light retroreflected and focused by the second objective lens. Then, the relative movement mechanism moves so as to photograph different cross sections of the sample, and the arithmetic unit performs the deconvolution process, whereby a clear image with high resolution in the optical axis direction can be acquired.

  The focal point may be scanned on a plane orthogonal to the optical axis direction.

  By scanning the focal point on a plane orthogonal to the optical axis direction, it is possible to obtain cross-sectional information on a predetermined cross-section of the sample. The cross-sectional information is a clear image with high resolution in the optical axis direction because the arithmetic unit performs the deconvolution process.

  A pinhole disk in which a plurality of pinholes are arranged; and a rotating unit that rotates the pinhole disk. The illumination light is passed through the pinhole and the light that has passed through the pinhole is projected onto the sample. 3. The microscope according to claim 2, wherein a focal point is formed on the sample, and the rotating unit rotates the pinhole disk to cause the focal point to scan on a plane orthogonal to the optical axis direction. apparatus.

  By rotating the pinhole disk, the focal point can be scanned in a direction orthogonal to the optical axis direction. Thereby, the cross-section information in the predetermined cross section of the sample is acquired.

  Moreover, the component which passed the said pinhole among the fluorescence from the said focus is detected, It is characterized by the above-mentioned.

  Pinholes pass only light within the focal range, and by extracting the components that have passed through the pinhole from the fluorescence from the focal point, only the light within the focal range is computerized, and high resolution in the optical axis direction Can be generated.

  The focal point of the sample may be scanned on a plane orthogonal to the optical axis by a variable mirror that can rotate about one axis.

  The focal point can be scanned on a plane perpendicular to the optical axis direction by rotating the variable mirror instead of rotating the pinhole disk.

  The computing unit may perform the deconvolution process on all pixels in a horizontal plane direction orthogonal to the optical axis direction.

  As a result, an image with high resolution can be acquired for a predetermined region in the horizontal plane direction of the sample. The image in the horizontal plane direction can be acquired by means having a predetermined pixel in the horizontal plane direction like a camera, or can be acquired by scanning illumination light in the horizontal plane direction.

  In addition, an optical path length changing mechanism that changes an optical path length of the illumination light by changing an interval between the second objective lens and the retroreflective optical system, and the arithmetic unit is configured to change the optical path length when the optical path length changes. The calculation is performed based on the return light.

  By changing the optical path length, the phase of interference fringes in the sample changes, and more sample information can be obtained. Thereby, the precision of the calculation performed by the calculation unit is improved, and an image with higher resolution can be obtained.

  Further, a pupil relay lens system for relaying a pupil position of the second objective lens is provided between the second objective lens and the retroreflective optical system.

  Even when the second objective lens and the retroreflective optical system are too close together, the retroreflective optical system can be provided at the pupil position of the second objective lens by providing the pupil relay lens system, and the first A predetermined interval can be provided between the two objective lenses and the retroreflective optical system.

  In the present invention, the illumination light focused by the first objective lens interferes with the illumination light retroreflected and focused by the second objective lens. Then, the relative movement mechanism moves so as to photograph different cross sections of the sample, and the arithmetic unit performs the deconvolution process, whereby a clear image with high resolution in the optical axis direction can be acquired.

It is a block diagram of the microscope apparatus of embodiment. It is a block diagram of a pinhole disk and a micro lens disk. It is a figure explaining the intensity distribution of the focus of a sample. It is a figure explaining the other example of intensity distribution of the focus of a sample. It is a block diagram of the microscope apparatus of the modification 1. FIG. 10 is a configuration diagram of a microscope optical system according to Modification 2. It is a figure of the 1st objective lens and the 2nd objective lens of modification 3 and 4. It is a block diagram of the light source of the modification 6. FIG. It is a figure explaining the delay of phase distribution of the phase control board of the modification 6. FIG. It is a figure explaining the pattern of the focus of the STED laser of modification 6.

  Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 shows a microscope apparatus 1 according to an embodiment. The microscope apparatus 1 includes a scanning optical system 2 and a microscope optical system 3. As shown in the figure, the scanning optical system 2 includes a light source 4, a fiber 5, a collimating lens 6, a micro lens disk 7, a pinhole disk 8, a connecting drum 9, a motor 10, a dichroic mirror 11, a first relay lens 12, and a mirror. 13, a fluorescent filter 14, a second relay lens 15, a camera 16, a control unit 17, and a monitor 18.

  The light source 4 is a light source that oscillates the laser light L. The laser light L becomes illumination light for observing the sample S that is an observation target. The fiber 5 is an optical fiber and guides the laser light L. A collimating lens 6 is disposed on the emission side of the fiber 5, and the laser light L emitted from the fiber 5 becomes parallel light by the collimating lens 6.

  A microlens disk 7 is disposed in front of the collimating lens 6. The micro lens disk 7 and the pinhole disk 8 are connected by a connecting drum 9, and the connecting drum 9 is connected to a motor 10. The microlens disk 7 and the pinhole disk 8 are rotating disks, and when the rotational force is applied to the connecting drum 9 by the motor 10, the microlens disk 7 and the pinhole disk 8 rotate integrally. The connecting drum 9 and the motor 10 constitute a rotating part.

  As shown in FIG. 2, the pinhole disk 8 is formed with a large number of spiral pinholes 8 </ b> P arranged in multiple rows (four in the figure). The pinhole 8P is a minute opening that allows only light within the focal range of the sample S to pass through. The microlens disk (lens disk) 7 is formed by arranging a large number of spiral microlenses 7M in the same pattern as the pinhole 8P. The micro lens 7M has a function of condensing the laser light L in the corresponding pinhole 8P.

  As shown in FIGS. 1 and 2, a dichroic mirror 11 is provided between the microlens disk 7 and the pinhole disk 8. The dichroic mirror 11 is an optical element having a characteristic of transmitting the wavelength of the laser light L oscillated by the light source 4 and reflecting the wavelength of the fluorescence of the sample S. A first relay lens 12 is provided at a position where light is reflected by the dichroic mirror 11.

  The light relayed by the first relay lens 12 is reflected by the mirror 13 and guided to the fluorescent filter 14. The fluorescent filter 14 is a filter that selectively transmits only the wavelength of the fluorescent component generated by the sample S. The light that has passed through the fluorescent filter 14 enters the second relay lens 15.

  Then, this light is condensed on the camera 16 by the second relay lens 15. The camera 16 converts the received light into an electrical signal and outputs it to the control unit 17. The control unit 17 is a computer that controls the entire microscope apparatus 1, and generates an image of the sample S based on an electrical signal input from the camera 16. The control unit 17 operates as a calculation unit that performs a predetermined calculation. The generated image is displayed on the monitor 18.

  Next, the microscope optical system 3 will be described. As shown in FIG. 1, the microscope optical system 3 includes a photographing lens 19, a mirror 20, a first objective lens 21, a dish 22, a second objective lens 23, a corner cube 24, and a dish driving mechanism 25. Yes.

  The taking lens 19 converts the laser light L that has passed through the pinhole 8P into parallel light. The parallel light is reflected by the mirror 20 and guided to the first objective lens 21. The laser beam L is focused on the sample S mounted on the dish 22 by the first objective lens 21. The dish 22 is a mounting portion on which the sample S is mounted.

  The laser light L is transmitted through the sample S, and the second objective lens 23 is disposed at the transmitted position. The first objective lens 21 and the second objective lens 23 are arranged so that their focal planes coincide. The laser light L is converted into parallel light by the second objective lens 23 and is incident on the corner cube 24. The corner cube 24 is a retroreflective optical system in which a cube is formed by combining three mirrors at right angles to each other.

  A dish driving mechanism 25 is attached to the dish 22. The dish drive mechanism 25 is a relative movement mechanism that minutely moves the dish 22 in the optical axis direction of the laser light L. This relative movement mechanism relatively moves the focal point of the laser beam L and the sample S. The relative movement mechanism is controlled by the control unit 17, and the dish 22 moves in the optical axis direction.

  Next, the operation will be described. The controller 17 oscillates the laser light L from the light source 4. The laser light L is condensed on the fiber 5 and emitted from the end of the fiber 5 with a spread. The laser light L is converted into parallel light by the collimating lens 6 and enters the microlens 7M of the microlens disk 7.

  The microlens 7M has a function of a condensing lens. The laser light L passes through the dichroic mirror 11 and then focuses on the pinhole 8P corresponding to the microlens 7M. The laser beam L that has passed through the pinhole 8P is incident on the taking lens 19. The laser light L is converted by the photographing lens 19 into parallel light having an inclination corresponding to the position of the pinhole 8P. The parallel light is reflected by the mirror 20, the optical path is converted by 90 degrees, and enters the first objective lens 21.

  Then, the first objective lens 21 focuses on the sample S mounted on the dish 22. At this time, the laser light L that has passed through the sample S is converted by the second objective lens 23 into parallel light having an inclination corresponding to the focal position. That is, when the focal point is at the center of the optical axis, the parallel light is parallel to the optical axis, and as the focal point is shifted from the optical axis center, the parallel light is inclined with respect to the optical axis.

  The laser light L converted into parallel light by the second objective lens 23 is retroreflected by the corner cube 24 in the same direction as the incident direction. Therefore, the light travels back along the same optical path as the incident light, and enters the second objective lens 23 again. Then, the laser light L is focused on the sample S again by the action of the second objective lens 23.

  The sample S is introduced with a fluorescent dye or fluorescent protein that fluoresces light of the wavelength of the laser beam L. Therefore, when the laser beam L is focused from the first objective lens 21 and the second objective lens 23 by the sample S, the sample S is fluorescent. The generated fluorescence goes to the first objective lens 21 and the second objective lens 23.

  The fluorescent light traveling toward the second objective lens 23 is retroreflected by the corner cube 24 and focused on the sample S again. This fluorescence goes to the first objective lens 21. On the other hand, the fluorescence originally generated in the sample S goes to the first objective lens 21.

  Therefore, the fluorescence generated in the sample S is composed of two fluorescences, light directly toward the first objective lens 21 and light retroreflected by the corner cube 24. Thereby, the fluorescence generated in the sample S can be collected from the upper and lower directions, so that the fluorescence yield is improved. Two fluorescences heading toward the first objective lens 21 are referred to as return light R. Note that the two fluorescent lights have a short coherence distance and therefore hardly interfere with each other.

  The return light R becomes parallel light by the first objective lens 21 and is reflected by the mirror 20. The return light R reflected by the mirror 20 is condensed from the photographing lens 19 to the pinhole 8P. Since the pinhole 8P allows only light within the focal range of the sample S to pass, the image generated by the camera 16 is a confocal image. The return light R that has passed through the pinhole 8P is reflected by the dichroic mirror 11.

  Then, the return light R reflected by the dichroic mirror 11 is relayed by the first relay lens 12 and reflected by the mirror 13. The return light R reflected by the mirror 13 enters the fluorescent filter 14 and the components other than the fluorescent component of the sample S are removed. Thereby, only the fluorescence image is taken by the camera 16.

  The return light R transmitted through the fluorescent filter 14 is imaged on the camera 16 by the second relay lens 12. The camera 16 converts the formed return light R into an electric signal and outputs it to the control unit 17. The control unit 17 performs predetermined image processing based on the input electrical signal.

  Here, the control unit 17 controls the motor 10 to apply a rotational force. Thereby, the connecting drum 9 rotates and the microlens disk 7 and the pinhole disk 8 rotate integrally. A number of microlenses 7M are arranged on the microlens disk 7, and a number of pinholes 8P having the same pattern are arranged on the pinhole disk 8.

  Therefore, by rotating the microlens disk 7 and the pinhole disk 8 integrally, as shown in FIG. 2, the pinhole 8P through which the laser light L passes changes sequentially with rotation. Thereby, the laser beam L scans the focal plane of the sample S on a horizontal plane. The return light R passes through the pinhole 8P again, is reflected by the dichroic mirror 11, and is scanned on the imaging surface of the camera 16.

  Therefore, the laser light L can be scanned at high speed in the horizontal plane direction of the sample S by integrally rotating the microlens disk 7 and the pinhole disk 8. Thereby, since the camera 16 outputs the electrical signal of the return light R sequentially scanned, the control unit 17 can generate an image of the sample S at high speed. Then, the generated image of the sample S is displayed on the monitor 18.

  At this time, the laser light L applied to the sample S and the return light R imaged on the camera 16 pass through the pinhole 8P. Since the pinhole 8P passes only light within the focal point of the sample S and does not allow other light to pass through, the return light R imaged on the camera 16 is only light within the focal range of the sample S. Thereby, a high-resolution confocal image is generated in the depth direction.

  The camera 16 has predetermined pixels in the planar direction (X direction and Y direction). That is, it has a plurality of pixels in the X direction and a plurality of pixels in the Y direction, and this is an image of a predetermined region of the sample S in the horizontal plane direction. Attention is paid to one pixel of the pixels of the camera 16. This pixel is fluorescence information of one point of the sample S, but this fluorescence information is blurred in the optical axis direction (Z direction) of the laser light L.

  That is, the spatial information in the Z direction is imprinted in one plane image and is optically convolved information. In order to return this to the original information, a deconvolution integration (deconvolution) process is performed. When performing the deconvolution process, information on a plurality of cross sections in the Z direction is required.

  Therefore, the control unit 17 drives the dish driving mechanism 25. Thereby, the dish 22 is finely driven, and the sample S mounted on the dish 22 is finely moved along the optical axis (Z direction) of the laser light L. By this movement, information of different cross sections in the Z direction can be acquired. The control unit 17 continuously drives the dish drive mechanism 25 to acquire information on each cross section in the Z direction.

  Here, with respect to the sample S, two laser beams L, a laser beam L irradiated from the first objective lens 21 and a laser beam L retroreflected by the corner cube 24 and irradiated from the second objective lens 23. To focus on. Therefore, these two laser beams L interfere with each other in the sample S.

  The optical path length from when the laser beam L is first focused on the sample S to when it is retroreflected by the corner cube 24 and then focused again becomes an even multiple of half the wavelength λ of the laser beam L (λ / 2). In some cases, the light intensifies at the focal plane. Here, it is assumed that the optical path length takes into account the phase change due to reflection occurring in the corner cube 24. Further, at the position separated from the focal plane by λ / 2, the phases of the two lights are shifted by λ, so they strengthen each other. Since the intensity of light at a position where there is no interference effect decreases as the distance from the focal plane increases, the intensity distribution at the focal point is as shown in FIG.

  In other words, as shown in FIG. 3, when the optical path length is an even multiple of λ / 2 of the laser light L, the focus F intensifies in a narrow region F1 on the focal plane, and λ / The light intensifies in the region F2 separated by two. In addition, light intensifies in a region F3 that is separated from the focal plane by λ. However, the region where the light is strong is “F1> F2> F3”.

  On the other hand, when the optical path length is an odd multiple of half the wavelength λ of the laser light L (λ / 2), the light is weakened at the focal plane. Further, light is intensified in a region F1 separated by λ / 4 from the focal plane, and light is intensified in a region F2 separated by 3λ / 4. However, the area where the light is intensified is “F1> F2.”

  The region in which the light is intensified varies depending on the optical path length and varies depending on the refractive index distribution of the sample or the like on the optical path, but generally varies according to the distribution as shown in FIGS.

  Spatial information in the optical axis direction (Z direction) is imprinted on one pixel of the camera 16, that is, the information of FIG. 3 or FIG. 4 is imprinted as blur information. The return light R received by the camera 16 is converted into an electrical signal and output to the control unit 17. The control unit 17 performs a deconvolution process based on the electrical signal.

  The control unit 17 has a PSF pattern for performing the deconvolution process. The PSF pattern may be measured in advance, or may be estimated from an image obtained by observing the sample S. For example, as a method for obtaining a PSF pattern by actual measurement in advance, there are an inverse filter method and an asymptotic method, and as a method for estimating from an observed image, there is a blind method.

  The control unit 17 has a PSF pattern corresponding to the optical path length, and each of the samples S when the dish 22 is finely moved by the dish driving mechanism 25 (that is, the sample S is finely moved in the optical axis direction). Deconvolution processing is performed based on the cross-sectional image. Thereby, the spatial information imaged on one pixel of the camera 16 can be returned to the original information.

  That is, it is possible to remove the information of the overlapping components. Thereby, the information of the component of high resolution can be acquired. For example, referring to FIG. 3 or FIG. 4, a clear image without blurring can be generated without the information of only the region F1 in the focal point F being imprinted with the information of other cross sections (tomographic sections). it can.

  For this reason, the information of the image of the area | region F1 very narrow in the Z direction (optical axis direction) in the focus F can be acquired as a clear image without blur. For this reason, a clear image with high resolution can be acquired. Moreover, since the corner cube 24 is used for retroreflection, the optical axis adjustment in the direction orthogonal to the optical axis does not require the accuracy of the optical axis adjustment as that of the 4Pi confocal microscope. In addition, it is not necessary to make adjustments so that the optical paths of the return lights coincide. Therefore, it can be realized by simple optical adjustment.

  The camera 16 has a predetermined number of pixels in the XY directions. The deconvolution process described above is performed for each pixel. Thereby, a horizontal tomographic image of a predetermined region of the sample S can be acquired. This tomographic image has a high resolution in the Z direction and is a clear image.

  In the above, the corner cube 24 is an optical system in which a cube is formed by combining three mirrors at right angles to each other. As the mirror, a mirror that is made by polishing glass to be totally reflected may be used, or a mirror that is provided with a reflective film may be used. In short, any optical element can be used as long as it performs retroreflection to return incident light in the incident direction.

  Further, the dish driving mechanism 25 as a relative movement mechanism moves the dish 22 in the optical axis direction, thereby moving the sample S relative to the focal point of the laser light L. In this regard, the dish 22 (that is, the sample S) may be fixed and the focal point of the laser light L may be moved. In this case, other optical systems (such as the first objective lens 21 and the second objective lens 23) other than the dish 22 are moved relative to the dish 22.

  Next, Modification 1 will be described with reference to FIG. In the first modification, the configurations of the scanning optical system 2 and the microscope optical system 3 are different from those of the embodiment. First, the scanning optical system 2 will be described. As shown in FIG. 5, the scanning optical system 2 includes a light source 31, a dichroic mirror 32, a scanning optical unit 33, a scanning system pupil relay lens 34, a fluorescent filter 35, a focusing lens 36, a pinhole 37, a detector 38, and a control unit 17. And a monitor 18. The control unit 17 and the monitor 18 are the same as those in the embodiment.

  Moreover, the microscope optical system 3 adds a first pupil relay lens 41 and a second pupil relay lens 42 in addition to the configuration of the embodiment. The first pupil relay lens 41 and the second pupil relay lens 42 constitute a pupil relay lens system, and the pupil relay lens system is provided between the second objective lens 23 and the corner cube 24. The corner cube 24 is provided with a cube driving mechanism 43 that moves the corner cube 24 in the optical axis direction of the laser light L.

  In the above configuration, the control unit 17 oscillates the parallel laser beam L from the light source 31. The laser light L is reflected by the dichroic mirror 32 and guided to the scanning optical unit 33. The scanning optical unit 33 includes a first variable mirror 33a that can rotate around one axis, and a second variable mirror 33b that can rotate around an axis substantially orthogonal to the axis of the first variable mirror 33a.

  The laser light L is scanned in the horizontal plane direction of the sample S by the first variable mirror 33a and the second variable mirror 33b. The light passing through the scanning optical unit 33 exits the scanning optical system 2 from the scanning system pupil relay lens 34 and enters the photographing lens 19 of the microscope optical system 3. Then, the first objective lens 21 focuses on the sample S.

  The laser light L that has passed through the sample S is converted into parallel light by the second objective lens 23 and guided to the corner cube 24 by the pupil relay lens system of the first pupil relay lens 41 and the second pupil relay lens 42. Then, the sample is retroreflected by the corner cube 24, passes through the first pupil relay lens 41 and the second pupil relay lens 42, and is focused again on the sample S by the second objective lens 23.

  As a result, the sample S is irradiated with the laser light L from the upper and lower directions, and the two laser lights L interfere as described above. When the laser beam L focused by the sample S interferes, the sample S generates fluorescence and the return light R is generated. The return light R is reflected by the mirror 20 and enters the dichroic mirror 32 from the photographing lens 19 through the scanning system pupil relay lens 34 and the scanning optical unit 33.

  Then, the light passes through the dichroic mirror 32, and only the wavelength of the fluorescent component is selected by the fluorescent filter 35 and converges on the detector 38 by the focusing lens 36. At this time, a pinhole 37 is provided on the optical path, and the pinhole 37 is arranged in a positional relationship conjugate with the focal point of the first objective lens 21. Therefore, only the light within the focal range of the sample S of the return light R passes. Thereby, the generated image becomes a confocal image.

  At this time, the dish drive mechanism 25 finely moves the dish 22 to acquire images of different cross sections of the sample S. Thus, the deconvolution processing is performed from the PSF pattern of the image having the focal intensity distribution as shown in FIG. 3 or FIG. 4, and the target intensity component (for example, the focal plane region F1 in the case of FIG. 3). Remove components other than). For this reason, it is possible to eliminate blur caused by information on a plurality of cross sections in the Z direction being imprinted in one image, and to acquire a clear image with high resolution in the optical axis direction.

  Here, in the first modification, a cube driving mechanism 43 that moves the corner cube 24 in the optical axis direction is provided. The cube drive mechanism 43 moves the corner cube 24 in the optical axis direction, so that the optical path length from when the laser light L is focused on the sample S until it is retroreflected by the corner cube 24 and focused on the sample S again. Can be changed.

  That is, by changing the optical path length, both the intensity distribution pattern as shown in FIG. 3 and the intensity distribution pattern as shown in FIG. 4 can be adopted, and an appropriate pattern therebetween is also adopted. be able to. That is, the pattern can be changed to an arbitrary intensity distribution pattern.

  As a result, the phase of interference fringes at the focal point can be arbitrarily changed. At this time, the control unit 17 performs deconvolution processing in consideration of image information with different phases of interference fringes (performs calculation processing in consideration of different image information as shown in FIGS. 3 and 4). As a result, the deconvolution process can be performed using image information of interference fringes having different phases, so that the calculation accuracy can be improved. That is, since an image of the sample S can be obtained based on a larger amount of information, an image with higher resolution in the optical axis direction than in the embodiment can be obtained.

  Here, the pupil position of the second objective lens 23 may be very close to the second objective lens 23 or may be located inside the lens barrel of the second objective lens 23. Here, the corner cube 24 can be arranged at the pupil position (rear focus position) of the second objective lens 23 to obtain the highest light utilization efficiency. However, the pupil position of the second objective lens 23 is within the lens barrel. The corner cube 24 cannot be placed at the pupil position of the second objective lens 23.

  Therefore, a first pupil relay lens 41 and a second pupil relay lens 42 that relay the pupil position of the second objective lens 23 are provided. Thereby, the pupil position of the second objective lens 23 is relayed, and the pupil position can be relayed to the corner cube 24 located at a position away from the second objective lens 23. As a result, almost all of the light emitted from the pupil of the second objective lens 23 is incident on the second objective lens 23 again by retroreflection, so that high light utilization efficiency can be obtained.

  In the first modification, the detector 38 receives the return light R at one point. That is, information on one point of the sample S is received. For this reason, the scanning optical unit 33 scans the laser beam L in the X direction and the Y direction of the sample S. Thereby, the image of the predetermined area | region of the horizontal direction of the sample S is acquirable.

  Next, Modification 2 will be described. FIG. 6 shows a microscope apparatus 1 according to the second modification. In FIG. 6, only the microscope optical system 3 is shown, and the scanning optical system 2 is omitted. The scanning optical system 2 is the same as that in the above-described embodiment. The microscope optical system 3 of Modification 2 includes a telecentric imaging lens 51 and a reflection mirror 52 instead of the corner cube 24.

  The laser light L converted into parallel light by the second objective lens 23 is focused on the reflection mirror 52 by the telecentric imaging lens 51 and returns from the telecentric imaging lens 51 to the second objective lens 23. Therefore, the telecentric imaging lens 51 and the reflection mirror 52 may constitute a retroreflection optical system that retroreflects the incident laser light L.

  Next, Modification 3 will be described. FIG. 7 shows a first objective lens 21, a dish 22, and a second objective lens 23 in Modification 3. In the third modification, the first objective lens 21 uses a dry objective lens. Here, a 20 × dry objective lens, a numerical aperture of 0.75, and a working distance of about 0.6 mm are used. Of course, numerical values other than these may be used.

  Since the working distance of the first objective lens 21 is very short, when a similar dry objective lens is used as the second objective lens 23, the dish in which the sample S is mounted between the first objective lens 21 and the second objective lens 23 is used. It becomes difficult to arrange 22.

  Therefore, a water immersion objective lens is used as the second objective lens 23. As the water immersion objective lens, it is desirable to use, for example, an electrophysiological objective lens that does not require a cover glass. For example, it is desirable to use a 40 × water immersion lens, a numerical aperture of 0.8, and a working distance of about 3.3 mm.

  By using such a water immersion objective lens as the second objective lens 23, a sufficient working distance can be ensured, and the dish 22 on which the sample S is mounted can be easily disposed. Further, when the sample S is a biological sample of a cell, since it is often in the culture medium W, it is preferable to use a water immersion objective lens.

  Next, Modification 4 will be described. The configurations of the first objective lens 21, the dish 22, and the second objective lens 23 are the same as those in FIG. In Modification 4, the first objective lens 21 uses an oil immersion objective lens. Here, a 100 × oil immersion objective lens having a numerical aperture of 1.4 and a working distance of about 0.1 mm is used. Of course, lenses other than these may be used.

  As in the third modification, the working distance of the first objective lens 21 is very short. Therefore, when a similar oil immersion objective lens is used as the second objective lens 23, the first objective lens 21 and the second objective lens 23 It becomes difficult to arrange the dish 22 on which the sample S is mounted.

  Therefore, a water immersion objective lens is used as the second objective lens 23. As the water immersion objective lens, it is desirable to use, for example, an electrophysiological objective lens that does not require a cover glass. For example, if a 60-times water immersion lens, a numerical aperture of 1.1, and a working distance of about 1.1 mm are used, a sufficient working distance can be secured.

  In Modification 4, the numerical aperture of the second objective lens 23 is smaller than that of the first objective lens 21. For this reason, the focal point which is retroreflected by the corner cube 24 and is connected to the sample S by the second objective lens 23 is slightly increased, and the density of interference is also decreased.

  Therefore, the control unit 17 performs a process of extracting only a high frequency component in the optical axis direction of the laser light L when performing the deconvolution process. As a result, it is possible to avoid degradation of resolution due to the numerical aperture of the second objective lens 23 being reduced.

  Next, Modification 5 will be described. In Modification 5, the microscope apparatus 1 of Modification 1 is applied to a saturation excitation microscope. In the microscope apparatus 1 of the modified example 5, the oscillation intensity of the laser light L from the light source 31 is increased. Further, the oscillation frequency of the laser light L is modulated at a constant frequency f. This causes saturation of fluorescence, particularly only near the top of the intensity of the point spread (PSF).

  As a result, distortion occurs in the temporal change of the fluorescence signal from the region where the fluorescence occurs, and harmonics including twice and three times the frequency appear in the distorted wave. By detecting a signal component including this harmonic, an image with high resolution in the horizontal direction (XY direction) and the optical axis direction (Z direction) can be obtained.

  At this time, the laser beam L focused by the first objective lens 21 and the laser beam L retroreflected and focused by the second objective lens 23 interfere with each other in the sample S. In addition, the dish driving mechanism 25 moves the dish 22 in the optical axis direction to acquire images of different cross sections of the sample S. Then, by performing the deconvolution process based on the image acquired by the control unit 17, it is possible to acquire a clear image with high resolution in the optical axis direction.

  Therefore, in the fifth modification, by applying a microscope apparatus capable of obtaining high resolution in the optical axis direction to the saturation microscope, high resolution can be obtained not only in the optical axis direction but also in the horizontal direction. It becomes possible to obtain an image with a high dimensional resolution.

  Next, Modification 6 will be described. In Modification 6, the microscope apparatus 1 of Modification 1 is applied to a stimulated emission suppression (STED) microscope. FIG. 8 shows a light source 31 used in the sixth modification. As shown in this figure, the light source 31 has an excitation laser light source 60, a STED laser light source 61, a phase control plate 62, a mirror 63, and a dichroic mirror 64.

  The excitation laser light source 60 is a light source that oscillates the laser light L. The STED laser light source 61 is a light source that oscillates the STED laser LS. The STED laser LS is a laser beam for stimulated emission. The phase control plate 62 is an optical element that generates a phase distribution delay as shown in FIG.

  The mirror 63 reflects the STED laser LS. The dichroic mirror 64 is an optical element having a characteristic of transmitting the laser light L and reflecting the STED laser LS. The dichroic mirror 32 shown in FIG. 3 has a characteristic of reflecting the laser light L and the STED laser LS and transmitting the fluorescence.

  When the light source 31 shown in FIG. 8 is used, the STED laser LS is reflected by the dichroic mirror 64 under the action of the phase control plate 62. Thereby, the optical paths of the laser beam L and the STED laser LS are the same. When the sample S is focused by the first objective lens 21, the focus of the STED laser LS is a ring-shaped pattern as shown in FIG. 10 on the XY plane.

  By reducing the center area of the ring-shaped pattern, the area where fluorescence is generated can be made extremely small. This makes it possible to obtain an image with high resolution in the XY directions.

  At this time, the laser beam L focused by the first objective lens 21 and the laser beam L retroreflected and focused by the second objective lens 23 interfere with each other in the sample S. In addition, the dish driving mechanism 25 moves the dish 22 in the optical axis direction to acquire images of different cross sections of the sample S. Then, by performing the deconvolution process based on the image acquired by the control unit 17, it is possible to acquire a clear image with high resolution in the optical axis direction.

  Therefore, in the fifth modification, by applying a microscope apparatus capable of obtaining high resolution in the optical axis direction to the STED microscope, high resolution can be obtained not only in the optical axis direction but also in the horizontal direction. It becomes possible to obtain an image with a high dimensional resolution.

DESCRIPTION OF SYMBOLS 1 Microscope apparatus 2 Scan optical system 3 Microscope optical system 4 Light source 7 Micro lens disk 8 Pinhole disk 11 Dichroic mirror 16 Camera 17 Control part 21 1st objective lens 22 Dish 23 2nd objective lens 24 Corner cube 25 Dish drive mechanism 41 1st 1 pupil relay lens 42 2 pupil relay lens 43 Cube drive mechanism L Laser light R Return light S Sample

Claims (8)

A light source that oscillates the illumination light applied to the sample;
A first objective lens that focuses the illumination light on the sample;
A retroreflective optical system that retroreflects the illumination light transmitted through the sample;
A second objective lens that focuses the illumination light retroreflected by the retroreflective optical system on the sample;
A detection unit that detects return light that the sample is fluorescent by the illumination light; and
A relative movement mechanism for relatively moving the sample and the focal point in the optical axis direction of the illumination light;
An arithmetic unit for performing a deconvolution process based on cross-sectional information of the sample obtained by relative movement by the relative movement mechanism;
A microscope apparatus comprising: The microscope apparatus according to claim 1, wherein the focal point is scanned on a plane orthogonal to the optical axis direction. A pinhole disk with a plurality of pinholes,
A rotating part for rotating the pinhole disk;
With
The illumination light is allowed to pass through the pinhole, and the light passing through the pinhole is projected onto the sample to form a focal point on the sample, and the rotating unit rotates the pinhole disk to thereby cause the focal point to rotate. The microscope apparatus according to claim 2, wherein the scanning is performed on a plane orthogonal to the optical axis direction. The microscope apparatus according to claim 3, wherein a component that has passed through the pinhole is detected in the fluorescence from the focal point. The microscope apparatus according to claim 2, wherein the focal point of the sample is scanned on a plane orthogonal to the optical axis by a variable mirror that can rotate around one axis. 6. The microscope apparatus according to claim 1, wherein the calculation unit performs the deconvolution processing on all pixels in a horizontal plane direction orthogonal to the optical axis direction. An optical path length changing mechanism that changes an optical path length of the illumination light by changing an interval between the second objective lens and the retroreflective optical system;
The microscope apparatus according to claim 6, wherein the calculation unit performs the calculation based on the return light when the optical path length changes. The microscope apparatus according to claim 7, wherein a pupil relay lens system that relays a pupil position of the second objective lens is provided between the second objective lens and the retroreflective optical system.

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