JP2009157084A - Microscope apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microscope apparatus capable of acquiring information on a super-resolution image, at a high speed. <P>SOLUTION: In the microscope device which includes an illumination optical system for illuminating a sample with a spatially modulated illuminating light, the illumination optical system has a beam-splitting part which splits a beam into a plurality of beams; a heater 44 which selects at least two beams from among the plurality of beams; and a waveguide type phase modulating element 42, which performs phase-modulation of at least one beam from among the beams selected by the heater 44. The beam-splitting part is equipped with a waveguide type splitter 43 that splits the beam into plurality of beams, by branching to a plurality of branched waveguides 51a to 51f from an input waveguide 50. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a microscope, and more particularly to a high-resolution microscope that can realize super-resolution in the in-plane direction.

  In the field of observation and measurement of sample microstructure, observation with higher spatial resolution is required. As a method of increasing the in-plane resolution of the sample, light of a high spatial frequency component is imaged out of the light from the sample, so that light is emitted near the sample surface or at a position conjugate with the sample surface in the illumination optical system. Alternatively, there is a technique in which a certain modulation is applied to the illumination light and demodulation corresponding to the given modulation is performed at a position substantially conjugate with the sample surface in the imaging optical system. As an example, there are methods disclosed in Patent Documents 1 and 2.

US Pat. No. 6,239,909 US Reissue Patent No. 38307 Specification

  When changing the phase of the structured illumination on the specimen, if the position of the grating used as the light splitting means is highly accurately translated in the direction perpendicular to the optical axis by the translation stage, this translation stage has a large It takes time to stop positioning due to inertia, and high-speed driving is difficult. Also, if the rotation of the grating in the direction of the structured illumination is performed by rotating the grating by the rotation mechanism, it takes time to stop the rotation due to the moment of inertia of the rotation mechanism, and it is difficult to rotate the structured illumination at a high speed. In particular, when the observation target is a living biological specimen, it is essential to acquire an image at high speed, but there is a problem that the method of mechanical positioning as described above is not practical.

  The present invention has been made in view of such problems, and an object of the present invention is to provide a microscope apparatus capable of acquiring information of a super-resolution image at high speed.

  According to a first aspect illustrating the present invention, in a microscope apparatus including an illumination optical system that illuminates spatially modulated illumination light onto a specimen, the illumination optical system includes a light beam dividing unit that divides a light beam into a plurality of light beams. A light beam selection unit that selects at least two light beams out of a plurality of light beams, and a phase modulation unit that phase-modulates at least one light beam among the light beams selected by the light beam selection unit. What is claimed is: 1. A microscope apparatus comprising a multi-divided waveguide element (for example, a waveguide type demultiplexer 43 in the embodiment) that splits a light beam into a plurality of light beams by branching from the waveguide into a plurality of branch waveguides. Provided.

  According to the second aspect of the present invention, in the microscope apparatus including the illumination optical system that illuminates the spatially modulated illumination light onto the specimen, the illumination optical system splits the light beam into a plurality of light beams. And a phase modulation unit that phase-modulates at least one of the light beams selected by the light beam selection unit. A microscope apparatus comprising a waveguide type phase modulation element having at least a pair of control electrodes is provided.

  In the present invention, it is possible to realize a microscope apparatus that can acquire information of a super-resolution image at high speed by using a waveguide element.

  Embodiments of the present invention will be described below with reference to the drawings. The microscope apparatus of the present embodiment acquires a super-resolution image by irradiating a specimen with structured illumination light using a waveguide element.

  First, a first embodiment of the microscope apparatus will be described with reference to FIG. FIG. 1 is a schematic configuration diagram of a microscope apparatus according to the first embodiment of the present invention. As shown in FIG. 1, the microscope apparatus includes an optical fiber (light source) 1, a waveguide duplexer 43, a waveguide phase modulator 42, a lens 41, a lens 4, a light shielding plate 6, a lens 7, and a field stop. 8, lens 9, excitation filter 10, dichroic mirror 11, objective lens 12, specimen (biological specimen, etc.) 13, barrier filter 14, second objective lens 15, imaging device (CCD camera, etc.) 21, control / arithmetic unit 22, An image display device 23 is arranged.

  Among these, the optical fiber 1, the waveguide type demultiplexer 43, the waveguide type phase modulation element 42, the lens 41, the lens 4, the light shielding plate 6, the lens 7, the field stop 8, the lens 9, the excitation filter 10, and the dichroic mirror 11. The objective lens 12 constitutes an illumination optical system, and the objective lens 12, the dichroic mirror 11, the barrier filter 14, and the second objective lens 15 constitute an observation optical system (imaging optical system). The illumination optical system and the observation optical system share an optical path from the objective lens 12 to the dichroic mirror 11.

  Light from a coherent light source (not shown) of the illumination optical system is guided by the optical fiber 1 and is incident on a waveguide type splitter 43 connected to the output end of the optical fiber 1.

  As shown in FIG. 2, the waveguide duplexer 43 includes branch waveguides 51 a to 51 f branched from one input waveguide 50 into six, and input light incident on the input waveguide 50. Are respectively emitted to the branching waveguides 51a to 51f as six lights having the same light intensity. Each of the branching waveguides 51a to 51f is provided with a heater 44. The heater 44 is energized by applying a predetermined voltage according to an output signal from the control / arithmetic unit 22, and a corresponding waveguide portion (output) Part) is heated locally. As a result, the resistance of the heated waveguide portion increases, whereby the light is attenuated, and the output light from the waveguide heated by the heater 44 can be blocked. By providing the heater 44 in this way, the waveguide type demultiplexer 43 can switch on and off the respective output lights from the branched waveguides 51a to 51f branched into six. Only the waveguide can emit light. The on / off switching circuit 44a of the heater 44 is disposed only in the branch waveguide 51f in FIG. 2, and the illustration of the other branch waveguides is omitted, but the on / off switching circuit 44a is 6 Each of the six branch waveguides 51a to 51f is provided, and each of the six branch waveguides 51a to 51f can be switched on / off independently of the light output.

  The waveguide duplexer 43 may include an interference waveguide switch instead of the heater 44 in the branch waveguides 51a to 51f as described above.

  Six waveguide phase modulation elements 42 respectively corresponding to the six branch waveguides 51 a to 51 f are connected to the output side of the waveguide duplexer 43. As shown in FIG. 3, each of the waveguide type phase modulation elements 42 is parallel to the optical axis at the position of each vertex of a regular hexagon formed around the optical axis in the annular member 70 that fixes the waveguide. Be placed. For this reason, the waveguide type phase modulation elements 42 are arranged at intervals of 60 ° in a plane perpendicular to the optical axis. Each waveguide type phase modulation element 42 includes a pair (two) of electrodes 45 disposed so as to sandwich the branch waveguides 52a to 52f, and a predetermined voltage is applied between the electrodes from the controller 22. By applying, the refractive index (optical path length) of the branched waveguides 52a to 52f can be changed, and the phase of the output light can be changed. The voltage application circuit 45a of the electrode 45 is disposed only in the branch waveguide 52f and the other illustration is omitted, but the voltage application circuit 45a is provided in each of the six branch waveguides 52a to 52f. A voltage can be applied independently and independently to each electrode 45 provided in the branching waveguides 52a to 52f.

Here, the phase change amount Δφ of the light having the wavelength λ when the voltage V is applied is approximately determined by a constant A determined by the physical properties of the substance forming the waveguide,
Δφ = −AVL / λ
Therefore, it is possible to obtain an applied voltage at which a desired phase change amount Δφ can be obtained in advance. In this embodiment, a waveguide element that performs phase modulation by applying a voltage between the electrodes is used. However, a waveguide element that performs phase modulation by heating the waveguide or applying mechanical pressure is used. May be configured.

  Here, in the first embodiment, as will be described in detail later, the structured illumination that irradiates the specimen 13 is formed by interference by two light beams that are symmetrical with respect to the optical axis. Therefore, it is necessary to output light from the two waveguide phase modulation elements 42 that are directly opposed to the optical axis among the six waveguide phase modulation elements 42. For this reason, on / off control of the optical output is performed by the waveguide type demultiplexer 43, and the optical output is generated from the two waveguide type phase modulation elements 42 facing the optical axis. Two symmetrical light beams.

  The light emitted from the waveguide type phase modulation element 42 is converted into parallel light by the lens 41, and a light source image is formed on the pupil conjugate plane 31 by the lens 4. A light shielding plate 6 is disposed at the pupil conjugate position, and unnecessary light such as stray light is shielded and removed. The light that has passed through the light-shielding plate 6 passes through a field stop 8 disposed at a position where a sample conjugate plane is formed by a lens 7, and then dichroic divides and synthesizes the lens 9, excitation filter 10, epi-illumination system, and imaging system. Two spots are formed on the pupil P of the objective lens 12 via the mirror 11. These two spots are formed almost at the outermost periphery of the objective lens 12 and, when exiting from the objective lens 12, illuminate the sample surface as parallel light beams having an angle of maximum NA facing each other. At this time, since the two light beams are coherent, the sample surface is irradiated with the structure of the equally spaced interference fringes. The illumination light having the stripe structure is called structured illumination.

  The spot position on the pupil plane of the objective lens 12 can be adjusted by changing the magnification of the lens 41 and the lens 4, but this interval determines the period of structured illumination on the specimen 13, and this structure. Determining the degree of super-resolution of a standardized illumination microscope relative to a normal microscope.

  When the specimen 13 is irradiated with this structured illumination, the periodic structure of the illumination light interferes with the periodic structure of the specimen 13 to generate moire interference fringes. At this time, the moire interference fringes contain high-frequency shape information of the specimen 13, but are converted into a lower spatial frequency band corresponding to the spatial frequency of the structured illumination, and thus have a structure with a high spatial frequency exceeding the resolution limit. Even the light is captured by the objective lens 12. The principle of the structured illumination super-resolution microscope is to acquire an imaged image, calculate the periodic structure of known illumination light, and obtain and visualize the unknown sample shape by calculating and restoring the periodic structure.

  Then, the light from the specimen 13 is converted into parallel light through the objective lens 12, passes through the dichroic mirror 11, enters the single optical path of the observation optical system, passes through the barrier filter 14, and then passes through the second objective lens. 15, a modulated image of the sample is formed on the imaging surface 16 of the imaging device 21 such as a CCD camera. This modulated image is picked up by the image pickup device 21 to generate image data. This image data includes information for super-resolution observation of the specimen 13 with structured illumination.

  However, since this image data is an image as a result of illumination with illumination light modulated by the waveguide type phase modulation element 43 as described above, the image data is processed by a known image calculation means by the control / calculation device 22. The specimen image can be obtained by restoring by applying reverse modulation (remodulation), and the super-resolution image of the specimen 13 can be displayed on the image display device 23.

  When restoring the original image by image processing, it is preferable to shoot the same specimen by modulating the phase of the interference fringes of the illumination three or more times. This is because the modulated image includes components unnecessary for the demodulated image. In other words, the modulation image has three unknown parameters of the 0th-order light component, the + 1st-order light component, and the −1st-order light component in the information obtained by the structured illumination of the frequency component of the specimen 13. This is because more information than the number of unknowns is required to obtain the unknown.

  Therefore, first, a current is applied by applying a predetermined voltage from the control / arithmetic unit 22 to energize the heater 44 of the waveguide type demultiplexer 43, and the waveguide type phase modulation element 42 corrects the optical axis with respect to the optical axis. Of the branching waveguides (51a and 51d, 51b and 51e, 51c and 51f) of the waveguide type duplexer 43 corresponding to the branching waveguides (52a and 52d, 52b and 52e, 52c and 52f) to be opposed, Only the pair of branch waveguides (for example, 51a and 51d) turns on the light output, and the light output from the remaining branch waveguides (51b and 51e, 51c and 51f) is blocked. At this time, the amount of current to be passed may be equal to or greater than a certain threshold current. This is because the light is thermally turned on and off by heating the waveguide to a certain temperature or higher.

  Furthermore, the electrode 45 is connected to one of the branching waveguides 52a and 52d of the waveguide type phase modulation element 42 connected to the branching waveguides 51a and 51d that output light according to a signal from the control / arithmetic unit 22. A predetermined voltage is applied. Thereby, by changing the phase of the light corresponding to this one waveguide, the phase of the structured illumination on the sample 13 is changed, and a modulated image of the sample with respect to each phase is acquired. The amount of phase change is made as uniform as possible every 1/3 period (that is, relatively 0, 2π / 3, 4π / 3). This is because when the amount of change in phase is uneven, the specimen fades into a striped pattern. In this way, three image data are acquired by changing the phase of structured illumination.

  Since only one of the two lights may be subjected to phase modulation, the waveguide type phase modulator 42 may be provided only in one of the waveguides that performs phase modulation. In order to align the size and the optical path length, it is preferable to provide the same ones symmetrically.

  Next, the direction of the structured illumination is changed by changing the waveguide that outputs light from the waveguide type phase modulation element 42, and the above processing is performed again to obtain the image data of the modulated image of the sample. This is because information for super-resolution observation in multiple directions can be obtained if image data is acquired each time the direction of structured illumination is changed in multiple directions (usually three directions). This enables (two-dimensional) super-resolution observation of the specimen 13. Therefore, image data is acquired by changing the direction of structured illumination in three directions of 0 °, 120 °, and 240 °.

  Specifically, if three pairs of branching waveguides (52a and 52d, 52b and 52e, 52c and 52f) arranged at positions facing the optical axis are sequentially turned on, these waveguides In terms of arrangement (each waveguide is arranged at intervals of 60 °), it can be changed in three directions of 0 °, 120 °, and 240 °. For this reason, the light of the two branch waveguides 52a and 52d, which has been optically output so far, is turned off, and only the branch waveguides 52b and 52e out of the other waveguides facing each other with respect to the optical axis. Turn on and obtain the image data of the modulated image of the specimen when the direction of structured illumination is changed by 120 °. Finally, the optical outputs of the branched waveguides 52b and 52e are turned off, and the optical outputs of only the remaining two branched waveguides 52c and 52f facing each other with respect to the optical axis are turned on. The image data of the modulated image of the sample when the angle is further changed by 120 ° is acquired.

  In this way, the control / arithmetic unit 22 repeats image data acquisition until the structured illumination direction is set to three directions of 0 °, 120 °, and 240 °, and finally acquires a total of nine image data. To do.

  The control / arithmetic unit 22 takes in the nine pieces of image data obtained as described above, and performs calculations by known means as described in Patent Document 1 to obtain a super-resolution image of the specimen 13. be able to. Then, the control / arithmetic unit 22 sends this image data to the image display unit 23 and displays a super-resolution image.

  As described above, in the microscope apparatus according to the first embodiment, the light output is turned on / off by the heater 44 provided in the waveguide of the waveguide type demultiplexer 43 to rotate the direction of the interference fringes (structured illumination). Further, voltage can be applied to the electrode 45 of the waveguide type phase modulation element 42 to perform phase modulation of structured illumination. Therefore, this microscope apparatus can acquire a super-resolution image at high speed.

  In the first embodiment described above, a case has been described in which a super-resolution image is obtained using structured illumination formed by two-beam interference. However, in the second embodiment, three beams are used. A case will be described in which a super-resolution image is acquired using structured illumination formed by interference. In the second embodiment, the same components as those in the first embodiment are denoted by the same reference numerals, and the difference from the first embodiment will be mainly described.

  In the following description, an XYZ orthogonal coordinate system is set in FIG. 4, and the description will be made with reference to this coordinate system. The XYZ orthogonal coordinate system shown in FIG. 4 is set so that the X axis and the Y axis are parallel to the sample surface, and the Z axis is set in a direction orthogonal to the sample surface.

  FIG. 4 is a schematic configuration diagram of a microscope apparatus according to the second embodiment of the present invention. As shown in FIG. 4, the microscope apparatus according to the second embodiment includes an optical fiber (light source) 1, a waveguide duplexer 43 ′, a waveguide phase modulator 42 ′, a lens 41, a lens 4, Shading plate 6 ', lens 7, field stop 8, lens 9, excitation filter 10, dichroic mirror 11, objective lens 12, specimen (biological specimen etc.) 13, barrier filter 14, second objective lens 15, imaging device (CCD camera) 21), a control / arithmetic unit 22, and an image display unit 23 are arranged. Among these, the configurations of the waveguide duplexer 43 ′, the waveguide phase modulation element 42 ′, and the light shielding plate 6 ′ are different from those of the first embodiment.

  As shown in FIG. 5, the waveguide type demultiplexer 43 ′ includes branch waveguides 61 a to 61 g branched from one input waveguide 60 into seven, and an input incident on the input waveguide 60. The light can be divided and emitted to the branched waveguides 61a to 61g as seven lights having the same light intensity. Each of the seven branching waveguides 61a to 61g is provided with a heater 44, and output light can be switched on and off. And on the output side of the waveguide type demultiplexer 43 ', there are provided waveguide type phase modulation elements 42' respectively corresponding to the branched waveguides 61a to 61g.

  As shown in FIG. 6, the waveguide type phase modulation element 42 ′ is arranged in parallel to the optical axis at the position of each vertex of a regular hexagon formed with the optical axis as the center, and is the same as this optical axis. It is also placed on the line. The waveguide type phase modulation element 42 ′ is provided with a pair of electrodes 45 so as to sandwich each of the branched waveguides 62 a to 62 g, and by applying a predetermined voltage between the electrodes from the control / arithmetic unit 22, The refractive index of the branching waveguides 62a to 62g is changed, and the phase of the output light is changed.

  The light shielding plate 6 ′ is provided with a pinhole through which the light emitted from the waveguide type phase modulation element 42 ′ and condensed through the lens 41 and the lens 4 can pass. For this reason, in the first embodiment, a pinhole is also opened at the central portion of the light shielding plate 6 ′ located on the optical axis that has been shielded from light, and the light condensed on the optical axis (disposed on the optical axis). Light emitted from the branching waveguide 62g) can pass therethrough.

  On / off control of light output is performed by applying a predetermined voltage from the controller / arithmetic unit 22 and energizing the heater 44 of the waveguide duplexer 43 '. In the second embodiment, the optical axis is controlled. The optical output is turned on for the two waveguides that face each other, and the optical output is always turned on for the branching waveguide 62g disposed on the optical axis. As described above, the waveguide type demultiplexer 43 'always outputs light on the optical axis and two lights facing the optical axis.

  Of the three light beams output from the waveguide type demultiplexer 43 'in this way, a predetermined voltage is applied to the electrode 45 for the two waveguides facing the optical axis, and one of the waveguides The phases of the light and the light of the other waveguide are always changed so that their absolute values are equal and opposite in sign, and are emitted from the waveguide type phase modulation element 42 '. The light of the waveguide disposed on the optical axis is emitted from the waveguide type phase modulation element 42 'without changing the phase.

  The light emitted from the waveguide type phase modulation element 42 ′ is converted into parallel light by the lens 41, and a light source image is formed on the pupil conjugate plane 31 by the lens 4. The light that has passed through the light shielding plate 6 'disposed at the pupil conjugate position forms a sample conjugate plane at the position of the field stop 8 by the lens 7, and then the lens 9, the excitation filter 10, the epi-illumination system, and the image formation. After condensing on the pupil P of the objective lens 12 through the dichroic mirror 11 that divides and synthesizes the system, it becomes a parallel light beam from the objective lens 12 and enters the sample surface at a predetermined angle and interferes with each other (three-beam interference). . Thus, the specimen 13 is structured and illuminated with spatially modulated illumination light.

  In this structured illumination, interference fringes due to two light fluxes of ± first-order light, interference fringes due to two light fluxes of zero-order light and primary light, and two light fluxes of zero-order light and −1st-order light on the specimen 13. Interference fringes due to are combined and exist. At this time, the interference fringes of the 0th-order light and the primary light and the interference fringes of the 0th-order light and the −1st-order light are the same interference fringes, so that they are completely overlapped, and ± 1st order It has a period twice that of interference fringes due to light. Thus, on the specimen 13, interference fringes due to two light fluxes of ± 1st order light and interference fringes of 0th order light and 1st order light (0th order light and −1st order light) are present. .

  In the interference between the 0th order light and the 1st order light (0th order light and −1st order light), the wave number vector in the optical axis direction (Z direction) perpendicular to the specimen 13 is different. appear. Since the phase of the interference fringes in the optical axis direction changes due to defocusing, that is, depending on the surface inside the specimen being observed, the interference fringes synthesized by the three light beams of the zero-order light and the ± first-order light are the specimen 13. The interference fringes are structured not only in the XY direction but also in the Z direction (optical axis direction). For this reason, the sample that is structured and illuminated with the interference fringes of the three light beams is modulated in the Z direction in addition to the modulation in the XY direction as in the first embodiment. Therefore, by obtaining this modulated image data and performing image processing by a known image calculation means by the control / calculation device 22, a so-called sectioning image with improved resolution in the Z direction can be obtained.

  When restoring the original image by image processing, it is preferable to shoot the same specimen by modulating the phase of the interference fringes of the illumination five times or more. This is because the modulated image includes components unnecessary for the demodulated image. In other words, there are five unknown parameters as components of sample information in the modulated image, and in order to obtain the unknowns in the calculation process, more information than the number of unknowns is required.

  Therefore, the phase modulation by the waveguide phase modulation element 42 ′ is performed every ± π / 5 so that the absolute values of the phases are equal and opposite to each other with respect to the two waveguides facing the optical axis. By doing so, it is preferable that the phase modulation of the structured illumination is performed uniformly in increments of 2π / 5. In this way, five image data are acquired by changing the phase of structured illumination.

  As in the first embodiment, the direction of structured illumination is changed to three directions (0 °, 120 °, and 240 °) by changing the waveguide that outputs light from the waveguide type phase modulation element 42 ′. Change the image data. At this time, as described above, for the branching waveguide 62g arranged on the optical axis, the optical output is always turned on, and the branching waveguides (62a, 62d, 62b By sequentially changing 62e, 62c and 62f), three light beams are always emitted.

  In this way, the control / arithmetic unit 22 repeats the acquisition of image data until the structured illumination direction is set to three directions, and finally acquires a total of 15 pieces of image data. Then, the control / arithmetic unit 22 takes in the 15 pieces of image data obtained as described above, performs calculation by a known means as described in Patent Document 1, and performs super-resolution of the specimen 13. An image can be obtained. Then, the control / arithmetic unit 22 sends this image data to the image display unit 23 and displays a super-resolution image.

  As described above, in the microscope apparatus according to the second embodiment, a super-resolution image can be acquired at high speed, and not only the two-dimensional super-resolution observation of the specimen 13 but also the resolution in the optical axis direction (Z direction). Also improved so-called sectioning images can be obtained.

It is a figure which shows the structure of the microscope apparatus which is 1st Embodiment. It is a figure which shows the waveguide type splitter and waveguide type phase modulation element which are used for 1st Embodiment. It is a figure which shows the waveguide type phase modulation element used for 1st Embodiment. It is a figure which shows the structure of the microscope apparatus which is 2nd Embodiment. It is a figure which shows the waveguide type splitter and waveguide type phase modulation element which are used for 2nd Embodiment. It is a figure which shows the waveguide type phase modulation element used for 2nd Embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Optical fiber 4 Lens 6 Light-shielding plate 8 Field stop 12 Objective lens 13 Sample 41 Lens 42 Waveguide type | mold phase modulation element (phase modulation part) 43 Waveguide type | mold splitter (beam splitting part)
44 Heater (light flux selection unit, heating unit) 21 Imaging device (imaging unit)
22 control / arithmetic unit (image generation unit) 50, 60 input waveguides 51a to 51f, 61a to 61g branching waveguide

Claims (7)

In a microscope apparatus including an illumination optical system for illuminating spatially modulated illumination light on a specimen,
The illumination optical system includes a light beam dividing unit that divides a light beam into a plurality of light beams, a light beam selection unit that selects at least two light beams among the plurality of light beams, and at least one of the light beams selected by the light beam selection unit. A phase modulation unit for phase-modulating two light beams,
The microscope apparatus, wherein the light beam splitting unit includes a multi-split waveguide element that splits the light beam into a plurality of light beams by branching from an input waveguide into a plurality of branch waveguides.   The microscope apparatus according to claim 1, wherein the phase modulation unit includes a waveguide type phase modulation element having at least a pair of control electrodes sandwiching the branching waveguide.   The microscope apparatus according to claim 1, wherein the light beam selection unit includes a heating unit capable of heating each of the plurality of branch waveguides formed in the multi-divided waveguide element. In a microscope apparatus including an illumination optical system for illuminating spatially modulated illumination light on a specimen,
The illumination optical system includes a light beam dividing unit that divides a light beam into a plurality of light beams, a light beam selection unit that selects at least two light beams among the plurality of light beams, and at least one of the light beams selected by the light beam selection unit. A phase modulation unit for phase-modulating two light beams,
The microscope apparatus, wherein the phase modulation unit includes a waveguide type phase modulation element having at least a pair of control electrodes. The light beam selection unit selects three light beams from the plurality of light beams,
5. The microscope apparatus according to claim 1, wherein the phase modulation unit is configured to phase-modulate at least two light beams among the three light beams selected by the light beam selection unit.   The light beam selection unit is configured to change a direction of spatial modulation of illumination light by changing a light beam to be selected among the plurality of light beams divided by the light beam splitting unit. Item 6. The microscope apparatus according to any one of Items 1 to 5. An imaging optical system that forms a modulated image from the specimen illuminated with the spatially modulated illumination light;
An imaging unit that captures the modulated image;
And an image generation unit that generates a sample image by performing arithmetic processing on the plurality of modulated images captured by the imaging unit each time the phase of the spatially modulated illumination light is modulated. The microscope apparatus according to any one of claims 1 to 6.

Images