Microscope device

Abstract

A microscope device (1) comprises: a first microscope unit (10) that irradiates a sample (SA) with first illumination light in a first direction to detect light emitted from the sample (SA) in response to the irradiation with the first illumination light; a second microscope unit (50) that irradiates the sample (SA) with second illumination light in a second direction orthogonal to the first direction to detect light emitted from the sample (SA) in response to the irradiation with the second illumination light; and a data processing unit that, on the basis of a detection signal of the light detected by the first microscope unit (10) and a detection signal of the light detected by the second microscope unit (50), generates a three-dimensional refractive index distribution of the sample (SA).

Description

Technical Field

[0001] The present invention relates to a microscope apparatus in a position and is concerned with

Background Art

[0002] In recent years, methods for obtaining a three-dimensional refractive index distribution in a sample such as a phase object have been devised (see, for example, Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

[0004] The microscope apparatus according to the present invention includes a first light source that generates first illumination light, a first illumination optical system that irradiates a sample with first illumination light directed in a first direction, and a first detection optical system that receives light from the sample in response to the irradiation of the first illumination light the and a first microscope unit including of ; a first detector that detects light from the sample via the first detection optical system, a second illumination optical system that irradiates the sample with second illumination light directed in a second direction different from the first direction, and a second detection optical system that receives light from the sample in response to the irradiation of the second illumination light a second light source that generates second illumination light, and a second microscope unit including the ; of and has a second detector that detects light from the sample via the second detection optical system, ; a data processing unit that generates a three-dimensional refractive index distribution in the sample based on a detection signal of light detected by the first detector and a detection signal of light detected by the second detector, and includes any one of the following (1) to (4). (1) the first illumination optical system is provided and At the position of the pupil of the first illumination optical system or at a position conjugate to the pupil and the intensity distribution of the first illumination light includes a first modulation element that changes in the plane of the pupil or in a plane conjugate to the pupil and the second detection optical system is provided and at the position of the pupil of the second detection optical system or at a position conjugate to the pupil is provided and the intensity distribution of the light from the sample in response to the irradiation of the second illumination lightand within the plane of the pupil or within a plane conjugate to the pupil includes a second modulation element that changes , The first detector is provided at a position conjugate to the sample in the first detection optical system, and the second detector is provided at a position conjugate to the pupil in the second detection optical system. (2) the first illumination optical system at the position of the pupil of the first illumination optical system or at a position conjugate to the pupil is provided and the intensity distribution of the first illumination light and within the plane of the pupil or within a plane conjugate to the pupil includes a first modulation element that changes , the second illumination optical system at the position of the pupil of the second illumination optical system or at a position conjugate to the pupil is provided and the intensity distribution of the second illumination light and within the plane of the pupil or within a plane conjugate to the pupil includes a second modulation element that changes , The first detector is provided at a position conjugate to the sample in the first detection optical system, and the second detector is provided at a position conjugate to the sample in the second detection optical system. (3) the first detection optical system at the position of the pupil of the first detection optical system or at a position conjugate to the pupil is provided and the intensity distribution of the light from the sample in response to the irradiation of the first illumination light and within the plane of the pupil or within a plane conjugate to the pupil includes a first modulation element that changes , the second detection optical system at the position of the pupil of the second detection optical system or at a position conjugate to the pupil is provided and the intensity distribution of the light from the sample in response to the irradiation of the second illumination light and within the plane of the pupil or within a plane conjugate to the pupil includes a second modulation element that changes , The first detector is provided at a position conjugate to the pupil in the first detection optical system, and the second detector is provided at a position conjugate to the pupil in the second detection optical system. (4) the first detection optical system at the position of the pupil of the first detection optical system or at a position conjugate to the pupil is provided and the intensity distribution of the light from the sample in response to the irradiation of the first illumination light andin the plane of the pupil or in a plane conjugate to the pupil includes a first modulation element that changes , the second illumination optical system at the position of the pupil of the second illumination optical system or at a position conjugate to the pupil is provided and the intensity distribution of the second illumination light and in the plane of the pupil or in a plane conjugate to the pupil includes a second modulation element that changes , The first detector is provided at a position conjugate to the pupil in the first detection optical system, The second detector is provided at a position conjugate to the sample in the second detection optical system.

Brief Description of the Drawings

[0006]

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Embodiments for Carrying Out the Invention

[0007] Hereinafter, a microscope apparatus according to each embodiment will be described. In the drawings used in the following description, for the sake of easy understanding of the features, the components may be shown enlarged for convenience, and the dimensional ratios and the like of each component are not necessarily the same as the actual ones.

[0008] <First Embodiment> First, the microscope apparatus 1 according to the first embodiment will be described with reference to FIGS. 1 and 2. As shown in FIGS. 1 and 2, the microscope apparatus 1 according to the first embodiment includes a first microscope unit 10 and a second microscope unit 50. Further, as shown in FIG. 2, the microscope apparatus 1 includes a stage 2, a control unit 90, and an image processing unit 91. The stage 2 supports a sample SA. The sample SA is a phase object such as a cell, for example. A stage driving unit (not shown) is provided on the stage 2. The stage driving unit moves the stage 2 along the optical axis AX1 of the first microscope unit 10.

[0009] As shown in FIG. 2, a coordinate axis extending in the optical axis direction (vertical direction) of the first microscope unit 10 is defined as the z-axis, and coordinate axes perpendicular to the z-axis are defined as the x-axis and the y-axis. By moving the stage 2 in the z-direction by the stage driving unit, as shown in FIG. 3, it is possible to acquire image data of the cross-section of the sample SA at a predetermined position Z0, a position Z0 + Δz separated from the position Z0 by +Δz, a position Z0 - Δz separated from the position Z0 by -Δz, a position Z0 + 2Δz separated from the position Z0 by +2Δz, a position Z0 - 2Δz separated from the position Z0 by -2Δz, and so on.

[0010] The first microscope unit 10 includes, as shown in FIGS. 1 and 2, a first light source 11, a first illumination optical system 20, a first detection optical system 30, and a first detector 40. The first light source 11 is configured using a white light source such as a halogen lamp or an LED (Light Emitting Diode). Further, the first light source 11 may be configured using a near-infrared light source such as a halogen lamp or an LED. The first light source 11 generates illumination light in a predetermined wavelength band (hereinafter referred to as the first illumination light).

[0011] The first illumination optical system 20 irradiates the sample SA by directing the first illumination light L1 emitted from the first light source 11 in the -z direction (the first direction). As shown in FIG. 2, the first illumination optical system 20 includes, in order from the first light source 11 side, a collector lens 21, a field stop 23, a relay lens 24, a first modulation element 25, an aperture stop 26, and a condenser lens 27. When a white light source is used as the first light source 11, it is preferable to provide an element for narrowing the wavelength band of the first illumination light. For example, by inserting a band-pass filter 22 having predetermined spectral transmittance characteristics in the optical path between the collector lens 21 and the field stop 23 in the first illumination optical system 20, it is possible to narrow the wavelength band of the first illumination light. By narrowing the wavelength band of the first illumination light, the accuracy of calculated values such as POTF, which will be described in detail later, can be improved. The spectral transmittance characteristics of the band-pass filter 22 are set based on the wavelength band of the illumination light according to the purpose of observation such as bright-field observation. Note that the band-pass filter 22 may be inserted in the optical path between the field stop 23 and the relay lens 24 in the first illumination optical system 20.

[0012] The first modulation element 25 and the aperture stop 26 are arranged in a plane perpendicular to the optical axis AX1 of the first microscope unit 10 (first illumination optical system 20) at the position P1 of the pupil (hereinafter sometimes referred to as the illumination pupil) between the relay lens 24 and the condenser lens 27 in the first illumination optical system 20. The first modulation element 25 is arranged adjacent to the aperture stop 26 (for example, above the aperture stop 26 as shown in FIG. 2). The plane perpendicular to the optical axis AX1 of the first microscope unit 10 at the position P1 of the illumination pupil is referred to as the illumination pupil plane. The first modulation element 25 is, for example, a flat plate having light transmissivity, and the light transmittance changes within the plane of this flat plate. This flat plate is formed, for example, by depositing a film (a light-shielding film) capable of reducing the light transmittance on a parallel flat plate such as a glass substrate. As an example, a metal film is deposited. For example, by changing the film thickness according to the part of the parallel flat plate where the film is deposited, the light transmittance can be changed according to the part of the parallel flat plate (the thicker the film thickness, the lower the transmittance). By arranging this first modulation element 25 on the illumination pupil plane, the light transmittance can be changed within the illumination pupil plane. Therefore, it can be said that the light transmittance of the first modulation element 25 changes within the illumination pupil plane. The light transmittance of the first modulation element 25 changes continuously (or discretely) within the illumination pupil plane.

[0013] Note that, by changing the light transmittance according to the part of the first modulation element 25, the distribution of the light transmittance of the first modulation element 25 (in other words, the distribution of the light transmittance on the illumination pupil plane) is determined. As the first modulation element 25, it is possible to select any one of a plurality of first modulation elements 25 having different light transmittance changes, that is, different light transmittance distributions, and arrange it at the position P1 of the illumination pupil. Details of the light transmittance of the first modulation element 25 will be described later. Note that the position where the first modulation element 25 is arranged is not limited to the position P1 of the illumination pupil. For example, the first modulation element 25 may be arranged in a plane perpendicular to the optical axis AX1 (in other words, a plane conjugate to the illumination pupil) at a position conjugate to the illumination pupil. Also, the first light source 11 is arranged at a position conjugate to the illumination pupil.

[0014] The condenser lens 27 is disposed to face above the stage 2. As the condenser lens 27, any one of a plurality of condenser lenses 27 having different optical characteristics can be selected and disposed above the stage 2.

[0015] The first detection optical system 30 receives light from the sample SA in response to the irradiation of the first illumination light L1 from the side opposite to the first illumination optical system 20 with the sample SA interposed therebetween. As shown in FIG. 2, the first detection optical system 30 includes, in order from the sample SA side, an objective lens unit 31, an imaging lens 36, and a mirror 37. The objective lens unit 31 includes a plurality of first objective lenses 32, a lens holding unit 33, and a unit driving unit 34. The first objective lens 32 is disposed to face below the stage 2. The lens holding unit 33 holds a plurality of first objective lenses 32 having different focal lengths. The lens holding unit 33 is configured using, for example, a revolver or a turret. The unit driving unit 34 can drive the lens holding unit 33 to select any one of the plurality of first objective lenses 32 and dispose it below the stage 2. Note that the unit driving unit 34 may move the lens holding unit 33 along the z-axis. In this case, the aforementioned stage driving unit may be used in combination, or the stage driving may not be used.

[0016] Light from the sample SA in response to the irradiation of the first illumination light is incident on the first objective lens 32 disposed below the stage 2. The light transmitted through the first objective lens 32 is incident on the imaging lens 36. The light transmitted through the imaging lens 36 is reflected by the mirror 37 and forms an image on a predetermined image plane I. Here, the position of the predetermined image plane I is a position conjugate to the focal position of the first objective lens 32 in the sample SA. Note that instead of the mirror 37, a half mirror may be provided, and an observation optical system (not shown) having an eyepiece lens (not shown) may be provided on the optical path of the light transmitted through the half mirror. Thereby, the observer can observe an image of the sample SA using the eyepiece lens.

[0017] A first detector 40 is arranged on an image plane I of a first detection optical system 30. The first detector 40 is configured using an imaging device such as a CCD (Charge Coupled Device) or a CMOS (Complementary Metal Oxide Semiconductor). The first detector 40 detects light from a sample SA via the first detection optical system 30.

[0018] As shown in FIGS. 1 and 2, a second microscope unit 50 includes a light source unit 51, a second illumination optical system 70, a second detection optical system 75, and a second detector 80. The light source unit 51 is also referred to as a beam steering unit. As shown in FIG. 2, the light source unit 51 includes a second light source 52, a first lens 53, a first galvanometer mirror 54, a second lens 55, a third lens 56, a second galvanometer mirror 57, a liquid lens 59, a fourth lens 60, and a fifth lens 61. Further, the light source unit 51 has a cylindrical lens (not shown) that can be inserted and removed in an optical path between the first lens 53 and the first galvanometer mirror 54.

[0019] The second light source 52 is configured using a laser light source. The second light source 52 emits illumination light in a predetermined wavelength band (hereinafter referred to as second illumination light). The first lens 53 collimates the second illumination light emitted from the second light source 52. The first galvanometer mirror 54 reflects the second illumination light from the first lens 53 toward the second lens 55. The first galvanometer mirror 54 can change the traveling direction of the second illumination light by changing the orientation of the reflection surface. By changing the traveling direction of the second illumination light by the first galvanometer mirror 54, the condensing position of the second illumination light on the sample SA is changed in the y direction. The second lens 55 and the third lens 56 make the second illumination light reflected by the first galvanometer mirror 54 incident on the second galvanometer mirror 57. Note that when the first galvanometer mirror 54 and the second galvanometer mirror 57 are arranged at positions conjugate to the pupil, the second lens 55 and the third lens 56 may not be provided.

[0020] The second galvanometer mirror 57 reflects the second illumination light from the third lens 56 toward the liquid lens 59. The second galvanometer mirror 57 can change the traveling direction of the second illumination light by changing the orientation of the reflecting surface. By changing the traveling direction of the second illumination light by the second galvanometer mirror 57, the focusing position of the second illumination light on the sample SA is changed in the z direction.

[0021] The liquid lens 59 can change the focal length of the liquid lens 59 by changing the radius of curvature of the lens surface. By changing the focal length of the liquid lens 59 by the liquid lens 59, the focusing position of the second illumination light on the sample SA is changed in the x direction. The fourth lens 60 and the fifth lens 61 cause the second illumination light transmitted through the liquid lens 59 to enter the second illumination optical system 70. The first galvanometer mirror 54, the second galvanometer mirror 57, and the liquid lens 59 can change the focusing position of the second illumination light on the sample SA in three-dimensional directions (three directions: the x direction, the y direction, and the z direction) to three-dimensionally scan the sample SA.

[0022] From the perspective of deconvolution, it is preferable to adjust in advance so that the irradiation range of the first illumination light (the three-dimensional observation range on the sample SA) by the first illumination optical system 20 and the irradiation range of the second illumination light (the three-dimensional observation range on the sample SA) by the second illumination optical system 70 coincide. The adjustment to make the irradiation ranges (observation ranges) of the first illumination light and the second illumination light coincide can be realized, for example, by controlling the swing angle of at least one of the first galvanometer mirror 54 and the second galvanometer mirror 57 in the light source unit 51. Also, without performing the adjustment to make the irradiation ranges (observation ranges) of the first illumination light and the second illumination light coincide, by specifying the common irradiation range (observation range) of the first illumination light and the second illumination light, image construction by three-dimensional scanning of the sample SA may be performed.

[0023] The second illumination optical system 70 condenses and irradiates the sample SA with the second illumination light L2 emitted from the light source unit 51 (second light source 52) in the +x direction (a second direction orthogonal to the first direction). In FIGS. 2, 15, 16, 18, 19, and 21, the second illumination light L2 is shown by a dashed line for easy distinction from the first illumination light L1. As shown in FIGS. 2 and 3, the second illumination optical system 70 has a second objective lens 71 for illumination. The second objective lens 71 for illumination is arranged to face the left side of the sample SA. The second objective lens 71 for illumination condenses the second illumination light L2 emitted from the light source unit 51 (second light source 52) onto the sample SA. The optical axis AX2 between the second illumination optical system 70 and the second detection optical system 75 in the second microscope unit 50 is orthogonal to the optical axis AX1 between the first illumination optical system 20 and the first detection optical system 30 in the first microscope unit 10.

[0024] The second detection optical system 75 receives light from the sample SA corresponding to the irradiation of the second illumination light from the side opposite to the second illumination optical system 70 with the sample SA interposed therebetween. As shown in FIGS. 2 and 3, the second detection optical system 75 has, in order from the sample SA side, a second objective lens 76 for detection and a second modulation element 77. The second objective lens 76 for detection is provided on the side opposite to the second objective lens 71 for illumination with the sample SA interposed therebetween. Light from the sample SA corresponding to the irradiation of the second illumination light is incident on the second objective lens 76 for detection. Note that the second objective lens 71 for illumination and the second objective lens 76 for detection may be the same and have a high numerical aperture (NA).

[0025] The second modulation element 77 is arranged on a plane perpendicular to the optical axis AX2 of the second microscope unit 50 (second detection optical system 75) at a position P2 conjugate to the pupil of the second objective lens 76 for detection in the second detection optical system 75 (hereinafter may be referred to as the detection pupil). A plane perpendicular to the optical axis AX2 of the second microscope unit 50 at the position P2 conjugate to the detection pupil is referred to as a plane conjugate to the detection pupil. The second modulation element 77 is formed, for example, in the same manner as the first modulation element 25, by depositing a film capable of reducing the light transmittance on a parallel flat plate such as a glass substrate. By arranging the second modulation element 77 on the plane conjugate to the detection pupil, the light transmittance can be changed within the plane conjugate to the detection pupil. Therefore, it can be said that the light transmittance of the second modulation element 77 changes within the plane conjugate to the detection pupil. The light transmittance of the second modulation element 77 changes continuously (or discretely) within the plane conjugate to the detection pupil. As the second modulation element 77, any one of a plurality of second modulation elements 77 having different light transmittance distributions can be selected and arranged at the position P2 conjugate to the detection pupil. Details of the light transmittance of the second modulation element 77 will be described later. Note that the position where the second modulation element 77 is arranged is not limited to the position P2 conjugate to the detection pupil. For example, the second modulation element 77 may be arranged on a plane perpendicular to the optical axis AX2 at the position of the detection pupil (in other words, the plane of the detection pupil). In this case, for example, the second modulation element 77 may be built in the second objective lens 76 for detection.

[0026] The second detector 80 is arranged adjacent to the second modulation element 77 at a position P2 conjugate to the detection pupil in the second detection optical system 75. The second detector 80 is configured using a PMT (Photomultiplier tube), NDD (Non-Descanned Detection), or the like. The second detector 80 detects light from the sample SA through the second detection optical system 75.

[0027] An example of the change in the light transmittance of the first modulation element 25 and the second modulation element 77 in the plane of the pupil (in other words, the distribution of the light transmittance in the plane of the pupil) will be described. FIG. 4 is a graph showing an example of the distribution of the light transmittance of the first modulation element 25 and the second modulation element 77. In FIG. 4, X is the coordinate in the x direction with the coordinate position through which the optical axis (the optical axis AX1 of the first microscope unit 10 or the optical axis AX2 of the second microscope unit 50) passes as the origin, and Y is the coordinate in the y direction with the coordinate position through which the optical axis passes as the origin.

[0028] In the example shown in FIG. 4, the light transmittance of the first modulation element 25 or the second modulation element 77 changes in one direction according to a continuous function. Specifically, the light transmittance of the first modulation element 25 or the second modulation element 77 monotonically decreases according to a sine function in the X direction (for example, the -X direction) (the portions with equal light transmittance are distributed linearly extending in the Y direction). That is, the light transmittance of the first modulation element 25 or the second modulation element 77 changes according to a sine function. Note that it can also be said that the light transmittance of the first modulation element 25 or the second modulation element 77 monotonically increases according to a sine function in the X direction (for example, the +X direction) within the plane of the pupil, or that it monotonically decreases or increases in the X direction.

[0029] Note that the light transmittance of the first modulation element 25 or the second modulation element 77 may monotonically decrease or increase according to a sine function in the Y direction (the portions with equal light transmittance may be distributed linearly extending in the X direction). Also, the light transmittance of the first modulation element 25 or the second modulation element 77 may monotonically decrease or increase according to a sine function not only in the X direction and the Y direction but also in any direction in the XY coordinate system (the portions with equal light transmittance may be distributed linearly extending in a direction perpendicular to any direction in the above XY coordinate system).

[0030] In the example shown in FIG. 4, the light transmittance of the first modulation element 25 or the second modulation element 77 may monotonically decrease according to a linear function in the X direction (for example, the -X direction). That is, the light transmittance of the first modulation element 25 or the second modulation element 77 is not limited to a sine function and may change according to a linear function.

[0031] In the example shown in FIG. 4, the above continuous function may be any one of a linear function, a quadratic function, a Gaussian function, a sine function, and a cosine function. Note that the above continuous function is not limited to a linear function, a quadratic function, a Gaussian function, a sine function, and a cosine function, and may be other functions such as a cubic function. Further, the range in which the light transmittance changes in the first modulation element 25 and the second modulation element 77 may be set according to the size (diameter) of the pupil (illumination pupil, detection pupil). For example, in the case shown in FIG. 4, the first modulation element 25 and the second modulation element 77 are formed so that the region where the light transmittance becomes 0 coincides with the outer peripheral portion of the pupil (illumination pupil, detection pupil).

[0032] Note that when the light transmittance of the first modulation element 25 or the second modulation element 77 changes according to a cosine function or a sine function, it is desirable that the change according to the cosine function or the sine function be within a range smaller than one cycle in the plane of the pupil (illumination pupil, detection pupil). If the range is larger than one cycle, the value of the POTF also exhibits periodic behavior, which is not preferable from the viewpoint of performing deconvolution. In this case, there will be a plurality of frequencies at which the value of the POTF becomes 0, and the noise generated in the process of deconvolution increases, so the accuracy of the refractive index distribution of the obtained sample SA decreases. Further, when the light transmittance of the first modulation element 25 or the second modulation element 77 changes according to a cosine function, it is designed so that the light transmittance becomes 0 (the value of the cosine function becomes 0) at the outer peripheral portion of the pupil (illumination pupil, detection pupil). This is in consideration of the fact that the discontinuity of the light transmittance at the outer peripheral portion of the pupil causes artifacts such as ringing in the image.

[0033] In the example shown in FIG. 4, the light transmittance of the first modulation element 25 or the second modulation element 77 changes along one direction in the plane of the pupil or in the plane conjugate to the pupil according to a continuous function, and becomes 0 (zero) at a part of the outer periphery in the plane of the pupil or in the plane conjugate to the pupil, but is not limited thereto. For example, the light transmittance of the first modulation element 25 or the second modulation element 77 may change according to a continuous function as it moves away from the optical axis in the plane of the pupil or in the plane conjugate to the pupil, and may become 0 (zero) over the entire circumference of the outer peripheral portion in the plane of the pupil or in the plane conjugate to the pupil.

[0034] In this embodiment, when performing bright-field observation of the sample SA, the first illumination light L1 emitted from the first light source 11 of the first microscope unit 10 enters the collector lens 21 of the first illumination optical system 20. The first illumination light L1 that has passed through the collector lens 21 becomes parallel light and passes through the field stop 23 (when a white light source is used as the first light source 11, the band-pass filter 22 and the field stop 23), and enters the relay lens 24. The first illumination light L1 that has passed through the relay lens 24 enters the condenser lens 27 through the first modulation element 25 and the aperture stop 26. The first illumination light L1 that has passed through the condenser lens 27 becomes parallel light and irradiates the sample SA on the stage 2. Thereby, the first illumination optical system 20 irradiates the sample SA with the first illumination light L1 emitted from the first light source 11 in the -z direction (the first direction).

[0035] The light transmitted through the sample SA from the first illumination optical system 20 (hereinafter sometimes referred to as the first detection light) enters the first objective lens 32 of the first detection optical system 30. The first detection light that has passed through the first objective lens 32 enters the imaging lens 36. The first detection light that has passed through the imaging lens 36 is reflected by the mirror 37 and forms an image at a predetermined image plane I where the first detector 40 is disposed. The first detector 40 detects the light (first detection light) from the sample SA via the first detection optical system 30 and outputs a detection signal of the light. The detection signal of the light (first detection light) output from the first detector 40 is transmitted to the image processing unit 91 via the control unit 90. It should be noted that the first detector 40 can be described as capturing an image of the sample SA via the detection optical system 40. Here, the detection signal is a signal indicating the signal intensity detected by the first detector 40 or the second detector 80 according to the intensity of the light (detection light). For example, when the first detector 40 is configured using a CCD, it is the signal at each pixel of the CCD. It should be noted that the detection signal of the first detector 40 can be described as a signal indicating the signal intensity detected by the first detector 40 according to the intensity of the image of the sample SA.

[0036] When performing bright-field observation of the sample SA, the cylindrical lens (not shown) of the light source unit 51 of the second microscope unit 50 is retracted from the optical path between the first lens 53 and the first galvanometer mirror 54 in the light source unit 51. The second illumination light emitted from the second light source 52 of the light source unit 51 enters the first lens 53. The second illumination light transmitted through the first lens 53 becomes parallel light and is reflected by the first galvanometer mirror 54. The second illumination light reflected by the first galvanometer mirror 54 passes through the second lens 55 and the third lens 56 and is reflected by the second galvanometer mirror 57. The second illumination light reflected by the second galvanometer mirror 57 enters the liquid lens 59. The second illumination light transmitted through the liquid lens 59 passes through the fourth lens 60 and the fifth lens 61 and is emitted outside the light source unit 51. Thereby, the light source unit 51 emits the second illumination light.

[0037] The second illumination light L2 emitted from the light source unit 51 (the second light source 52) enters the second objective lens 71 for illumination of the second illumination optical system 70. The second illumination light L2 transmitted through the second objective lens 71 for illumination is condensed and irradiated onto the sample SA on the stage 2. Thereby, the second illumination optical system 70 condenses and irradiates the second illumination light L2 emitted from the light source unit 51 (the second light source 52) onto the sample SA in the +x direction (the second direction orthogonal to the first direction).

[0038] The light transmitted through the sample SA from the second illumination optical system 70 (hereinafter sometimes referred to as the second detection light) enters the second objective lens 76 for detection of the second detection optical system 75. The second detection light transmitted through the second objective lens 76 for detection passes through the second modulation element 77 and enters the second detector 80. The second detector 80 detects the light (the second detection light) from the sample SA via the second detection optical system 75 and outputs a detection signal of the light. The detection signal of the light (the second detection light) output from the second detector 80 is transmitted to the image processing unit 91 via the control unit 90.

[0039] In addition, it is also possible to perform fluorescence observation of the sample SA by using the microscope apparatus 1 as a selective plane illumination microscope (SPIM). When performing fluorescence observation of the sample SA, a cylindrical lens (not shown) of the light source unit 51 of the second microscope unit 50 is inserted into the optical path between the first lens 53 and the first galvanometer mirror 54 in the light source unit 51.

[0040] When performing fluorescence observation of the sample SA, the excitation light emitted from the second light source 52 of the light source unit 51 is incident on the first lens 53. The excitation light transmitted through the first lens 53 passes through a cylindrical lens (not shown), and is reflected by the first galvanometer mirror 54. The excitation light reflected by the first galvanometer mirror 54 passes through the second lens 55 and the third lens 56, and is reflected by the second galvanometer mirror 57. The excitation light reflected by the second galvanometer mirror 57 is incident on the liquid lens 59. The excitation light transmitted through the liquid lens 59 passes through the fourth lens 60 and the fifth lens 61, and is emitted to the outside of the light source unit 51. Thereby, the light source unit 51 emits excitation light that is sheet light.

[0041] The excitation light emitted from the light source unit 51 (second light source 52) is incident on the second objective lens 71 for illumination of the second illumination optical system 70. The excitation light transmitted through the second objective lens 71 for illumination is condensed and irradiated onto the sample SA on the stage 2. Thereby, the second illumination optical system 70 irradiates the sample SA with the excitation light that is sheet light emitted from the light source unit 51 (second light source 52).

[0042] Due to the irradiation of the excitation light, the fluorescent substance contained in the sample SA is excited and emits fluorescence. The fluorescence from the sample SA is incident on the first objective lens 32 of the first detection optical system 30 of the first microscope unit 10. The fluorescence transmitted through the first objective lens 32 is incident on the imaging lens 36. The fluorescence transmitted through the imaging lens 36 is reflected by the mirror 37 and forms an image at a predetermined image plane I where the first detector 40 is disposed. The first detector 40 detects the fluorescence from the sample SA via the first detection optical system 30, and outputs a detection signal of the fluorescence. The detection signal of the fluorescence output from the first detector 40 is transmitted to the image processing unit 91 via the control unit 90.

[0043] The control unit 90 controls the overall operation of the microscope apparatus 1. The control unit 90 is electrically connected to a stage drive unit (not shown), a unit drive unit 34, a first detector 40, a light source unit 51, a second detector 80, an image processing unit 91, an operation input unit (not shown), an image display unit (not shown), and the like.

[0044] When performing bright-field observation of the sample SA, the image processing unit 91 generates refractive index data regarding the sample SA based on the detection signal of the light (first detection light) output from the first detector 40 and the detection signal of the light (second detection light) output from the second detector 80. Here, the refractive index data regarding the sample SA is data representing the refractive index of the sample SA, for example, data on the refractive index at each position in the sample SA, that is, data indicating the refractive index distribution in the sample SA. Further, the refractive index data regarding the sample SA is stored, for example, in a storage unit (not shown) as a look-up table. In addition, the image processing unit 91 generates image data (hereinafter sometimes referred to as image data of the refractive index distribution of the sample SA) in which the luminance value of each pixel is set according to the refractive index value at each position of the refractive index distribution in the sample SA. Also, the image processing unit 91 generates image data (hereinafter sometimes referred to as image data of the sample SA by bright-field observation) in which the luminance value of each pixel is set according to the signal intensity value of the detection signal at each position (each pixel of the first detector 40) in the sample SA based on the detection signal of the light (first detection light) output from the first detector 40 and the detection signal of the light (second detection light) output from the second detector 80.

[0045] When performing fluorescence observation of the sample SA, the image processing unit 91 generates image data (hereinafter sometimes referred to as image data of the sample SA by fluorescence observation) in which the luminance value of each pixel is set according to the signal intensity value of the detection signal at each position in the sample SA based on the detection signal of the fluorescence output from the first detector 40.

[0046] Accordingly, based on the image data of the refractive index distribution of the sample SA generated by the image processing unit 91, it is possible to display an image of the refractive index distribution in the sample SA on an image display unit (not shown). Also, based on the image data of the sample SA obtained by bright-field observation generated by the image processing unit 91, it is possible to display an image of the sample SA by bright-field observation on the image display unit. Based on the image data of the sample SA obtained by fluorescence observation generated by the image processing unit 91, it is possible to display an image of the sample SA by fluorescence observation.

[0047] Next, a known method for obtaining a three-dimensional refractive index distribution in the sample SA as refractive index data regarding the sample SA by the image processing unit 91 will be described. As a typical example of obtaining a three-dimensional refractive index distribution in the sample SA, there is a method using a theory called PC-ODT (Partially Coherent-Optical Diffraction Tomography). Hereinafter, the theory of PC-ODT will be briefly described. From the equation of partially coherent imaging, the intensity I(x, y, z) of the image of a three-dimensional object can be expressed as in the following equation (1).

[0048]

Equation

[0049] In Equation (1), o represents the complex amplitude transmittance of the object. TCC represents the Transmission Cross Coefficient. (ξ, η, ζ) represents the direction cosine of the diffracted light (or direct light). Also, in this case, the image refers to the image of the sample SA obtained by imaging the light (detection light) that has passed through at least a part of the sample SA by illumination. Therefore, the intensity I(x, y, z) of the image of the three-dimensional object, that is, the image of the three-dimensional sample SA, can be replaced by the signal intensity of the detection signal output from the first detector 40 or the like in image processing (for example, the signal intensity at each pixel of the first detector 40 when imaging the sample SA with the first detector 40). As shown in FIG. 2, the coordinate axis extending in the optical axis direction (vertical direction) of the first microscope unit 10 is defined as the z-axis, and the coordinate axes perpendicular to the z-axis are defined as the x-axis and the y-axis. The mutual transmission coefficient TCC can be expressed as in the following Equation (2).

[0050] [Number]

[0051] In Equation (2), S represents the illumination pupil. G represents the detection pupil. Since the mutual transmission coefficient TCC is a Hermitian conjugate, it has the property shown in the following Equation (3).

[0052] [Number]

[0053] In the case of a thin sample such as a cell, since the influence of scattering is small, the first Born approximation (low contrast approximation) holds. At this time, it is only necessary to consider the interference between the direct light (0th order diffracted light) that has passed through the sample and the diffracted light (1st order diffracted light) diffracted by the sample. Therefore, by the first Born approximation, the following Equation (4) is obtained from the above Equations (1) to (3).

[0054] [Number]

[0055] Also, the complex amplitude transmittance o of the object can be approximated as in the following formula (5).

[0056]

Number

[0057] In formula (5), P represents the real part of the scattering potential. Φ represents the imaginary part of the scattering potential. The above formula (4) is expressed as the following formula (6) using formula (5).

[0058]

Number

[0059] Here, TCC is renamed as WOTF (Weak Object Transfer Function). WOTF is defined by the following formula (7).

[0060]

Number

[0061] From the above formulas (6) and (7), the intensity I(x, y, z) of the three-dimensional object image obtained by the transmission illumination microscope is expressed as the following formula (8).

[0062]

Number

[0063] Here, it is assumed that the amplitude change of the sample is small enough to be ignored. That is, P = 0. In this case, when the above formula (8) is expressed in real space, the following formula (9) is obtained.

[0064]

Number

[0065] In Equation (9), EPSF represents the Effective Point Spread Function. EPSF is equal to the inverse Fourier transform of WOTF. EPSF is generally a complex function. The first term in Equation (9) represents the background intensity. The second term in Equation (9) represents that the imaginary part Im[EPSF] of the EPSF is multiplied by the imaginary part Φ of the scattering potential of the sample. Using this Equation (9), the imaginary part Φ of the scattering potential of the sample can be obtained.

[0066] As a first method for obtaining Φ(x, y, z), there is a method of directly performing deconvolution using Im[EPSF]. Figure 5 schematically shows the process of obtaining the intensities (signal intensities of the detection signals output from the first detector 40, etc.) of images of a plurality of cross-sections (xy cross-sections) with different positions of the sample SA in the z direction (i.e., the optical axis direction) by moving the stage 2 in the z direction, and then performing deconvolution. Note that a plurality of images of cross-sections with different positions of the sample SA in the z direction (i.e., the optical axis direction) may be collectively referred to as the z-stack image of the sample SA. The first term in Equation (9) is a constant term representing the background intensity. First, both sides of Equation (9) are divided by this constant term for normalization, and then the first term of the normalized Equation (9) is removed in real space (or frequency space). Then, by performing deconvolution using Im[EPSF], the following Equation (10) is obtained.

[0067]

Number

[0068] In Equation (10), let POTF (Phase Optical Transfer Function) be the three-dimensional Fourier transform of Im[EPSF]. Since Im[EPSF] can take values from positive to negative, the value of POTF can also take values from positive to negative. Here, POTF is an index representing the contrast and resolution of the image (image) of the sample SA in bright-field observation. Specifically, the absolute value of POTF represents the contrast of the image, and the higher the absolute value of POTF, the higher the contrast of the image (image) of the sample SA in bright-field observation. Also, the wider the region where the value of POTF is not zero in the frequency space, the higher the resolution of the image (image) of the sample SA in bright-field observation. Also, let I’ be the intensity I (for example, I1 to I6 in FIG. 5) of each cross-section image of the sample SA in the z-stack image of the sample SA normalized by the constant term of Equation (9). γ takes an arbitrary small value.

[0069] As a second method for obtaining Φ(x, y, z), there is a method of obtaining the difference in intensity between images of two cross-sections with different positions of the sample SA in the z direction (that is, the position in the optical axis direction), removing the constant term of Equation (9), and then performing deconvolution using Im[EPSF] of the obtained intensity difference. This method is also described in WO 2021 / 064807 pamphlet, and the description is omitted.

[0070] Note that the scattering potential Φ is defined by the following Equation (11) when P = 0.

[0071]

Equation

[0072] In Equation (11), n(x, y, z) represents the three-dimensional refractive index distribution in the sample SA, k0 represents the wave number in vacuum, and n mrepresents the refractive index of the medium. Using Equation (11), the scattering potential Φ obtained by the method described above can be converted into a three-dimensional refractive index distribution. The image processing unit 91 calculates the three-dimensional refractive index distribution n(x, y, z) in the sample SA from the signal intensity of the detection signal output from the first detector 40 or the like, that is, the intensity I(x, y, z) of the image of the three-dimensional sample SA, using the above Equation (10) and Equation (11). As an example, the image processing unit 91 generates image data in which the luminance value of each pixel is set according to the value of the refractive index at each position (coordinate) of the calculated three-dimensional refractive index distribution in the sample SA, that is, the image data of the three-dimensional refractive index distribution of the sample SA. Note that the intensity of the image of the three-dimensional sample SA can be expressed as the intensity of each cross-sectional image of the sample SA in the z-stack image of the sample SA. That is, the intensity of the image of the three-dimensional sample SA can also be said to be the intensity of a plurality of images with different positions in the z direction (that is, positions in the optical axis direction) in the sample SA.

[0073] When detecting light from the sample SA using only the first microscope unit 10 as in the conventional method, since the POTF has a region where information is missing in the z direction (hereinafter referred to as the missing cone region), an error occurs in the change of the refractive index in the z direction. Therefore, it is difficult to generate an image of the three-dimensional refractive index distribution of the sample SA or an image of the sample SA by bright-field observation. FIG. 6 shows an example of an image of the sample SA (xz cross-section) by bright-field observation generated by the conventional method. FIG. 7 shows an example of an image of the three-dimensional refractive index distribution (xz cross-section) of the sample SA generated by the conventional method. As the sample SA shown in FIGS. 6 and 7, a substantially spherical phantom cell with a known refractive index or the like is used. In the examples shown in FIGS. 6 and 7, the refractive index of the sample SA (phantom cell) is set to ~1.35, and the refractive index of the medium is set to 1.33. Also, the illumination-side NA (numerical aperture) and the detection-side NA of the microscope used in the examples shown in FIGS. 6 and 7 are set to 0.95.

[0074] FIG. 8 shows the distribution of the conventional POTF. In FIG. 8, white (background) indicates that the value of the POTF is 0, and black indicates that the value of the POTF is a positive or negative value. Therefore, in FIG. 8, the darker the black, the larger the absolute value of the POTF. As shown in FIG. 8, there is a large missing cone region on the central side of the distribution of the conventional POTF. Therefore, the image of the sample SA by bright-field observation deviates from the original substantially spherical shape and extends in the z direction, as shown in FIG. 6 for example.

[0075] Also, since the POTF has a missing cone region, when obtaining the three-dimensional refractive index distribution using the above equations (10) and (11), correction of the refractive index is required. Therefore, correction of the refractive index is performed using a missing cone estimation method such as the Gerchberg-Papoulis method, the Edge-Preserving Regularization method, or the Total Variation Regularization method. Specifically, a missing cone estimation algorithm is used to set a constraint condition so that the minimum refractive index value becomes a predetermined refractive index value (for example, the refractive index value of the medium in the sample SA that is known), and the missing cone region is estimated.

[0076] FIG. 9 shows the distribution of the spectrum obtained by estimating and complementing the missing cone region of the conventional POTF. As shown in FIG. 9, even if the missing cone region of the POTF is complemented, the image of the three-dimensional refractive index distribution of the sample SA deviates from the original substantially spherical shape and extends in the z direction, as shown in FIG. 7 for example.

[0077] In the present embodiment, the image processing unit 91 generates image data of the three-dimensional refractive index distribution of the sample SA based on the detection signal of the light output from the first detector 40 when the first illumination optical system 20 irradiates the sample SA with the first illumination light L1 in the -z direction (the first direction), and the detection signal of the light output from the second detector 80 when the second illumination optical system 70 condenses and irradiates the sample SA with the second illumination light L2 in the +x direction (the second direction orthogonal to the first direction). Thus, according to the present embodiment, based on the detection signal of the light output from the second detector 80, it becomes possible to reduce the missing cone region in the POTF, and thus it becomes possible to more accurately generate an image of the three-dimensional refractive index distribution of the sample SA and an image of the sample SA by bright-field observation.

[0078] FIG. 10 shows an example of an image of the sample SA (xz cross-section) by bright-field observation generated by the method according to the present embodiment. FIG. 11 shows an example of an image of the three-dimensional refractive index distribution (xz cross-section) of the sample SA generated by the method according to the present embodiment. As the sample SA shown in FIGS. 10 and 11, a substantially spherical phantom cell with a known refractive index and the like is used. In the examples shown in FIGS. 10 and 11, the refractive index of the sample SA (phantom cell) is set to ~1.35, and the refractive index of the medium is set to 1.33. Also, the illumination-side NA (numerical aperture) and the detection-side NA of the first microscope unit 10 are set to 0.95, and the illumination-side NA and the detection-side NA of the second microscope unit 50 are set to 0.5.

[0079] Fig. 12 shows the distribution of POTF in this embodiment. In Fig. 12, similar to Fig. 8, the darker the black color, the larger the absolute value of POTF. As shown in Fig. 12, there is a POTF based on the detection signal of the light output from the second detector 80 so as to fill the missing cone region on the central side of the POTF distribution in this embodiment. Therefore, the image of the sample SA by bright-field observation becomes closer to the original substantially spherical shape, for example, as shown in Fig. 10. Fig. 13 shows the distribution of the spectrum obtained by estimating and complementing the missing cone region of POTF in this embodiment. As shown in Fig. 13, the missing cone region of POTF can be complemented in a wider range than before, and the image of the three-dimensional refractive index distribution of the sample SA becomes closer to the original substantially spherical shape, for example, as shown in Fig. 11.

[0080] Also, let f be the spatial frequency, the pupil function of the lens (objective lens / condenser lens) that determines the illumination-side NA (numerical aperture) be Pill(f), and the pupil function of the lens (objective lens / condenser lens) that determines the detection-side NA be Pcol(f). POTF is obtained by the convolution of the illumination system effective pupil function and the detection system effective pupil function. In the first microscope unit 10 which is a Köhler illumination microscope, the illumination system effective pupil function is |Pill(f)| 2 P * col(f), and it is known that the detection system effective pupil function is Pcol(f). On the other hand, in the second microscope unit 50 which is a non-confocal laser microscope, the illumination system effective pupil function is Pill(f), and the detection system effective pupil function is |Pcol(f)| 2 Pill * (f). Since the convolution does not change even if the order is switched, the same POTF can be obtained for the Köhler illumination microscope (the first microscope unit 10) and the non-confocal laser microscope (the second microscope unit 50) when the conditions such as NA are the same.

[0081] When a first-order function intensity transmission mask (for example, a first modulation element 25 in which the light transmittance monotonically increases or decreases along one direction in the pupil plane according to a first-order function) is arranged at the position of the illumination pupil in a Köhler illumination microscope (first microscope unit 10), |Pill(f)| 2 becomes a first-order function, so the illumination system effective pupil function becomes a first-order function. Therefore, the distribution of the POTF becomes large symmetrically left and right in the example shown in FIG. 12, and the resolution of the sample SA in the direction orthogonal to the optical axis is improved. On the other hand, when a first-order function intensity transmission mask (for example, a second modulation element 77 in which the light transmittance monotonically increases or decreases along one direction in the plane conjugate to the pupil according to a first-order function) is arranged at the position conjugate to the detection pupil in a non-confocal laser microscope (second microscope unit 50), |Pcol(f)| 2 becomes a first-order function, so the detection system effective pupil function becomes a first-order function. Therefore, the distribution of the POTF becomes large symmetrically up and down in the example shown in FIG. 12, and the resolution of the sample SA in the direction orthogonal to the optical axis is improved. By arranging the Köhler illumination microscope and the non-confocal laser microscope orthogonally to each other, the resolution in two directions (in the example shown in FIG. 12, the x direction and the z direction) can be improved, and for the sample SA which is a transparent phase object, an accurate three-dimensional image can be formed. Therefore, based on the light detection signal by the Köhler illumination microscope (first microscope unit 10) and the calculation based on the light detection signal by the non-confocal laser microscope (second microscope unit 50), it becomes possible to more accurately obtain the three-dimensional refractive index distribution in the sample SA.

[0082] Note that the Köhler illumination microscope (first microscope unit 10) and the non-confocal laser microscope (second microscope unit 50) may satisfy the following formula (12) when the illumination side NA and the detection side NA are the same.

[0083]

Equation

[0084] Here, NA_1 is the NA (numerical aperture) of the Köhler illumination microscope (the first microscope unit 10). NA_2 is the NA (numerical aperture) of the non-confocal laser microscope (the second microscope unit 50). n_1 is the refractive index of the immersion liquid in the sample SA in the Köhler illumination microscope (the first microscope unit 10). n_2 is the refractive index of the immersion liquid in the sample SA in the non-confocal laser microscope (the second microscope unit 50). By satisfying formula (12), the missing cone region in the POTF is completely filled, so that it becomes possible to more accurately obtain the three-dimensional refractive index distribution in the sample SA.

[0085] Also, although it is ideal to arrange the first microscope unit 10 and the second microscope unit 50 so as to be orthogonal to each other, it is not limited to this. Even if the first microscope unit 10 and the second microscope unit 50 are arranged at different angles, it is possible to more accurately obtain the three-dimensional refractive index distribution in the sample SA as compared with the case where only the first microscope unit 10 is used.

[0086] If the first microscope unit 10, which is a Köhler illumination microscope, is arranged vertically and the second microscope unit 50, which is a non-confocal laser microscope, is arranged horizontally, the configuration of a conventional Köhler illumination microscope can be utilized, so that the practicality as a microscope apparatus is enhanced. However, the combination of the first microscope unit and the second microscope unit is not limited to the combination of a Köhler illumination microscope and a non-confocal laser microscope. For example, even a combination of Köhler illumination microscopes or a combination of non-confocal laser microscopes can similarly more accurately obtain the three-dimensional refractive index distribution in the sample SA.

[0087] It is ideal that the light transmittance of the first modulation element 25 and the second modulation element 77 monotonically increases or decreases along one direction in the in-pupil plane or the plane conjugate to the pupil according to a linear function, but it is not limited to this. The light transmittance of either the first modulation element 25 or the second modulation element 77 may be monotonically increased or decreased along one direction in the in-pupil plane or the plane conjugate to the pupil according to a linear function. As described above, the light transmittance of at least one of the first modulation element 25 and the second modulation element 77 may monotonically increase or decrease along one direction in the in-pupil plane or the plane conjugate to the pupil according to a continuous function, and the continuous function may be any one of a sine function, a cosine function, a quadratic function, and a Gaussian function. Even with such a configuration, it is possible to more accurately obtain the three-dimensional refractive index distribution in the sample SA.

[0088] As described above, as the first modulation element 25, it is possible to select any one of a plurality of first modulation elements 25 having different changes in light transmittance, that is, different distributions of light transmittance, and arrange it at the illumination pupil position P1. In this case, a turret (not shown) holding a plurality of first modulation elements 25 may be provided, and the first modulation element 25 arranged at the illumination pupil position P1 may be selected by rotating the turret. Note that the element selection unit capable of selecting any one of the plurality of first modulation elements 25 and arranging it at the illumination pupil position P1 is not limited to a turret, and an existing mechanism such as a slider may be used. Thereby, the control unit 90 controls the element selection unit so as to switch to any one of the plurality of first modulation elements 25 and arrange it at the illumination pupil position P1, thereby changing the distribution of the light transmittance in the in-pupil plane of the illumination pupil.

[0089] Further, as the second modulation element 77, it is possible to select any one of a plurality of second modulation elements 77 having different light transmittance distributions and arrange it at a position P2 conjugate to the detection pupil. In this case, a turret (not shown) holding a plurality of second modulation elements 77 may be provided, and the second modulation element 77 arranged at the position P2 conjugate to the detection pupil may be selected by rotating the turret. Note that, as a means for selecting any one of a plurality of second modulation elements 77 and arranging it at the position P2 conjugate to the detection pupil, the same means (element selection unit) as the means for selecting any one of a plurality of first modulation elements 25 and arranging it at the position P1 of the illumination pupil can be used. Thereby, the control unit 90 controls the element selection unit so as to switch to any one of a plurality of second modulation elements 77 and arrange it at the position P2 conjugate to the detection pupil, thereby changing the light transmittance distribution in the plane conjugate to the detection pupil.

[0090] Next, a refractive index data generation method in the microscope apparatus 1 according to the first embodiment will be described. FIG. 14 is a flowchart showing the data generation method according to the first embodiment. Note that it is assumed that the sample SA is placed on the stage 2 in advance. The control unit 90 includes, for example, a computer system. The control unit 90 reads out a control program stored in the storage unit and executes various processes according to this control program.

[0091] First, the first illumination optical system 20 of the first microscope unit 10 irradiates the sample SA with first illumination light directed in a first direction (step ST1). Next, the first detection optical system 30 receives light from the sample SA in response to the irradiation with the first illumination light (step ST2). Next, the first detector 40 detects the light from the sample SA via the first detection optical system 30 and outputs a detection signal of the light (step ST3). Next, the second illumination optical system 70 of the second microscope unit 50 irradiates the sample SA with second illumination light directed in a second direction orthogonal to the first direction (step ST4). Next, the second detection optical system 75 receives light from the sample SA in response to the irradiation with the second illumination light (step ST5). Next, the second detector 80 detects the light from the sample SA via the second detection optical system 85 and outputs a detection signal of the light (step ST6). Note that the processes of steps ST1 to ST6 are repeated so as to detect light from each cross-section of the sample SA corresponding to a plurality of cross-sectional images of the sample SA having different positions in the z direction (positions in the optical axis direction), that is, the z-stack image of the sample SA. For example, in steps ST1 to ST3, the light from each cross-section of the sample SA may be detected by the first microscope unit 10 (the first detector 40) by moving the stage 2 (the sample SA) in the z direction by a stage drive unit (not shown). In steps ST4 to ST6, the sample SA may be scanned three-dimensionally by the first galvanometer mirror 54, the second galvanometer mirror 57, and the liquid lens 59, and the light from each cross-section of the sample SA may be detected by the second microscope unit 50 (the second detector 80). Then, the image processing unit 91 generates a three-dimensional refractive index distribution in the sample SA (for example, image data of the three-dimensional refractive index distribution of the sample SA) based on the detection signal of the light output from the first detector 40 and the detection signal of the light output from the second detector 80 (step ST7). At this time, for example, the image processing unit 91 merges the intensity of the image of the sample SA corresponding to the signal intensity of the detection signal output from the first detector 40 and the intensity of the image of the sample SA corresponding to the signal intensity of the detection signal output from the second detector 80, and based on the intensity of the image of the sample SA obtained thereby, calculates the three-dimensional refractive index distribution in the sample SA using the above formulas (10) and (11). Thereby, the three-dimensional refractive index distribution in the sample SA can be obtained more accurately.

[0092] In the flow shown in FIG. 14, the first microscope unit 10 irradiates the sample SA with first illumination light directed in a first direction and detects light from the sample SA in response to the irradiation of the first illumination light (steps ST1 to ST3), and the second microscope unit 50 irradiates the sample SA with second illumination light directed in a second direction orthogonal to the first direction and detects light from the sample SA in response to the irradiation of the second illumination light (steps ST4 to ST6) are performed in this order, but it is not limited thereto. For example, each step (steps ST1 to ST3) by the first microscope unit 10 and each step (steps ST4 to ST6) by the second microscope unit 50 may be performed simultaneously. Thereby, the three-dimensional refractive index distribution in the sample SA can be obtained in a short time.

[0093] In the above-described first embodiment, the image processing unit 91 performs three-dimensional deconvolution based on the detection signal of the light detected by the first detector 40 and the detection signal of the light detected by the second detector 80 for each cross section of the sample SA corresponding to the z-stack image of the sample SA, and obtains the three-dimensional refractive index distribution in the sample SA (for example, image data of the three-dimensional refractive index distribution of the sample SA), but it is not limited thereto. For example, the image processing unit 91 performs two-dimensional deconvolution (for example, two-dimensional deconvolution based on the POTF in the cross section where Fz(Fx) = 0 in the distribution of the three-dimensional POTF illustrated in FIG. 12) based on the detection signal of the light detected by the first detector 40 and the detection signal of the light detected by the second detector 80 for each cross section of the sample SA corresponding to the z-stack image of the sample SA, and obtains the refractive index distribution in each cross section of the sample SA, thereby obtaining the three-dimensional refractive index distribution in the sample SA.

[0094] In the above-described first embodiment, the light source unit 51 of the second microscope unit 50 has the first galvanometer mirror 54 and the second galvanometer mirror 57, but is not limited thereto. For example, as in the microscope apparatus 1a shown in FIG. 15, the light source unit 51a of the second microscope unit 50a may have only one galvanometer mirror 64. In this case, in the light source unit 51a, a mirror 67 is disposed instead of the second galvanometer mirror 57. Instead of the second galvanometer mirror 57, the stage 2 is moved in the z direction by a stage drive unit (not shown), thereby changing the condensing position of the second illumination light on the sample SA in the z direction. The galvanometer mirror 64 has the same configuration as the first galvanometer mirror 54, and changes the traveling direction of the second illumination light, thereby changing the condensing position of the second illumination light on the sample SA in the y direction. The galvanometer mirror 64, the liquid lens 59, and the stage drive unit can change the condensing position of the second illumination light on the sample SA in three-dimensional directions to three-dimensionally scan the sample SA. In this case, by moving the stage 2 (sample SA) in the z direction by the stage drive unit, in accordance with detecting the light from each cross section of the sample SA by the first microscope unit 10 (first detector 40), the sample SA is scanned by the galvanometer mirror 64 and the liquid lens 59, and the light from each cross section of the sample SA may be detected by the second microscope unit 50a (second detector 80). Thereby, it is possible to simultaneously perform each process (steps ST1 to ST3) by the first microscope unit 10 and each process (steps ST4 to ST6) by the second microscope unit 50a.

[0095] <Second Embodiment> Next, with reference to FIG. 16, the microscope apparatus 101 according to the second embodiment will be described. The microscope apparatus 101 according to the second embodiment has a main part in common with the microscope apparatus 1 according to the first embodiment, except for the second microscope unit. Therefore, the same components as those in the first embodiment are denoted by the same reference numerals as those in the first embodiment, and detailed description thereof is omitted. The microscope apparatus 101 according to the second embodiment includes a first microscope unit 110 and a second microscope unit 150. Further, the microscope apparatus 101 according to the second embodiment includes a stage 2, a control unit 90, and an image processing unit 91.

[0096] The first microscope unit 110 includes a first light source 11, a first illumination optical system 20, a first detection optical system 130, and a first detector 40. The first light source 11, the first illumination optical system 20, and the first detector 40 are configured in the same manner as in the first embodiment. The first detection optical system 130 includes, in order from the sample SA side, an objective lens unit 31, an imaging lens 36, and a mirror 37, as in the first embodiment. Further, the first detection optical system 130 includes the half mirror 172 of the second microscope unit 150. The objective lens unit 31, the imaging lens 36, and the mirror 37 are configured in the same manner as in the first embodiment.

[0097] The second microscope unit 150 includes a light source unit 51, a second illumination optical system 170, a second detection optical system 175, and a second detector 80. The light source unit 51 and the second detector 80 are configured in the same manner as in the first embodiment.

[0098] The second illumination optical system 170 condenses and irradiates the sample SA with the second illumination light L2 emitted from the light source unit 51 (second light source 52) in the -x direction (a second direction orthogonal to the first direction). As shown in FIGS. 16 and 17, the second illumination optical system 170 includes a half mirror 172 and an illumination mirror 174. Further, the second illumination optical system 170 includes the objective lens unit 31 (first objective lens 32) of the first microscope unit 110 (first detection optical system 130).

[0099] The half mirror 172 is disposed in the optical path between the first objective lens 32 and the imaging lens 36 in the first microscope unit 110 (first detection optical system 130). The ratio of the transmittance to the reflectance of the half mirror 172 is set, for example, to 1:1. The half mirror 172 reflects a part of the second illumination light L2 emitted from the light source unit 51 (second light source 52) toward the detection pupil (rear focal plane) in the first objective lens 32. The first objective lens 32 condenses the second illumination light L2 reflected by the half mirror 172. Note that since the second illumination light L2 reflected by the half mirror 172 passes through the detection pupil (rear focal plane) in the first objective lens 32 from a direction inclined with respect to the central axis of the first objective lens 32, it advances in the +z direction offset with respect to the central axis of the first objective lens 32 when passing through the first objective lens 32. The illumination mirror 174 is disposed to face the right side of the sample SA above the first objective lens 32 (and the stage 2). The illumination mirror 174 reflects the second illumination light L2 that has passed through the first objective lens 32 and advances in the +z direction offset with respect to the central axis of the first objective lens 32 in the -x direction (second direction). The optical axis AX2 between the second illumination optical system 170 and the second detection optical system 175 in the second microscope unit 150 is orthogonal to the optical axis AX1 between the first illumination optical system 20 and the first detection optical system 130 in the first microscope unit 110.

[0100] The second detection optical system 175 receives light from the sample SA corresponding to the irradiation of the second illumination light from the side opposite to the second illumination optical system 170 with the sample SA interposed therebetween. As shown in FIGS. 16 and 17, the second detection optical system 175 includes a second objective lens 176 for detection and a second modulation element 177 in order from the sample SA side. The second objective lens 176 for detection is provided on the side opposite to the illumination mirror 174 with the sample SA interposed therebetween. Light from the sample SA corresponding to the irradiation of the second illumination light is incident on the second objective lens 176 for detection. The second modulation element 177 is disposed on a plane conjugate with the detection pupil of the second objective lens 176 for detection in the second detection optical system 175 (a plane perpendicular to the optical axis AX2 of the second microscope unit 150 (second detection optical system 175) at a position P2 conjugate with the detection pupil). The second modulation element 177 is configured in the same manner as the second modulation element 77 according to the first embodiment.

[0101] In the second embodiment, when performing bright-field observation of the sample SA, the first illumination optical system 20 of the first microscope unit 110 irradiates the sample SA by directing the first illumination light L1 emitted from the first light source 11 in the -z direction (the first direction), similar to the first embodiment.

[0102] The first detection light transmitted through the sample SA from the first illumination optical system 20 enters the first objective lens 32 of the first detection optical system 130. The first detection light transmitted through the first objective lens 32 enters the half mirror 172. A part of the first detection light incident on the half mirror 172 passes through the half mirror 172 and enters the imaging lens 36. The first detection light transmitted through the imaging lens 36 is reflected by the mirror 37 and forms an image at a predetermined image plane I where the first detector 40 is disposed. The first detector 40 detects the light (the first detection light) from the sample SA via the first detection optical system 130 and outputs a detection signal of the light. The detection signal of the light (the first detection light) output from the first detector 40 is transmitted to the image processing unit 91 via the control unit 90.

[0103] The second illumination light L2 emitted from the light source unit 51 (the second light source 52) of the second microscope unit 150 enters the half mirror 172 of the second illumination optical system 170. A part of the second illumination light L2 incident on the half mirror 172 is reflected by the half mirror 172 and enters the first objective lens 32, passing through the detection pupil in the first objective lens 32 from a direction inclined with respect to the central axis of the first objective lens 32. The second illumination light L2 transmitted through the first objective lens 32 is reflected by the illumination mirror 174, condensed, and irradiated onto the sample SA on the stage 2. Thereby, the second illumination optical system 170 condenses and irradiates the sample SA by directing the second illumination light L2 emitted from the light source unit 51 (the second light source 52) in the -x direction (the second direction orthogonal to the first direction).

[0104] The second detection light that has passed through the sample SA from the second illumination optical system 170 is incident on the second objective lens 176 for detection of the second detection optical system 175. The second detection light that has passed through the second objective lens 176 for detection is incident on the second detector 80 through the second modulation element 177. The second detector 80 detects the light (second detection light) from the sample SA via the second detection optical system 175 and outputs a detection signal of the light. The detection signal of the light (second detection light) output from the second detector 80 is transmitted to the image processing unit 91 via the control unit 90.

[0105] When the fluorescence of the sample SA is observed by the second microscope unit 150, a cylindrical lens (not shown) of the light source unit 51 of the second microscope unit 150 is inserted into the optical path between the first lens 53 and the first galvanometer mirror 54 in the light source unit 51. Further, a fluorescence filter cube 173 is inserted into the optical path between the first objective lens 32 and the imaging lens 36 instead of the half mirror 172. The fluorescence filter cube 173 includes a dichroic mirror 173a, an excitation filter 173b, and an absorption filter 173c.

[0106] The excitation light emitted from the light source unit 51 (second light source 52) passes through the excitation filter 173b of the fluorescence filter cube 173 and is incident on the dichroic mirror 173a. The excitation light incident on the dichroic mirror 173a of the fluorescence filter cube 173 is reflected by the dichroic mirror 173a and is incident on the first objective lens 32, and passes through the detection pupil in the first objective lens 32 from a direction inclined with respect to the central axis of the first objective lens 32. The excitation light that has passed through the first objective lens 32 is reflected by the illumination mirror 174, collected, and irradiated onto the sample SA on the stage 2. Thereby, the second illumination optical system 170 irradiates the sample SA with the excitation light, which is sheet light emitted from the light source unit 51 (second light source 52).

[0107] Upon irradiation with excitation light, the fluorescent substance contained in the sample SA is excited to emit fluorescence. The fluorescence from the sample SA enters the first objective lens 32 of the first detection optical system 130. The fluorescence that has passed through the first objective lens 32 enters the dichroic mirror 173a of the fluorescence filter cube 173. The fluorescence that has entered the dichroic mirror 173a of the fluorescence filter cube 173 passes through the dichroic mirror 173a, passes through the absorption filter 173c, and enters the imaging lens 36. The fluorescence that has passed through the imaging lens 36 is reflected by the mirror 37 and forms an image on a predetermined image plane I where the first detector 40 is disposed. The first detector 40 detects the fluorescence from the sample SA via the first detection optical system 130 and outputs a detection signal of the fluorescence. The detection signal of the fluorescence output from the first detector 40 is transmitted to the image processing unit 91 via the control unit 90.

[0108] In the second embodiment, a three-dimensional refractive index distribution (for example, image data of the three-dimensional refractive index distribution of the sample SA) in the sample SA can be generated by the same method as the refractive index data generation method according to the first embodiment. Therefore, according to the second embodiment, the same effects as those of the first embodiment can be obtained.

[0109] In the above-described second embodiment, the light source unit 51 of the second microscope unit 150 has the first galvanometer mirror 54 and the second galvanometer mirror 57, but is not limited thereto. For example, as in the microscope apparatus 101a shown in FIG. 18, the light source unit 51a of the second microscope unit 150a may have only one galvanometer mirror 64. In this case, similar to the microscope apparatus 1a according to the modification of the first embodiment, in the light source unit 51a, a mirror 67 is disposed instead of the second galvanometer mirror 57.

[0110] <Third Embodiment> Next, the microscope apparatus 201 according to the third embodiment will be described with reference to FIG. 19. The microscope apparatus 201 according to the third embodiment has the same main components as the microscope apparatus 1 according to the first embodiment, except for the second microscope unit. Therefore, for the components that are the same as those in the first embodiment, the same reference numerals as in the first embodiment are used, and detailed descriptions thereof are omitted. The microscope apparatus 201 according to the third embodiment includes a first microscope unit 210 and a second microscope unit 250. Further, the microscope apparatus 201 according to the third embodiment includes a stage 2, a control unit 90, and an image processing unit 91.

[0111] The first microscope unit 210 includes a first light source 11, a first illumination optical system 20, a first detection optical system 230, and a first detector 40. The first light source 11, the first illumination optical system 20, and the first detector 40 are configured in the same manner as in the first embodiment. The first detection optical system 230 includes, in order from the sample SA side, an objective lens unit 31, an imaging lens 36, and a polarization beam splitter 237. Further, the first detection optical system 230 includes the half mirror 272 of the second microscope unit 250. The objective lens unit 31 and the imaging lens 36 are configured in the same manner as in the first embodiment. The polarization beam splitter 237 reflects the light (s-polarized light) from the sample SA in response to the irradiation of the first illumination light and transmits the light (p-polarized light) from the sample SA in response to the irradiation of the second illumination light.

[0112] The second microscope unit 250 includes a light source unit 51, a second illumination optical system 270, a second detection optical system 275, and a second detector 80. The light source unit 51 and the second detector 80 are configured in the same manner as in the first embodiment.

[0113] The second illumination optical system 270 condenses and irradiates the second illumination light L2 emitted from the light source unit 51 (second light source 52) in the -x direction (a second direction orthogonal to the first direction) onto the sample SA. As shown in FIGS. 19 and 20, the second illumination optical system 270 includes a half mirror 272 and an illumination mirror 274. Further, the second illumination optical system 270 includes the objective lens unit 31 (first objective lens 32) of the first microscope unit 210 (first detection optical system 230).

[0114] The half mirror 272 is disposed in the optical path between the first objective lens 32 and the imaging lens 36 in the first microscope unit 210 (first detection optical system 230). The ratio of the transmittance to the reflectance of the half mirror 272 is set to, for example, 1:1. The half mirror 272 reflects a part of the second illumination light L2 emitted from the light source unit 51 (second light source 52) toward the detection pupil (rear focal plane) in the first objective lens 32. The first objective lens 32 condenses the second illumination light L2 reflected by the half mirror 272. Note that since the second illumination light L2 reflected by the half mirror 272 passes through the detection pupil (rear focal plane) in the first objective lens 32 from a direction inclined with respect to the central axis of the first objective lens 32, it proceeds in the +z direction offset with respect to the central axis of the first objective lens 32 when passing through the first objective lens 32. The illumination mirror 274 is disposed to face the right side of the sample SA above the first objective lens 32 (and the stage 2). The illumination mirror 274 reflects the second illumination light L2 that passes through the first objective lens 32 and proceeds in the +z direction offset with respect to the central axis of the first objective lens 32 toward the -x direction (second direction). The optical axis AX2 between the second illumination optical system 270 and the second detection optical system 275 in the second microscope unit 250 is orthogonal to the optical axis AX1 between the first illumination optical system 20 and the first detection optical system 230 in the first microscope unit 210.

[0115] [[ID=!4]] The second detection optical system 275 receives light from the sample SA corresponding to the irradiation of the second illumination light from the side opposite to the second illumination optical system 270 with the sample SA interposed therebetween. As shown in FIGS. 19 and 20, the second detection optical system 275 includes a detection mirror 276, a relay lens 278, and a second modulation element 277. Further, the second detection optical system 275 includes the objective lens unit 31 (first objective lens 32) of the first microscope unit 210 (first detection optical system 230), the imaging lens 36, the polarization beam splitter 237, and the half mirror 272 of the second illumination optical system 270.

[0116] The detection mirror 276 is provided above the first objective lens 32 (and the stage 2) on the side opposite to the illumination mirror 274 with the sample SA interposed therebetween. The illumination mirror 274 reflects the light from the sample SA in response to the irradiation of the second illumination light in the -z direction (i.e., the first objective lens 32) offset with respect to the central axis of the first objective lens 32. Light from the sample SA in response to the irradiation of the second illumination light enters the first objective lens 32 through the detection mirror 276. The relay lens 278 makes the light (p-polarized light) from the sample SA that has passed through the polarization beam splitter 237 enter the second modulation element 277. The second modulation element 277 is arranged on a plane conjugate with the detection pupil of the first objective lens 32 in the second detection optical system 275 (a plane perpendicular to the optical axis of the second microscope unit 250 (second detection optical system 275) at the position P2 conjugate with the detection pupil). The second modulation element 277 is configured in the same manner as the second modulation element 77 according to the first embodiment.

[0117] In the third embodiment, when performing bright-field observation of the sample SA, the first illumination optical system 20 of the first microscope unit 110 irradiates the sample SA with the first illumination light L1 emitted from the first light source 11 in the -z direction (the first direction) in the same manner as in the first embodiment. In the third embodiment, a polarizing plate (not shown) is arranged in the optical path in the first illumination optical system 20 (for example, the optical path between the collector lens 21 and the field stop 23) so that s-polarized light (the first detection light) enters the polarization beam splitter 237.

[0118] The first detection light (s-polarized light) that has passed through the sample SA from the first illumination optical system 20 is incident on the first objective lens 32 of the first detection optical system 230. The first detection light that has passed through the first objective lens 32 is incident on the half mirror 272. A part of the first detection light incident on the half mirror 272 passes through the half mirror 272 and is incident on the imaging lens 36. The first detection light (s-polarized light) that has passed through the imaging lens 36 is reflected by the polarization beam splitter 237 and forms an image on a predetermined image plane I where the first detector 40 is disposed. The first detector 40 detects the light (first detection light) from the sample SA via the first detection optical system 230 and outputs a detection signal of the light. The detection signal of the light (first detection light) output from the first detector 40 is transmitted to the image processing unit 91 via the control unit 90.

[0119] The second illumination light L2 emitted from the light source unit 51 (second light source 52) of the second microscope unit 250 is incident on the half mirror 272 of the second illumination optical system 270. In the third embodiment, for example, the light source unit 51 (second light source 52) emits the second illumination light L2 that is linearly polarized so that p-polarized light (second detection light) is incident on the polarization beam splitter 237. A part of the second illumination light L2 incident on the half mirror 272 is reflected by the half mirror 272 and is incident on the first objective lens 32, and passes through the detection pupil in the first objective lens 32 from a direction inclined with respect to the central axis of the first objective lens 32. The second illumination light L2 that has passed through the first objective lens 32 is reflected by the illumination mirror 274, condensed, and irradiated onto the sample SA on the stage 2. Thereby, the second illumination optical system 270 condenses and irradiates the second illumination light L2 emitted from the light source unit 51 (second light source 52) toward the sample SA in the -x direction (second direction orthogonal to the first direction).

[0120] The second detection light (p-polarized light) transmitted through the sample SA from the second illumination optical system 270 is reflected by the detection mirror 276 of the second detection optical system 275 and enters the first objective lens 32. The second detection light transmitted through the first objective lens 32 enters the half mirror 272. A part of the second detection light incident on the half mirror 272 passes through the half mirror 272 and enters the imaging lens 36. The second detection light (p-polarized light) transmitted through the imaging lens 36 passes through the polarization beam splitter 237 and enters the relay lens 278. The second detection light transmitted through the relay lens 278 passes through the second modulation element 277 and enters the second detector 80. The second detector 80 detects the light (second detection light) from the sample SA via the second detection optical system 275 and outputs a detection signal of the light. The detection signal of the light (second detection light) output from the second detector 80 is transmitted to the image processing unit 91 via the control unit 90.

[0121] In the third embodiment, a three-dimensional refractive index distribution (for example, image data of the three-dimensional refractive index distribution of the sample SA) in the sample SA can be generated by the same method as the refractive index data generation method according to the first embodiment. Therefore, according to the third embodiment, the same effects as those of the first embodiment can be obtained.

[0122] In the above-described third embodiment, the light source unit 51 of the second microscope unit 250 has the first galvanometer mirror 54 and the second galvanometer mirror 57, but is not limited thereto. For example, as in the microscope apparatus 201a shown in FIG. 21, the light source unit 51a of the second microscope unit 250a may have only one galvanometer mirror 64. In this case, similar to the microscope apparatus 1a according to the modification of the first embodiment, in the light source unit 51a, a mirror 67 is arranged instead of the second galvanometer mirror 57.

[0123] <Fourth Embodiment> Next, the microscope apparatus 301 according to the fourth embodiment will be described with reference to FIGS. 22 and 23. The microscope apparatus 301 according to the fourth embodiment has the same configuration as the microscope apparatus 1 according to the first embodiment, except for the first microscope unit and the second microscope unit. Therefore, for the same configurations as those in the first embodiment, the same reference numerals as those in the first embodiment are given and detailed descriptions thereof are omitted. As shown in FIGS. 22 and 23, the microscope apparatus 301 according to the fourth embodiment includes a first microscope unit 310 and a second microscope unit 350. Further, as shown in FIG. 23, the microscope apparatus 301 according to the fourth embodiment includes a stage 2, a control unit 90, and an image processing unit 91. In the fourth embodiment, as shown in FIG. 23, the coordinate axis extending in the optical axis direction (vertical direction) of the second microscope unit 350 is defined as the z-axis, and the coordinate axes perpendicular to the z-axis are defined as the x-axis and the y-axis.

[0124] As shown in FIGS. 22 and 23, the first microscope unit 310 includes a first light source 311, a first illumination optical system 320, a first detection optical system 330, and a first detector 340. The first light source 311 is configured in the same manner as the first light source 11 according to the first embodiment. The first light source 311 generates first illumination light. Further, the first light source 311 is disposed at a position conjugate to the illumination pupil.

[0125] The first illumination optical system 320 irradiates the sample SA by directing the first illumination light L1 emitted from the first light source 311 in the +x direction (the first direction). In FIG. 23, the first illumination light L1 is shown by a dashed line for easy distinction from the second illumination light L2. As shown in FIG. 23, the first illumination optical system 320 includes, in order from the first light source 311 side, a collector lens 321, a field stop (not shown), a relay lens 324, a first modulation element 325, an aperture stop (not shown), and a condenser lens 327. The collector lens 321, the field stop, the relay lens 324, the first modulation element 325, the aperture stop, and the condenser lens 327 are arranged side by side in the x direction and are otherwise configured in the same manner as the collector lens 21, the field stop 23, the relay lens 24, the first modulation element 25, the aperture stop 26, and the condenser lens 27 according to the first embodiment. The condenser lens 327 is arranged to face the left side of the stage 2. When a white light source is used as the first light source 311, an element (for example, a band-pass filter) for narrowing the wavelength band of the first illumination light may be provided as in the first embodiment.

[0126] The first detection optical system 330 receives light from the sample SA in response to the irradiation of the first illumination light L1 from the side opposite to the first illumination optical system 320 with the sample SA interposed therebetween. As shown in FIGS. 23 and 24, the first detection optical system 330 includes, in order from the sample SA side, a first objective lens 332 and an imaging lens 336. The first objective lens 332 is provided on the side opposite to the condenser lens 327 with the sample SA interposed therebetween. Light from the sample SA in response to the irradiation of the first illumination light is incident on the first objective lens 332. The light transmitted through the first objective lens 332 is incident on the imaging lens 336. The light transmitted through the imaging lens 336 forms an image on a predetermined image plane I (see FIG. 22).

[0127] A first detector 340 is arranged on the image plane I of the first detection optical system 330. The first detector 340 is configured in the same manner as the first detector 40 according to the first embodiment. The first detector 340 detects light from the sample SA via the first detection optical system 330.

[0128] As shown in FIGS. 22 and 23, the second microscope unit 350 includes a light source unit 351, a second illumination optical system 360, a second detection optical system 370, and a second detector 380. The light source unit 351 is also referred to as a Beam Steering unit. As shown in FIG. 23, the light source unit 351 includes a second light source 352, a first lens 353, a first galvanometer mirror 354, a second lens 355, a third lens 356, a second galvanometer mirror 357, and a fourth lens 358. Further, the light source unit 351 has a cylindrical lens (not shown) that can be inserted into and removed from the optical path between the first lens 353 and the first galvanometer mirror 354.

[0129] The second light source 352, the first lens 353, the first galvanometer mirror 354, the second lens 355, the third lens 356, and the second galvanometer mirror 357 are configured in the same manner as the second light source 52, the first lens 53, the first galvanometer mirror 54, the second lens 55, the third lens 56, and the second galvanometer mirror 57 according to the first embodiment. Note that the first galvanometer mirror 354 changes the traveling direction of the second illumination light, thereby changing the condensing position of the second illumination light on the sample SA in the y direction. The second galvanometer mirror 357 changes the traveling direction of the second illumination light, thereby changing the condensing position of the second illumination light on the sample SA in the x direction.

[0130] The fourth lens 358 condenses the second illumination light reflected by the second galvanometer mirror 357 at a predetermined intermediate image plane IM and makes it incident on the second illumination optical system 360. The position of the predetermined intermediate image plane IM is a position conjugate with the focal position of the second objective lens 366 on the sample SA. The first galvanometer mirror 354, the second galvanometer mirror 357, and the stage drive unit can change the condensing position of the second illumination light on the sample SA in three-dimensional directions (three directions of the x direction, the y direction, and the z direction) to three-dimensionally scan the sample SA.

[0131] The second illumination optical system 360 condenses and irradiates the sample SA with the second illumination light L2 emitted from the light source unit 351 (second light source 352) in the +z direction (a second direction orthogonal to the first direction). As shown in FIGS. 22 and 23, the second illumination optical system 360 includes, in order from the light source unit 351 side, a mirror 361, a collimator lens 362, and an objective lens unit 366. The mirror 361 reflects the second illumination light L2 emitted from the light source unit 351 (second light source 352) toward the collimator lens 362. The collimator lens 362 collimates the second illumination light L2 reflected by the mirror 361.

[0132] The objective lens unit 366 includes a plurality of second objective lenses 367 for illumination, a lens holding unit 368, and a unit driving unit 369. The second objective lenses 367 for illumination are arranged to face downward of the stage 2. The lens holding unit 368 holds a plurality of second objective lenses 367 for illumination having different optical characteristics. The lens holding unit 368 is configured using, for example, a revolver or a turret. The unit driving unit 369 can drive the lens holding unit 368 to select any one of the plurality of second objective lenses 367 for illumination and arrange it below the stage 2. Note that the unit driving unit 369 may move the lens holding unit 368 along the z-axis. In this case, the aforementioned stage driving unit may be used in combination, or the stage driving may not be used.

[0133] The second objective lens 367 for illumination arranged below the stage 2 condenses the second illumination light L2 that has passed through the collimator lens 362 onto the sample SA. As also shown in FIG. 24, the optical axis AX2 between the second illumination optical system 360 and the second detection optical system 370 in the second microscope unit 350 is orthogonal to the optical axis AX1 between the first illumination optical system 320 and the first detection optical system 330 in the first microscope unit 310.

[0134] The second detection optical system 370 receives light from the sample SA in response to the irradiation of the second illumination light from the side opposite to the second illumination optical system 360 with the sample SA interposed therebetween. As shown in FIGS. 22 and 23, the second detection optical system 370 includes, in order from the sample SA side, a second objective lens 371 for detection, a diaphragm 372, a second modulation element 373, a condenser lens 374, a mirror 376, and a relay lens 377. The second objective lens 371 for detection is provided on the side opposite to the second objective lens 367 for illumination with the sample SA interposed therebetween. As the second objective lens 371 for detection, any one of a plurality of second objective lenses 371 for detection having different optical characteristics can be selected and arranged above the stage 2. Light from the sample SA in response to the irradiation of the second illumination light is incident on the second objective lens 371 for detection.

[0135] The diaphragm 372 and the second modulation element 373 are arranged on a plane conjugate to the detection pupil of the second objective lens 371 for detection in the second detection optical system 370 (a plane perpendicular to the optical axis of the second microscope unit 350 (second detection optical system 370) at the position P2A conjugate to the detection pupil). The second modulation element 373 is arranged adjacent to the diaphragm 372 (above the diaphragm 372 as shown in FIG. 23, for example). The second modulation element 373 is configured in the same manner as the second modulation element 77 according to the first embodiment. The condenser lens 374 condenses the light that has passed through the diaphragm 372 and the second modulation element 373. The mirror 376 reflects the light that has passed through the condenser lens 374 toward the relay lens 377. The relay lens 377 causes the light reflected by the mirror 376 to be incident on the second detector 380.

[0136] The second detector 380 is arranged at a position P2B conjugate to the detection pupil in the second detection optical system 370. The second detector 380 is configured in the same manner as the second detector 80 according to the first embodiment. The second detector 380 detects light from the sample SA via the second detection optical system 370.

[0137] In the fourth embodiment, when performing bright-field observation of the sample SA, the first illumination light L1 emitted from the first light source 311 of the first microscope unit 310 is incident on the collector lens 321 of the first illumination optical system 320. The first illumination light L1 that has passed through the collector lens 321 becomes parallel light, passes through a field stop (not shown), and is incident on the relay lens 324. The first illumination light L1 that has passed through the relay lens 324 passes through the first modulation element 325 and an aperture stop (not shown) and is incident on the condenser lens 327. The first illumination light L1 that has passed through the condenser lens 327 becomes parallel light and irradiates the sample SA on the stage 2. Thereby, the first illumination optical system 320 irradiates the sample SA with the first illumination light L1 emitted from the first light source 311 in the +x direction (the first direction).

[0138] The first detection light that has passed through the sample SA from the first illumination optical system 320 is incident on the first objective lens 332 of the first detection optical system 330. The first detection light that has passed through the first objective lens 332 is incident on the imaging lens 336. The first detection light that has passed through the imaging lens 336 forms an image on a predetermined image plane I where the first detector 340 is disposed. The first detector 340 detects the light (the first detection light) from the sample SA via the first detection optical system 330 and outputs a detection signal of the light. The detection signal of the light (the first detection light) output from the first detector 340 is transmitted to the image processing unit 91 via the control unit 90.

[0139] Note that when performing bright-field observation of the sample SA, the cylindrical lens (not shown) of the light source unit 351 of the second microscope unit 350 is retracted from the optical path between the first lens 353 and the first galvanometer mirror 354 in the light source unit 351. The second illumination light emitted from the second light source 352 of the light source unit 351 is incident on the first lens 353. The second illumination light that has passed through the first lens 353 becomes parallel light and is reflected by the first galvanometer mirror 354. The second illumination light reflected by the first galvanometer mirror 354 passes through the second lens 355 and the third lens 356 and is reflected by the second galvanometer mirror 357. The second illumination light reflected by the second galvanometer mirror 357 passes through the fourth lens 358 and is emitted to the outside of the light source unit 351. Thereby, the light source unit 351 emits the second illumination light.

[0140] The second illumination light L2 emitted from the light source unit 351 (the second light source 352) is reflected by the mirror 361 of the second illumination optical system 360 and enters the collimator lens 362. The second illumination light L2 that has passed through the collimator lens 362 becomes parallel light and enters the second objective lens 367 for illumination. The second illumination light L2 that has passed through the second objective lens 367 for illumination is focused and irradiated onto the sample SA on the stage 2. As a result, the second illumination optical system 360 focuses and irradiates the second illumination light L2 emitted from the light source unit 351 (the second light source 352) onto the sample SA in the +z direction (the second direction orthogonal to the first direction).

[0141] The second detection light that has passed through the sample SA from the second illumination optical system 360 enters the second objective lens 371 for detection of the second detection optical system 370. The second detection light that has passed through the second objective lens 371 for detection enters the condenser lens 374 through the aperture stop 372 and the second modulation element 373. The second detection light that has passed through the condenser lens 374 is reflected by the mirror 376 and enters the relay lens 377. The second detection light that has passed through the relay lens 377 enters the second detector 380. The second detector 380 detects the light (the second detection light) from the sample SA via the second detection optical system 370 and outputs a detection signal of the light. The detection signal of the light (the second detection light) output from the second detector 380 is transmitted to the image processing unit 91 via the control unit 90.

[0142] When performing fluorescence observation of the sample SA with the second microscope unit 350, the cylindrical lens (not shown) of the light source unit 351 of the second microscope unit 350 is inserted into the optical path between the first lens 353 and the first galvanometer mirror 354 in the light source unit 351.

[0143] The excitation light emitted from the light source unit 351 (the second light source 352) is reflected by the mirror 361 of the second illumination optical system 360 and enters the collimator lens 362. The excitation light that has passed through the collimator lens 362 becomes parallel light and enters the second objective lens 367 for illumination. The excitation light that has passed through the second objective lens 367 for illumination is focused and irradiated onto the sample SA on the stage 2.

[0144] Due to the irradiation of the excitation light, the fluorescent substance contained in the sample SA is excited and emits fluorescence. The fluorescence from the sample SA enters the first objective lens 332 of the first detection optical system 330 of the first microscope unit 310. The fluorescence that has passed through the first objective lens 332 enters the imaging lens 336. The fluorescence that has passed through the imaging lens 336 forms an image on a predetermined image plane I where the first detector 340 is disposed. The first detector 340 detects the fluorescence from the sample SA via the first detection optical system 330 and outputs a detection signal of the fluorescence. The detection signal of the fluorescence output from the first detector 340 is transmitted to the image processing unit 91 via the control unit 90.

[0145] In the fourth embodiment, a three-dimensional refractive index distribution (for example, image data of the three-dimensional refractive index distribution of the sample SA) in the sample SA can be generated by the same method as the refractive index data generation method according to the first embodiment. Therefore, according to the fourth embodiment, the same effects as those of the first embodiment can be obtained.

[0146] <Fifth Embodiment> Next, with reference to FIG. 25, the microscope apparatus 401 according to the fifth embodiment will be briefly described. The microscope apparatus 401 according to the fifth embodiment has a main part in common configuration with the microscope apparatus 1 according to the first embodiment except for the second microscope unit. Therefore, for the same configuration as that in the first embodiment, the same reference numerals as those in the first embodiment are given and the detailed description is omitted. The microscope apparatus 401 according to the fifth embodiment includes a first microscope unit 410 and a second microscope unit 450. Further, although not shown, the microscope apparatus 401 according to the fifth embodiment includes a stage, a control unit, and an image processing unit. The stage, the control unit, and the image processing unit are configured in the same manner as the stage 2, the control unit 90, and the image processing unit 91 according to the first embodiment.

[0147] The first microscope unit 410 includes a first light source 11, a first illumination optical system 20, a first detection optical system 30, and a first detector 40. The first light source 11, the first illumination optical system 20, the first detection optical system 30, and the first detector 40 are configured in the same manner as in the first embodiment. Note that the first modulation element 25 of the first illumination optical system 20 is disposed at the position P1A of the illumination pupil. The first detector 40 is disposed on a predetermined image plane IA.

[0148] The second microscope unit 450 includes a second light source 451, a second illumination optical system 460, a second detection optical system 470, and a second detector 480. The second light source 451 is configured in the same manner as the first light source 311 according to the fourth embodiment. The second illumination optical system 460 is configured in the same manner as the first illumination optical system 320 according to the fourth embodiment. Note that the second modulation element 465 of the second illumination optical system 460 is configured in the same manner as the first modulation element 325 according to the fourth embodiment and is disposed at the position P1B of the illumination pupil. The second detection optical system 470 is configured in the same manner as the first detection optical system 330 according to the fourth embodiment. The second detector 480 is configured in the same manner as the first detector 340 according to the fourth embodiment and is disposed on a predetermined image plane IB.

[0149] In the fifth embodiment, a three-dimensional refractive index distribution (for example, image data of the three-dimensional refractive index distribution of the sample SA) in the sample SA can be generated by the same method as the refractive index data generation method according to the first embodiment. Therefore, according to the fifth embodiment, the same effects as those of the first embodiment can be obtained.

[0150] <Sixth Embodiment> Next, the microscope apparatus 501 according to the sixth embodiment will be briefly described with reference to FIG. 26. The microscope apparatus 501 according to the sixth embodiment has the same main components as the microscope apparatus 1 according to the first embodiment, except for the first microscope unit. Therefore, for the components having the same configuration as those in the first embodiment, the same reference numerals are given as in the first embodiment, and the detailed description thereof is omitted. The microscope apparatus 501 according to the sixth embodiment includes a first microscope unit 510 and a second microscope unit 550. Further, although not shown, the microscope apparatus 501 according to the sixth embodiment includes a stage, a control unit, and an image processing unit. The stage, the control unit, and the image processing unit are configured in the same manner as the stage 2, the control unit 90, and the image processing unit 91 according to the first embodiment.

[0151] The first microscope unit 510 includes a first light source unit 511 having a first light source 512, a first illumination optical system 520, a first detection optical system 530, and a first detector 540. The first light source unit 511 is configured in the same manner as the second light source unit 351 according to the fourth embodiment. The first illumination optical system 520 is configured in the same manner as the second illumination optical system 360 according to the fourth embodiment. The first detection optical system 530 is configured in the same manner as the second detection optical system 370 according to the fourth embodiment. Note that the first modulation element 533 of the first detection optical system 530 is configured in the same manner as the second modulation element 373 according to the fourth embodiment and is disposed at a position P2A conjugate with the detection pupil. The first detector 540 is configured in the same manner as the second detector 380 according to the fourth embodiment and is disposed at a position P2B conjugate with the detection pupil.

[0152] The second microscope unit 550 includes a light source unit 51, a second illumination optical system 70, a second detection optical system 75, and a second detector 80. The light source unit 51, the second illumination optical system 70, the second detection optical system 75, and the second detector 80 are configured in the same manner as those in the first embodiment. Note that the second modulation element 77 of the second detection optical system 75 is disposed at a position P2C conjugate with the detection pupil. The second detector 80 is also disposed at a position P2C conjugate with the detection pupil.

[0153] In the sixth embodiment, a three-dimensional refractive index distribution (for example, image data of the three-dimensional refractive index distribution of the sample SA) in the sample SA can be generated by the same method as the refractive index data generation method according to the first embodiment. Therefore, according to the sixth embodiment, the same effects as those of the first embodiment can be obtained.

[0154] <Modification Example> In the above-described first to third embodiments, the first detection optical systems 30, 130, 230 of the first microscope units 10, 110, 210 are provided separately from the first illumination optical system 20, but the present invention is not limited thereto, and a part of the first illumination optical system may be included. For example, as shown in FIG. 27, the first microscope unit 610 according to the modification example includes a first light source 11, a first illumination optical system 620, a first detection optical system 630, and a first detector 40. The first light source 11 and the first detector 40 are configured in the same manner as in the first embodiment.

[0155] The first illumination optical system 620 includes, in order from the first light source 11 side, a collector lens 621, a first relay lens 622, a first modulation element 623, a second relay lens 624, a condenser lens 625, a half mirror 626, an objective lens unit 631, and an illumination mirror 628. When a white light source is used as the first light source 11, an element (for example, a band-pass filter) for narrowing the wavelength band of the first illumination light may be provided in the same manner as in the first embodiment. The first modulation element 623 is disposed on a plane perpendicular to the optical axis AX1 of the first illumination optical system 620 at the position P1 of the illumination pupil between the first relay lens 622 and the second relay lens 624. The first modulation element 623 is configured in the same manner as the first modulation element 25 according to the first embodiment.

[0156] The half mirror 626 reflects part of the first illumination light from the first light source 11 toward the stage 2. The half mirror 626 transmits part of the light (first detection light) that has passed through the sample SA on the stage 2 toward the imaging lens 636 of the detection optical system 630. The ratio of the transmittance to the reflectance of the half mirror 626 is set to, for example, 1:1. The objective lens unit 631 includes a plurality of first objective lenses 632, a lens holding unit 633, and a unit driving unit 634. The first objective lens 632 is disposed to face downward of the stage 2. The lens holding unit 633 holds a plurality of first objective lenses 632 having different focal lengths. The lens holding unit 633 is configured using, for example, a revolver, a turret, or the like. The unit driving unit 634 can drive the lens holding unit 633 to select any one of the plurality of first objective lenses 632 and dispose it below the stage 2. The illumination mirror 628 is disposed to face upward of the stage 2.

[0157] The first detection optical system 630 includes the objective lens unit 631 and the half mirror 626. Further, the first detection optical system 630 has, in order from the half mirror 626 side, an imaging lens 636 and a mirror 637. The imaging lens 636 and the mirror 637 are configured in the same manner as the imaging lens 36 and the mirror 37 according to the first embodiment.

[0158] The first illumination light emitted from the first light source 11 of the first microscope unit 610 enters the collector lens 621 of the first illumination optical system 620. The first illumination light that has passed through the collector lens 621 becomes parallel light and enters the first relay lens 622. The first illumination light that has passed through the first relay lens 622 enters the second relay lens 624 through the first modulation element 623. The first illumination light that has passed through the second relay lens 624 enters the half mirror 626 through the condenser lens 625. A part of the first illumination light incident on the half mirror 626 is reflected by the half mirror 626 and enters the first objective lens 632. The first illumination light that has passed through the first objective lens 632 passes through the stage 2 and the sample SA and is reflected by the illumination mirror 628. The first illumination light reflected by the illumination mirror 628 is irradiated onto the sample SA on the stage 2. Thereby, the first illumination optical system 620 irradiates the sample SA with the first illumination light emitted from the first light source 11 in the -z direction (the first direction).

[0159] The first detection light that has been reflected by the illumination mirror 628 and passed through the sample SA enters the first objective lens 632 as the first detection optical system 630. The first detection light that has passed through the first objective lens 632 enters the half mirror 626. A part of the first detection light incident on the half mirror 626 passes through the half mirror 626 and enters the imaging lens 636. The first detection light that has passed through the imaging lens 636 is reflected by the mirror 637 and forms an image at a predetermined image plane I where the first detector 40 is disposed. The first detector 40 detects the light (the first detection light) from the sample SA via the first detection optical system 630 and outputs a detection signal of the light.

[0160] In each of the above-described embodiments, the image processing unit 91 obtains the three-dimensional refractive index distribution in the sample SA based on the detection signal of the light detected under one detection condition regarding the light transmittance. However, the present invention is not limited thereto. The image processing unit 91 may obtain the three-dimensional refractive index distribution in the sample SA based on the detection signals of the light detected under a plurality of detection conditions regarding the light transmittance. For example, the image processing unit 91 obtains the linear sum or difference of the POTFs based on the detection signals of the light detected under two detection conditions according to the user's settings or the like. Thereby, an absolute value of the POTF with a higher value can be obtained over a wider frequency band than in the case based on the detection signal of the light detected under one detection condition according to the user's settings or the like. Therefore, an image of the three-dimensional refractive index distribution in the sample SA with both high contrast and high resolution can be generated. Note that the image processing unit 91 can calculate the three-dimensional refractive index distribution n(x, y, z) in the sample SA using the above-described formulas (10) and (11) including the POTF.

[0161] For example, in the microscope apparatus according to the first to fourth embodiments, under the first detection condition, a first modulation element (first modulation element with a one-way change) in which the light transmittance changes according to a linear function in the example shown in FIG. 4 is disposed at the position of the illumination pupil. Under the second detection condition, a first modulation element (first modulation element with a reverse change) in which the light transmittance changes in the reverse direction to the example shown in FIG. 4 (first detection condition) according to a linear function is disposed at the position of the illumination pupil. In this modification, the difference between the POTF based on the detection signal of the light detected under the first detection condition and the POTF based on the detection signal of the light detected under the second detection condition is obtained. Then, since the value of the POTF under the first detection condition and the value of the POTF under the second detection condition have opposite signs to each other, although the width of the frequency band in which the absolute value of the POTF does not become zero does not change significantly, the absolute value of the POTF increases. Therefore, an image of the three-dimensional refractive index distribution in the sample SA with excellent contrast can be generated.

[0162] In each of the above embodiments, the first modulation element and the second modulation element are exemplified as elements in which the light transmittance changes within the plane of the flat plate, and are formed by depositing a film capable of reducing the light transmittance on a parallel flat plate such as a glass substrate. However, the present invention is not limited to this. For example, at least one of the first modulation element and the second modulation element may be formed by forming minute dot patterns (having light-shielding properties) capable of reducing the light transmittance on a parallel flat plate such as a glass substrate. In this case, it is possible to change the light transmittance by forming dot patterns with different densities on the parallel flat plate (glass substrate) using an existing lithography process or the like (the transmittance in the region where the dot pattern is dense is lower than that in the coarse region). At least one of the first modulation element and the second modulation element is not limited to the optical element as described above, and may be configured using an SLM (spatial light modulator) such as a transmissive liquid crystal element, a reflective liquid crystal element, or a DMD (digital micromirror device). When using an SLM, the SLM is arranged at the pupil (at least one of the illumination pupil and the detection pupil) or at a position conjugate to the pupil, in the same manner as the optical element in each of the above embodiments. For example, when using a transmissive liquid crystal element as the SLM, a desired light transmittance distribution can be set by controlling the transmittance of each pixel of the element. Also, when using a DMD as the SLM, a desired light transmittance distribution can be set by controlling the angle of each mirror.

[0163] In addition, when using the optical elements (i.e., flat plates having light transmissivity) in the above-described embodiments as the first modulation elements, the control unit 90 may change the distribution of the light transmittance within the illumination pupil plane by controlling the element selection unit to switch to any one of the plurality of first modulation elements and arrange it at the position of the illumination pupil. When using the optical elements (i.e., flat plates having light transmissivity) in the above-described embodiments as the second modulation elements, the control unit 90 may change the distribution of the light transmittance within the plane conjugate to the detection pupil by controlling the element selection unit to switch to any one of the plurality of second modulation elements and arrange it at the position conjugate to the detection pupil. When using an SLM as the first modulation element and the second modulation element, the control unit 90 changes the distribution of the light transmittance within the pupil plane or within the plane conjugate to the pupil by controlling the SLM. Therefore, in order to change the distribution of the light transmittance by the control unit 90, it is not necessary to provide a plurality of elements and an element selection unit.

[0164] In each of the above-described embodiments, the "lens" such as the collector lens 21 is described as a single lens in each figure for convenience of explanation, but it is not limited thereto. For example, the "lens" such as the collector lens 21 may be composed of a plurality of lenses, or may have a configuration in which a lens and an existing optical element other than the lens are combined.

Explanation of Reference Numerals

[0165] 1 Microscope device (First Embodiment) 2 Stage 10 First microscope unit 50 Second microscope unit 90 Control unit 91 Image processing unit (Data processing unit) 101 Microscope device (Second Embodiment) 110 First microscope unit 150 Second microscope unit 201 Microscope device (Third Embodiment) 210 First microscope unit 250 Second microscope unit 301 Microscope device (Fourth Embodiment) 310 First microscope unit 350 Second microscope unit 401 Microscope device (Fifth Embodiment) 410 First microscope unit 450 Second microscope unit 501 Microscope device (Sixth Embodiment) 510 First microscope unit 550 Second microscope unit

Claims

1. A first microscope unit including a first light source that generates first illumination light, a first illumination optical system that irradiates the sample with the first illumination light directed in a first direction, a first detection optical system that receives light from the sample in response to the irradiation of the first illumination light, and a first detector that detects the light from the sample via the first detection optical system. A second microscope unit including a second light source that generates second illumination light, a second illumination optical system that irradiates the sample with the second illumination light directed in a second direction different from the first direction, a second detection optical system that receives light from the sample in response to the irradiation of the second illumination light, and a second detector that detects the light from the sample via the second detection optical system. A data processing unit that generates a three-dimensional refractive index distribution in the sample based on a detection signal of the light detected by the first detector and a detection signal of the light detected by the second detector. A microscope apparatus comprising any one of the following (1) to (4). (1) The first illumination optical system includes a first modulation element provided at a position of the pupil of the first illumination optical system or a position conjugate with the pupil, and changes the intensity distribution of the first illumination light in the plane of the pupil or a plane conjugate with the pupil. The second detection optical system includes a second modulation element provided at a position of the pupil of the second detection optical system or a position conjugate with the pupil, and changes the intensity distribution of the light from the sample in response to the irradiation of the second illumination light in the plane of the pupil or a plane conjugate with the pupil. The first detector is provided at a position conjugate with the sample in the first detection optical system. The second detector is provided at a position conjugate with the pupil in the second detection optical system. (2) The first illumination optical system includes a first modulation element provided at a position of the pupil of the first illumination optical system or a position conjugate with the pupil, and changes the intensity distribution of the first illumination light in the plane of the pupil or a plane conjugate with the pupil. The second illumination optical system includes a second modulation element provided at a position of the pupil of the second illumination optical system or a position conjugate with the pupil, and changes the intensity distribution of the second illumination light in the plane of the pupil or a plane conjugate with the pupil. The first detector is provided at a position conjugate with the sample in the first detection optical system. The second detector is provided at a position conjugate with the sample in the second detection optical system. (3) The first detection optical system is provided at the position of the pupil of the first detection optical system or a position conjugate to the pupil, and includes a first modulation element that changes the intensity distribution of light from the sample in response to the irradiation of the first illumination light within the plane of the pupil or within a plane conjugate to the pupil. The second detection optical system is provided at the position of the pupil of the second detection optical system or a position conjugate to the pupil, and includes a second modulation element that changes the intensity distribution of light from the sample in response to the irradiation of the second illumination light within the plane of the pupil or within a plane conjugate to the pupil. The first detector is provided at a position conjugate to the pupil in the first detection optical system. The second detector is provided at a position conjugate to the pupil in the second detection optical system. (4) The first detection optical system is provided at the position of the pupil of the first detection optical system or a position conjugate to the pupil, and includes a first modulation element that changes the intensity distribution of light from the sample in response to the irradiation of the first illumination light within the plane of the pupil or within a plane conjugate to the pupil. The second illumination optical system is provided at the position of the pupil of the second illumination optical system or a position conjugate to the pupil, and includes a second modulation element that changes the intensity distribution of the second illumination light within the plane of the pupil or within a plane conjugate to the pupil. The first detector is provided at a position conjugate to the pupil in the first detection optical system. The second detector is provided at a position conjugate to the sample in the second detection optical system.

2. The first detection optical system has a first objective lens that receives light from the sample in response to the irradiation of the first illumination light, and the second illumination optical system has a second objective lens for illumination that condenses the second illumination light. The microscope apparatus according to claim 1, wherein the second detection optical system is provided on the side opposite to the second objective lens for illumination with the sample interposed therebetween, and has a second objective lens for detection that receives light from the sample in response to the irradiation of the second illumination light.

3. The first detection optical system has a first objective lens that receives light from the sample in response to the irradiation of the first illumination light. The second illumination optical system includes the first objective lens. The objective lens condenses the second illumination light. The microscope apparatus according to claim 1, wherein the second illumination optical system has an illumination mirror that reflects the second illumination light from the first objective lens in the second direction.

4. The microscope apparatus according to claim 3, wherein the second detection optical system is provided on a side opposite to the illumination mirror with the sample interposed therebetween, and includes a second objective lens for receiving light from the sample in response to irradiation of the second illumination light.

5. The second detection optical system includes the first objective lens, is provided on a side opposite to the illumination mirror with the sample interposed therebetween, and has a detection mirror that reflects light from the sample in response to irradiation of the second illumination light toward the first objective lens. The microscope apparatus according to claim 3, wherein the first objective lens receives light from the sample in response to irradiation of the second illumination light through the detection mirror.

6. The microscope apparatus according to claim 1, wherein the light transmittance of at least one of the first modulation element and the second modulation element changes along one direction in the plane of the pupil or in a plane conjugate with the pupil according to a continuous function.

7. The microscope apparatus according to claim 6, wherein the light transmittance of at least one of the first modulation element and the second modulation element monotonically increases or decreases along one direction in the plane of the pupil or in a plane conjugate with the pupil according to the continuous function, and becomes zero at a part of the outer periphery in the plane of the pupil or in a plane conjugate with the pupil.

8. The microscope apparatus according to claim 7, wherein the continuous function is any one of a linear function, a quadratic function, a Gaussian function, a sine function in a range smaller than one period, and a cosine function in a range smaller than one period.

9. The first modulation element includes a plurality of first modulation elements having different light transmittance distributions. The microscope apparatus according to claim 1, further comprising a switching control unit configured to perform control to switch to any one of the plurality of first modulation elements and arrange the selected element in the plane of the pupil or in a plane conjugate with the pupil.

10. The second modulation element includes a plurality of second modulation elements having different light transmittance distributions. The microscope apparatus according to claim 1, further comprising a switching control unit configured to perform control to switch to any one of the plurality of second modulation elements and arrange the selected element in the plane of the pupil or in a plane conjugate with the pupil.

11. At least one of the first modulation element and the second modulation element is a flat plate having light transmissivity and the light transmittance of which changes according to the continuous function. A dot pattern capable of reducing the light transmittance is formed on the flat plate. The microscope apparatus according to claim 6, wherein the light transmittance of the flat plate changes according to the continuous function depending on the density of the dot pattern.

12. At least one of the first modulation element and the second modulation element is a spatial light modulator capable of changing the distribution of the light transmittance that changes according to the continuous function. The spatial light modulator includes a transmissive liquid crystal element, a reflective liquid crystal element, or a digital micromirror device. The microscope apparatus according to claim 6, further comprising a modulator control unit configured to control the spatial light modulator to change the distribution of the light transmittance.

13. The first modulation element includes a first modulation element with a one-directional change in which the light transmittance changes along one direction in the pupil plane of the first illumination optical system or in a plane conjugate to the pupil according to the continuous function, and a first modulation element with a reverse-directional change in which the light transmittance changes along a direction opposite to the one direction in the pupil plane of the first illumination optical system or in a plane conjugate to the pupil according to the continuous function. It is possible to switch between the first modulation element with the one-directional change and the first modulation element with the reverse-directional change and arrange them in the pupil plane of the first illumination optical system or in a plane conjugate to the pupil. The data processing unit generates a three-dimensional refractive index distribution in the sample based on the detection signal of the light output from the first detector in a state where the first modulation element with the one-directional change is arranged in the pupil plane of the first illumination optical system or in a plane conjugate to the pupil, and the detection signal of the light output from the first detector in a state where the first modulation element with the reverse-directional change is arranged in the pupil plane of the first illumination optical system or in a plane conjugate to the pupil. The microscope apparatus according to claim 1.

14. The first detector detects light from a plurality of detection positions with different positions in the optical axis direction in the sample and outputs a detection signal of the light. The data processing unit generates a three-dimensional refractive index distribution in the sample based on the detection signals of the light from the plurality of detection positions output from the first detector. The microscope apparatus according to claim 1.

15. The second direction is a direction perpendicular to the first direction, and the optical axis between the second illumination optical system and the second detection optical system is orthogonal to the optical axis between the first illumination optical system and the first detection optical system. The microscope apparatus according to claim 1.

Images