microscope

Abstract

To provide a microscope which can more accurately determine a three-dimensional distribution of an index of refraction in a sample.SOLUTION: The microscope includes: an illumination optical system including an illumination objective lens, the illumination optical system scanning a sample by illumination light from a light source and irradiating the sample with the illumination light via a scanning unit in a plane which crosses a direction of an optical axis of the illumination objective lens; a detection optical system including a detection objective lens different from the illumination objective lens, the detection optical system guiding light which has passed through the sample to a detection unit; and an image processing unit for generating an image on the basis of a signal from the detection unit. The detection optical system is arranged in the pupil or its conjugate position of the detection objective lens and has an intensity modulation element having optical transmittance changing in the plane of the pupil or in the plane of the conjugate position. The detection unit is arranged in the conjugate position of the pupil of the detection objective lens.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a microscope. [Background technology]

[0002] In recent years, a method for determining the three-dimensional refractive index distribution in a sample such as a phase object has been devised (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2020 / 161826 Brochure Summary of the Invention

[0004] The microscope of the present invention comprises an illumination optical system that includes an illumination objective lens and scans and illuminates a specimen with illumination light from a light source via a scanning unit within a plane intersecting the optical axis direction of the illumination objective lens, a detection optical system that includes a detection objective lens different from the illumination objective lens and guides light that has passed through the specimen to a detection unit, and an image processing unit that generates an image based on a signal from the detection unit, wherein the detection optical system is positioned at the pupil of the detection objective lens or at a conjugate position thereof and has an intensity modulation element whose transmittance of the light changes within the plane of the pupil or within the plane of the conjugate position, and the detection unit is positioned at a conjugate position of the pupil of the detection objective lens. [Brief explanation of the drawings]

[0005] [Figure 1] 1 is a schematic diagram of a microscope apparatus according to a first embodiment. [Figure 2] 1 is a schematic configuration diagram showing a microscope apparatus according to a first embodiment. [Figure 3] FIG. 2 is an enlarged view of a stage in the first embodiment. [Figure 4] 10 is a graph showing the distribution of light transmittance of a modulation element. [Figure 5]FIG. 1 is a schematic diagram showing a method for performing deconvolution based on image data of multiple cross sections of a sample. [Figure 6] FIG. 1 shows a bright-field image of a sample generated by a conventional method. [Figure 7] FIG. 1 shows an image of the refractive index distribution of a sample generated by a conventional method. [Figure 8] FIG. 1 is a diagram showing a conventional distribution of POTF. [Figure 9] FIG. 10 is a diagram showing the distribution of a spectrum obtained by estimating and complementing the missing cone region of a conventional POTF. [Figure 10] FIG. 1 shows an image of a sample obtained by bright-field observation. [Figure 11] FIG. 1 is a diagram showing an image of the refractive index distribution of a sample. [Figure 12] FIG. 1 shows the distribution of POTF. [Figure 13] FIG. 10 is a diagram showing the distribution of a spectrum obtained by estimating and completing the missing cone region of the POTF. [Figure 14] 4 is a flowchart showing a data generating method according to the first embodiment. [Figure 15] FIG. 10 is a schematic configuration diagram showing a modified example of the microscope apparatus according to the first embodiment. [Figure 16] FIG. 10 is a schematic configuration diagram showing a microscope device according to a second embodiment. [Figure 17] FIG. 10 is an enlarged view of a stage in the second embodiment. [Figure 18] FIG. 10 is a schematic configuration diagram showing a modified example of the microscope apparatus according to the second embodiment. [Figure 19] FIG. 10 is a schematic configuration diagram showing a microscope device according to a third embodiment. [Figure 20] FIG. 11 is an enlarged view of a stage in a third embodiment. [Figure 21] FIG. 10 is a schematic configuration diagram showing a modified example of the microscope apparatus according to the third embodiment. [Figure 22] FIG. 10 is a schematic diagram of a microscope apparatus according to a fourth embodiment. [Figure 23] FIG. 10 is a schematic configuration diagram showing a microscope apparatus according to a fourth embodiment. [Figure 24] FIG. 10 is an enlarged view of a stage in a fourth embodiment. [Figure 25] FIG. 10 is a schematic diagram of a microscope apparatus according to a fifth embodiment. [Figure 26] FIG. 10 is a schematic diagram of a microscope apparatus according to a sixth embodiment. [Figure 27] FIG. 10 is a schematic configuration diagram showing a first microscope unit according to a modified example. DETAILED DESCRIPTION OF THE INVENTION

[0006] The microscope apparatus according to each embodiment will be described below. In the drawings used in the following description, components may be shown enlarged for the sake of convenience in order to make the features easier to understand, and the dimensional ratios of the components may not necessarily be the same as those in reality.

[0007] First Embodiment First, a 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 has a first microscope unit 10 and a second microscope unit 50. Furthermore, as shown in FIG. 2, the microscope apparatus 1 has 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. 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.

[0008] 2, the coordinate axis extending in the optical axis direction (vertical direction) of the first microscope unit 10 is the z-axis, and the coordinate axes perpendicular to the z-axis are the x-axis and y-axis. By moving the stage 2 in the z direction using the stage drive unit, it is possible to acquire image data of the cross section of the sample SA at a predetermined position Z0, a position Z0+Δz that is +Δz away from position Z0, a position Z0-Δz that is -Δz away from position Z0, a position Z0+2Δz that is +2Δz away from position Z0, a position Z0-2Δz that is -2Δz away from position Z0, and so on, as shown in FIG.

[0009] As shown in Figures 1 and 2, the first microscope unit 10 has 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). Alternatively, 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 first illumination light).

[0010] The first illumination optical system 20 irradiates the sample SA with first illumination light L1 emitted from the first light source 11 in the -z direction (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 that narrows the wavelength band of the first illumination light. For example, the wavelength band of the first illumination light can be narrowed by inserting a bandpass filter 22 with 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. Narrowing the wavelength band of the first illumination light can improve the accuracy of calculated values ​​such as the POTF (Potential Optimum Function), which will be described in detail later. The spectral transmittance characteristics of the bandpass filter 22 are set based on the wavelength band of the illumination light appropriate for the purpose of observation, such as bright-field observation. A 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 .

[0011] The first modulation element 25 and aperture stop 26 are disposed on a plane perpendicular to the optical axis AX1 of the first microscope unit 10 (first illumination optical system 20) at 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 disposed 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 position P1 of the illumination pupil is referred to as the illumination pupil plane. For example, the first modulation element 25 is a light-transmitting flat plate whose light transmittance changes within the plane of the plate. This flat plate is formed, for example, by evaporating a film capable of reducing light transmittance (having light-blocking properties) onto a parallel plate such as a glass substrate. For example, a metal film is evaporated. For example, by changing the film thickness depending on the location of the parallel plate on which the film is deposited, it is possible to change the light transmittance depending on the location of the parallel plate (the thicker the film, the lower the transmittance). By arranging this first modulation element 25 on the plane of the illumination pupil, it is possible to change the light transmittance within the plane of the illumination pupil. Therefore, it can be said that the light transmittance of the first modulation element 25 changes within the plane of the illumination pupil. The light transmittance of the first modulation element 25 changes continuously (or discretely) within the plane of the illumination pupil.

[0012] The light transmittance of the first modulation element 25 varies depending on the location of the first modulation element 25, thereby determining the distribution of the light transmittance of the first modulation element 25 (in other words, the distribution of the light transmittance on the plane of the illumination pupil). As the first modulation element 25, any one of a plurality of first modulation elements 25 having different variations in light transmittance, i.e., different distributions of light transmittance, can be selected and placed at position P1 of the illumination pupil. Details of the light transmittance of the first modulation element 25 will be described later. The position at which the first modulation element 25 is placed is not limited to position P1 of the illumination pupil. For example, the first modulation element 25 may be placed on a plane perpendicular to the optical axis AX1 at a position conjugate to the illumination pupil (in other words, a plane conjugate to the illumination pupil). Furthermore, the first light source 11 is placed at a position conjugate to the illumination pupil.

[0013] The condenser lens 27 is disposed above the stage 2 so as to face 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.

[0014] The first detection optical system 30 receives light from the sample SA in response to irradiation with the first illumination light L1 from the side opposite the first illumination optical system 20 across the sample SA. 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 multiple first objective lenses 32, a lens holder 33, and a unit driver 34. The first objective lenses 32 are disposed below and facing the stage 2. The lens holder 33 holds multiple first objective lenses 32 with different focal lengths. The lens holder 33 is configured using, for example, a revolver or a turret. The unit driver 34 can drive the lens holder 33 to select one of the multiple first objective lenses 32 and place it below the stage 2. The unit driver 34 may also move the lens holder 33 along the z-axis. In this case, the above-mentioned stage driving unit may be used in combination, or the stage driving may not be used.

[0015] Light from the sample SA in response to the irradiation of the first illumination light enters the first objective lens 32 arranged below the stage 2. The light that passes through the first objective lens 32 enters the imaging lens 36. The light that passes through the imaging lens 36 is reflected by a 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 with the focal position of the first objective lens 32 on the sample SA. Note that a half mirror may be provided instead of the mirror 37, and an observation optical system (not shown) having an eyepiece (not shown) may be provided on the optical path of the light that passes through the half mirror. This allows the observer to observe the image of the sample SA using the eyepiece.

[0016] The first detector 40 is disposed on the image plane I of the first detection optical system 30. The first detector 40 is configured using an imaging element such as a CCD (Charge Coupled Device) or a CMOS (Complementary Metal Oxide Semiconductor). The first detector 40 detects light from the sample SA via the first detection optical system 30.

[0017] As shown in FIGS. 1 and 2, the second microscope section 50 has 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 has 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. The light source unit 51 also has a cylindrical lens (not shown) that is insertable into and removable from the optical path between the first lens 53 and the first galvanometer mirror 54.

[0018] The second light source 52 is configured using a laser light source. The second light source 52 emits illumination light of 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 direction of travel of the second illumination light by changing the orientation of its reflecting surface. By changing the direction of travel of the second illumination light, the first galvanometer mirror 54 changes the focusing position of the second illumination light on the sample SA in the y direction. The second lens 55 and the third lens 56 direct the second illumination light reflected by the first galvanometer mirror 54 to the second galvanometer mirror 57. Note that if the first galvanometer mirror 54 and the second galvanometer mirror 57 are positioned conjugate with the pupil, the second lens 55 and the third lens 56 may not be provided.

[0019] 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, the second galvanometer mirror 57 changes the focusing position of the second illumination light on the sample SA in the z direction.

[0020] The focal length of the liquid lens 59 can be changed by changing the radius of curvature of the lens surface. By changing the focal length of 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 that has passed through the liquid lens 59 to be incident on 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 dimensions (the x direction, the y direction, and the z direction), thereby scanning the sample SA three-dimensionally.

[0021] From the viewpoint of deconvolution, it is preferable to adjust in advance the illumination range of the first illumination light from the first illumination optical system 20 (the three-dimensional observation range on the sample SA) and the illumination range of the second illumination light from the second illumination optical system 70 (the three-dimensional observation range on the sample SA) so that they coincide with each other. The adjustment to match the illumination ranges (observation ranges) of the first illumination light and the second illumination light can be achieved, 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. Alternatively, an image may be constructed by three-dimensional scanning of the sample SA by specifying a common illumination range (observation range) for the first illumination light and the second illumination light without adjusting the illumination ranges (observation ranges) of the first illumination light and the second illumination light to coincide with each other.

[0022] The second illumination optical system 70 focuses and irradiates the second illumination light L2 emitted from the light source unit 51 (second light source 52) onto the sample SA in the +x direction (a second direction perpendicular to the first direction). In FIGS. 2, 15, 16, 18, 19, and 21, the second illumination light L2 is indicated by a dashed line to easily distinguish it from the first illumination light L1. As shown in FIGS. 2 and 3, the second illumination optical system 70 has a second illumination objective lens 71. The second illumination objective lens 71 is disposed to the left of and opposite the sample SA. The second illumination objective lens 71 focuses 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 perpendicular 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.

[0023] The second detection optical system 75 receives light from the sample SA in response to irradiation with the second illumination light from the side opposite the second illumination optical system 70 across the sample SA. As shown in Figures 2 and 3, the second detection optical system 75 has, in order from the sample SA side, a second detection objective lens 76 and a second modulation element 77. The second detection objective lens 76 is provided on the opposite side of the sample SA from the second illumination objective lens 71. Light from the sample SA in response to irradiation with the second illumination light is incident on the second detection objective lens 76. Note that the second illumination objective lens 71 and the second detection objective lens 76 may have the same and high NA (numerical aperture).

[0024] The second modulation element 77 is disposed 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 with the pupil (hereinafter sometimes referred to as the detection pupil) of the second detection objective lens 76 in the second detection optical system 75. The plane perpendicular to the optical axis AX2 of the second microscope unit 50 at the position P2 conjugate with the detection pupil is referred to as the plane conjugate with the detection pupil. As an example, the second modulation element 77, like the first modulation element 25, is formed by depositing a film capable of reducing light transmittance on a parallel plate such as a glass substrate. By disposing this second modulation element 77 on a plane conjugate with the detection pupil, the light transmittance can be changed within the plane conjugate with the detection pupil. Therefore, it can be said that the light transmittance of the second modulation element 77 changes within the plane conjugate with the detection pupil. The light transmittance of the second modulation element 77 changes continuously (or discretely) within the plane conjugate with the detection pupil. As the second modulation element 77, any one of a plurality of second modulation elements 77 having different distributions of light transmittance can be selected and placed at a position P2 conjugate with the detection pupil. Details of the light transmittance of the second modulation element 77 will be described later. The position at which the second modulation element 77 is placed is not limited to the position P2 conjugate with the detection pupil. For example, the second modulation element 77 may be placed 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 into the second detection objective lens 76.

[0025] The second detector 80 is disposed adjacent to the second modulation element 77 at a position P2 conjugate with the detection pupil in the second detection optical system 75. The second detector 80 is configured using a PMT (Photomultiplier tube), an NDD (Non-Descanned Detection), or the like. The second detector 80 detects light from the sample SA via the second detection optical system 75.

[0026] An example of the change in light transmittance within the pupil plane of the first modulation element 25 and the second modulation element 77 (in other words, the distribution of light transmittance within the pupil plane) will be described. Fig. 4 is a graph showing an example of the distribution of light transmittance of the first modulation element 25 and the second modulation element 77. In Fig. 4, X is the x-coordinate with the coordinate position through which the optical axis (optical axis AX1 of the first microscope unit 10 or optical axis AX2 of the second microscope unit 50) passes as the origin, and Y is the y-coordinate with the coordinate position through which the optical axis passes as the origin.

[0027] 4, the light transmittance of the first modulation element 25 or the second modulation element 77 varies 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 in the X direction (for example, the −X direction) according to a sine function (portions of equal light transmittance are distributed in a straight line extending in the Y direction). In other words, the light transmittance of the first modulation element 25 or the second modulation element 77 varies in accordance with a sine function. Note that the light transmittance of the first modulation element 25 or the second modulation element 77 can be said to monotonically increase in the X direction (for example, the +X direction) according to a sine function within the pupil plane, or to monotonically decrease or increase in the X direction.

[0028] The light transmittance of the first modulation element 25 or the second modulation element 77 may monotonically decrease or increase in accordance with a sine function in the Y direction (portions with equal light transmittance may be distributed in a straight line 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 in accordance with a sine function in any direction in the XY coordinate system, not limited to the X or Y direction (portions with equal light transmittance may be distributed in a straight line extending in a direction perpendicular to the any direction in the XY coordinate system).

[0029] 4, the light transmittance of the first modulation element 25 or the second modulation element 77 may decrease monotonically in the X direction (for example, the −X direction) according to a linear function. That is, the light transmittance of the first modulation element 25 or the second modulation element 77 may change according to a linear function, not limited to a sine function.

[0030] In the example shown in FIG. 4, the 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 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 another function such as a cubic function. Furthermore, the range in which the light transmittance of the first modulation element 25 or the second modulation element 77 changes 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 or the second modulation element 77 is formed so that the region in which the light transmittance is 0 coincides with the outer periphery of the pupil (illumination pupil, detection pupil).

[0031] If the optical transmittance of the first modulation element 25 or the second modulation element 77 varies according to a cosine or sine function, it is desirable that the variation be within a range of less than one period within the plane of the pupil (illumination pupil, detection pupil). If the variation is within a range greater than one period, the POTF value will also behave periodically, which is undesirable from the perspective of deconvolution. In this case, there will be multiple frequencies at which the POTF value is zero, which increases the noise generated during the deconvolution process and reduces the accuracy of the refractive index profile of the sample SA. Furthermore, if the optical transmittance of the first modulation element 25 or the second modulation element 77 varies according to a cosine function, the optical transmittance is designed to be zero (i.e., the cosine function value is zero) at the outer periphery of the pupil (illumination pupil, detection pupil). This is because discontinuities in optical transmittance at the outer periphery of the pupil can cause artifacts such as ringing in the image.

[0032] 4, the optical transmittance of the first modulation element 25 or the second modulation element 77 varies in one direction within the pupil plane or a plane conjugate to the pupil according to a continuous function, and is 0 (zero) in a portion of the outer periphery within the pupil plane or a plane conjugate to the pupil, but is not limited to this. For example, the optical transmittance of the first modulation element 25 or the second modulation element 77 may vary in accordance with a continuous function with increasing distance from the optical axis within the pupil plane or a plane conjugate to the pupil, and be 0 (zero) along the entire outer periphery within the pupil plane or a plane conjugate to the pupil.

[0033] In this embodiment, when bright-field observation of the sample SA is performed, the first illumination light L1 emitted from the first light source 11 of the first microscope unit 10 is incident on the collector lens 21 of the first illumination optical system 20. The first illumination light L1 transmitted through the collector lens 21 becomes parallel light, 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 is incident on the relay lens 24. The first illumination light L1 transmitted through the relay lens 24 passes through the first modulation element 25 and the aperture stop 26 and is incident on the condenser lens 27. The first illumination light L1 transmitted through the condenser lens 27 becomes parallel light and is irradiated onto the sample SA on the stage 2. As a result, 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 (first direction).

[0034] Light transmitted through the sample SA from the first illumination optical system 20 (hereinafter, sometimes referred to as first detection light) is incident on the first objective lens 32 of the first detection optical system 30. The first detection light transmitted through the first objective lens 32 is incident on the imaging lens 36. The first detection light transmitted through the imaging lens 36 is reflected by a mirror 37 and forms an image on a predetermined image plane I where the first detector 40 is disposed. The first detector 40 detects 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. In other words, the first detector 40 captures 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, if the first detector 40 is configured using a CCD, it is a signal at each pixel of the CCD. The detection signal of the first detector 40 can be rephrased as a signal indicating the signal intensity detected by the first detector 40 in accordance with the intensity of the image of the sample SA.

[0035] When bright-field observation of the sample SA is performed, the cylindrical lens (not shown) of the light source unit 51 of the second microscope section 50 is retracted from the optical path between the first lens 53 and the first galvanometer mirror 54 of the light source unit 51. The second illumination light emitted from the second light source 52 of the light source unit 51 is incident on 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 is transmitted 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 is incident on the liquid lens 59. The second illumination light transmitted through the liquid lens 59 is transmitted through the fourth lens 60 and the fifth lens 61 and is emitted to the outside of the light source unit 51. This causes the light source unit 51 to emit the second illumination light.

[0036] The second illumination light L2 emitted from the light source unit 51 (second light source 52) is incident on the second illumination objective lens 71 of the second illumination optical system 70. The second illumination light L2 transmitted through the second illumination objective lens 71 is collected and irradiated onto the sample SA on the stage 2. As a result, the second illumination optical system 70 collects and irradiates the second illumination light L2 emitted from the light source unit 51 (second light source 52) onto the sample SA in the +x direction (a second direction perpendicular to the first direction).

[0037] Light transmitted through the sample SA from the second illumination optical system 70 (hereinafter, sometimes referred to as second detection light) is incident on a second detection objective lens 76 of the second detection optical system 75. The second detection light transmitted through the second detection objective lens 76 passes through a second modulation element 77 and is incident on a second detector 80. The second detector 80 detects the light (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 (second detection light) output from the second detector 80 is sent to an image processing unit 91 via a control unit 90.

[0038] It is also possible to perform fluorescence observation of the sample SA by using the microscope device 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 section 50 is inserted into the optical path between the first lens 53 and the first galvanometer mirror 54 in the light source unit 51.

[0039] 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 that has passed through the first lens 53 passes through a cylindrical lens (not shown) and is reflected by the first galvanometer mirror 54. The excitation light that has 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 that has reflected by the second galvanometer mirror 57 is incident on the liquid lens 59. The excitation light that has passed 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. In this way, the light source unit 51 emits excitation light that is a sheet light.

[0040] The excitation light emitted from the light source unit 51 (second light source 52) is incident on the second illumination objective lens 71 of the second illumination optical system 70. The excitation light transmitted through the second illumination objective lens 71 is collected and irradiated onto the sample SA on the stage 2. As a result, the second illumination optical system 70 irradiates the sample SA with the excitation light, which is sheet light emitted from the light source unit 51 (second light source 52).

[0041] Irradiation with the excitation light excites fluorescent substances contained in the sample SA, causing them to emit 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 that passes through the first objective lens 32 is incident on the imaging lens 36. The fluorescence that passes through the imaging lens 36 is reflected by a mirror 37 and forms an image on a predetermined image plane I where the first detector 40 is positioned. 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 fluorescence detection signal output from the first detector 40 is sent to an image processing unit 91 via the control unit 90.

[0042] The control unit 90 performs overall control of the microscope device 1. The control unit 90 is electrically connected to a stage driving unit (not shown), a unit driving 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.

[0043] When bright-field observation of the sample SA is performed, the image processing unit 91 generates refractive index data for 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 for the sample SA is data representing the refractive index of the sample SA, for example, data of the refractive index at each position on the sample SA, i.e., data representing the refractive index distribution on the sample SA. The refractive index data for the sample SA is stored in a storage unit (not shown), for example, as a look-up table. The image processing unit 91 also generates image data (hereinafter, sometimes referred to as image data of the refractive index distribution of the sample SA) in which the brightness value of each pixel is set according to the refractive index value at each position on the refractive index distribution on the sample SA. In addition, 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, the image processing unit 91 generates image data (hereinafter sometimes referred to as image data of the sample SA obtained by bright-field observation) in which the brightness value of each pixel is set according to the signal intensity value of the detection signal at each position on the sample SA (each pixel of the first detector 40).

[0044] When performing fluorescent observation of the sample SA, the image processing unit 91 generates image data (hereinafter sometimes referred to as image data of the sample SA observed by fluorescent observation) in which the brightness value of each pixel is set according to the signal intensity value of the detection signal at each position on the sample SA, based on the fluorescent detection signal output from the first detector 40.

[0045] This makes it possible to display an image of the refractive index distribution of the sample SA on the image display unit (not shown) based on the image data of the refractive index distribution of the sample SA generated by the image processing unit 91. Also, it is possible to display an image of the sample SA obtained by bright-field observation on the image display unit 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 obtained by fluorescence observation based on the image data of the sample SA obtained by fluorescence observation generated by the image processing unit 91.

[0046] Next, a known method for obtaining the three-dimensional refractive index distribution of the sample SA as refractive index data for the sample SA using the image processing unit 91 will be described. A typical example of obtaining the three-dimensional refractive index distribution of the sample SA is a method that uses a theory called PC-ODT (Partially Coherent-Optical Diffraction Tomography). The theory of PC-ODT will be briefly described below. From the equation for partial 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).

[0047]

number

[0048] 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). The image in this case refers to the image of the sample SA obtained by focusing light (detected light) that has been transmitted through at least a portion of the sample SA due to illumination. Therefore, the intensity I(x,y,z) of the image of the three-dimensional object, i.e., the three-dimensional image of the sample SA, can be replaced in image processing with the signal intensity of the detection signal output from the first detector 40, etc. (e.g., the signal intensity at each pixel of the first detector 40 when the sample SA is imaged by the first detector 40). As shown in Figure 2, the coordinate axis extending in the optical axis direction (vertical direction) of the first microscope unit 10 is the z-axis, and the coordinate axes perpendicular to the z-axis are the x-axis and y-axis. The transmission cross coefficient TCC can be expressed as Equation (2) below.

[0049]

number

[0050] In equation (2), S represents the illumination pupil, and G represents the detection pupil. The transmission cross coefficient TCC is a Hermite conjugate, and therefore has the properties shown in equation (3) below.

[0051]

number

[0052] For thin samples such as cells, the effect of scattering is small, so the first-order Born approximation (low contrast approximation) holds. In this case, we only need 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) that has been diffracted by the sample. Therefore, using the first-order Born approximation, we can obtain the following equation (4) from the above equations (1) to (3):

[0053]

number

[0054] Moreover, the complex amplitude transmittance o of an object can be approximated as in the following equation (5).

[0055]

number

[0056] In equation (5), P represents the real part of the scattering potential. Φ represents the imaginary part of the scattering potential. Using equation (5), the above equation (4) can be expressed as the following equation (6).

[0057]

number

[0058] Here, TCC is renamed to WOTF (Weak Object Transfer Function), which is defined by the following equation (7).

[0059]

number

[0060] From the above equations (6) and (7), the intensity I(x, y, z) of the image of a three-dimensional object obtained by a microscope using the transmitted illumination method is expressed as in the following equation (8).

[0061]

number

[0062] Here, the amplitude change of the sample is assumed to be small and negligible, i.e., P = 0. In this case, when the above equation (8) is expressed in real space, the following equation (9) is obtained.

[0063]

number

[0064] In equation (9), EPSF represents the effective point spread function. EPSF is equal to the inverse Fourier transform of the WOTF. EPSF is generally a complex function. The first term in equation (9) represents the background intensity. The second term in equation (9) represents the imaginary part Φ of the scattering potential of the sample multiplied by the imaginary part Im[EPSF] of the EPSF. Using equation (9), the imaginary part Φ of the scattering potential of the sample can be calculated.

[0065] The first method for calculating Φ(x, y, z) is to perform direct deconvolution using Im[EPSF]. Figure 5 shows a schematic diagram of the process of moving the stage 2 in the z direction (i.e., the optical axis direction) to acquire image intensities (signal intensities of detection signals output from the first detector 40, etc.) of multiple cross sections (xy cross sections) at different z-direction positions (i.e., positions along the optical axis) of the sample SA, and then performing deconvolution. Note that multiple cross-section images at different z-direction positions (i.e., positions along the optical axis) of the sample SA are sometimes collectively referred to as a 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 to normalize the image, and then the normalized first term of Equation (9) is removed in real space (or frequency space). Then, by performing deconvolution using Im[EPSF], the following Equation (10) is obtained.

[0066]

number

[0067] In equation (10), the 3D Fourier transform of Im[EPSF] is defined as POTF (Phase Optical Transfer Function). Since Im[EPSF] can range from positive to negative, the POTF value can also range from positive to negative. Here, POTF is an index that represents the contrast and resolution of the image of the sample SA observed in bright-field observation. Specifically, the absolute value of POTF represents the image contrast; the higher the absolute value of POTF, the higher the contrast of the image of the sample SA observed in bright-field observation. Furthermore, the wider the region in frequency space where the POTF value is not zero, the higher the resolution of the image of the sample SA observed in bright-field observation. Furthermore, I' is defined as the intensity I of each cross-section of the sample SA in the z-stack image of the sample SA (e.g., I1 to I6 in Figure 5) normalized by the constant term in equation (9). γ is assumed to be an arbitrarily small value.

[0068] A second method for calculating Φ(x, y, z) is to calculate the difference in intensity between two cross-sectional images at different positions in the z direction (i.e., the position in the optical axis direction) of the sample SA, remove the constant term in equation (9), and then perform deconvolution using Im[EPSF] of the calculated difference in intensity. This method is also described in the pamphlet of International Publication No. 2021 / 064807, and will not be described here.

[0069] When P=0, the scattering potential Φ is defined by the following equation (11).

[0070]

number

[0071] 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 calculated by the above-described method 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) of the sample SA from the signal intensity of the detection signal output from the first detector 40, etc., i.e., the intensity I(x, y, z) of the three-dimensional image of the sample SA, using Equations (10) and (11) above. As an example, the image processing unit 91 generates image data in which the brightness value of each pixel is set according to the refractive index value at each position (coordinate) of the calculated three-dimensional refractive index distribution of the sample SA, i.e., image data of the three-dimensional refractive index distribution of the sample SA. The intensity of the three-dimensional image of the sample SA can be expressed as the intensity of the image of each cross section of the sample SA in the z-stack image of the sample SA. In other words, the intensity of the three-dimensional image of the sample SA can also be considered as the intensity of multiple images at different positions in the z direction (i.e., positions in the optical axis direction) of the sample SA.

[0072] When light from the sample SA is detected using only the first microscope unit 10, as in the conventional method, the POTF has a region where information is missing in the z direction (hereinafter referred to as a missing cone region), resulting in errors in the change in refractive index in the z direction. This makes it difficult to generate an image of the three-dimensional refractive index distribution of the sample SA or an image of the sample SA obtained by bright-field observation. Figure 6 shows an example of an image of the sample SA (x-z cross section) obtained by bright-field observation using a conventional method. Figure 7 shows an example of an image of the three-dimensional refractive index distribution (x-z cross section) of the sample SA obtained by a conventional method. The sample SA shown in Figures 6 and 7 is a roughly spherical pseudo-cell with known refractive index and other properties. In the example shown in Figures 6 and 7, the refractive index of the sample SA (pseudo-cell) is set to approximately 1.35, and the refractive index of the medium is set to 1.33. The illumination-side NA (numerical aperture) and detection-side NA of the microscope used in the example shown in Figures 6 and 7 are set to 0.95.

[0073] Figure 8 shows the conventional POTF distribution. In Figure 8, white (background) indicates a POTF value of 0, and black indicates a POTF value that is positive or negative. Therefore, in Figure 8, the darker the black, the larger the absolute value of the POTF. As shown in Figure 8, there is a large missing cone region near the center of the conventional POTF distribution. Therefore, the image of sample SA obtained by bright-field observation deviates from its original approximately spherical shape and is elongated in the z direction, as shown in Figure 6, for example.

[0074] Furthermore, because the POTF has missing cone regions, refractive index correction is required when calculating the three-dimensional refractive index distribution using the above equations (10) and (11). Therefore, the refractive index is corrected using missing cone estimation methods such as the Gerchberg-Papoulis method, Edge-Preserving Regularization method, and Total Variation Regularization method. Specifically, a missing cone estimation algorithm is used to set constraints so that the minimum refractive index value is a predetermined refractive index value (for example, the refractive index value of the medium in the known sample SA), and the missing cone region is estimated.

[0075] Fig. 9 shows the spectral distribution obtained by estimating and interpolating the missing cone region of the conventional POTF. As shown in Fig. 9, even if the missing cone region of the POTF is interpolated, the image of the three-dimensional refractive index distribution of the sample SA deviates from the original approximately spherical shape and extends in the z direction, as shown in Fig. 7, for example.

[0076] In this embodiment, the image processing unit 91 generates image data of the three-dimensional refractive index distribution of the sample SA based on a detection signal of 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 (first direction) and a detection signal of 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 (second direction orthogonal to the first direction). As a result, according to this embodiment, it is possible to reduce the missing cone region in the POTF based on the detection signal of light output from the second detector 80, making it 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.

[0077] FIG. 10 shows an example of an image of sample SA (xz cross section) by bright-field observation generated by the method according to this embodiment. FIG. 11 shows an example of an image of the three-dimensional refractive index distribution (xz cross section) of sample SA generated by the method according to this embodiment. A roughly spherical pseudo-cell with a known refractive index is used as the sample SA shown in FIGS. 10 and 11. In the example shown in FIGS. 10 and 11, the refractive index of the sample SA (pseudo-cell) is set to about 1.35, and the refractive index of the medium is set to 1.33. Furthermore, the illumination-side NA (numerical aperture) and detection-side NA of the first microscope unit 10 are set to 0.95, and the illumination-side NA and detection-side NA of the second microscope unit 50 are set to 0.5.

[0078] FIG. 12 shows the distribution of the POTF in this embodiment. As in FIG. 8, the darker the black, the greater the absolute value of the POTF. As shown in FIG. 12, the POTF based on the detection signal of light output from the second detector 80 is present near the center of the POTF distribution in this embodiment, filling in the missing cone region. Therefore, the image of the sample SA obtained by bright-field observation resembles the original substantially spherical shape, as shown in FIG. 10, for example. FIG. 13 shows the spectral distribution obtained by estimating and interpolating the missing cone region of the POTF in this embodiment. As shown in FIG. 13, the missing cone region of the POTF can be interpolated over a wider range than in the past, and the image of the three-dimensional refractive index distribution of the sample SA resembles the original substantially spherical shape, as shown in FIG. 11, for example.

[0079] Furthermore, f is the spatial frequency, the pupil function of the lens (objective lens and condenser lens) that determines the illumination side NA (numerical aperture) is Pill(f), and the pupil function of the lens (objective lens and condenser lens) that determines the detection side NA is Pcol(f). The 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 Koehler illumination microscope, the illumination system effective pupil function is |Pill(f)| 2 P * It is known that the effective pupil function of the illumination system is Pill(f) and the effective pupil function of the detection system is |Pcol(f)| in the second microscope unit 50, which is a non-confocal laser microscope. 2 Pill * It is known that the result is (f). Note that since the order of convolution does not change even if the order is reversed, the Kohler illumination microscope (first microscope unit 10) and the non-confocal laser microscope (second microscope unit 50) will obtain the same POTF if the conditions such as NA are the same.

[0080] When a linear function intensity transmission mask (for example, a first modulation element 25 whose light transmittance monotonically increases or decreases in one direction in the pupil plane according to a linear function) is placed at the position of the illumination pupil in the Koehler illumination microscope (first microscope unit 10), |Pill(f)| 2 is a linear function, so the effective pupil function of the illumination system is also a linear function. Therefore, in the example shown in Fig. 12, the distribution of the POTF increases symmetrically, improving the resolution of the sample SA in the direction perpendicular to the optical axis. On the other hand, if a linear function intensity transmission mask (for example, a second modulation element 77 whose light transmittance monotonically increases or decreases in one direction in a plane conjugate to the pupil according to a linear function) is placed at a position conjugate to the detection pupil in the non-confocal laser microscope (second microscope unit 50), |Pcol(f)| 2 is a linear function, so the effective pupil function of the detection system is also a linear function. Therefore, in the example shown in FIG. 12, the POTF distribution increases symmetrically in the vertical direction, improving the resolution of the sample SA in the direction perpendicular to the optical axis. By orthogonally arranging the Köhler illumination microscope and the non-confocal laser microscope, the resolution can be improved in two directions (x and z directions in the example shown in FIG. 12), and an accurate three-dimensional image can be formed of the sample SA, which is a transparent phase object. Therefore, the three-dimensional refractive index distribution of the sample SA can be more accurately determined by calculation based on the light detection signals from the Köhler illumination microscope (first microscope unit 10) and the light detection signals from the non-confocal laser microscope (second microscope unit 50).

[0081] Note that the Koehler 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.

[0082]

number

[0083] Here, NA_1 is the NA (numerical aperture) of the Köhler illumination microscope (first microscope unit 10). NA_2 is the NA (numerical aperture) of the non-confocal laser microscope (second microscope unit 50). n_1 is the refractive index of the immersion liquid in the sample SA in the Köhler illumination microscope (first microscope unit 10). n_2 is the refractive index of the immersion liquid in the sample SA in the non-confocal laser microscope (second microscope unit 50). By satisfying equation (12), the missing cone region in the POTF is completely filled in, making it possible to more accurately determine the three-dimensional refractive index distribution in the sample SA.

[0084] Additionally, it is ideal to arrange the first microscope unit 10 and the second microscope unit 50 so that they are perpendicular to each other, but this is not a limitation. Even if the first microscope unit 10 and the second microscope unit 50 are arranged at different angles, it is possible to obtain the three-dimensional refractive index distribution of the sample SA more accurately than when only the first microscope unit 10 is used.

[0085] By vertically arranging the first microscope unit 10, which is a Köhler illumination microscope, and horizontally arranging the second microscope unit 50, which is a non-confocal laser microscope, the configuration of a conventional Köhler illumination microscope can be utilized, thereby increasing the practicality of the microscope device. However, the combination of the first microscope unit and the second microscope unit is not limited to a combination of a Köhler illumination microscope and a non-confocal laser microscope. For example, even when two Köhler illumination microscopes or two non-confocal laser microscopes are combined, the three-dimensional refractive index distribution of the sample SA can be determined more accurately in the same manner.

[0086] Ideally, the optical transmittance of the first modulation element 25 and the second modulation element 77 monotonically increases or decreases in one direction within the pupil plane or a plane conjugate to the pupil according to a linear function, but this is not limiting. The optical transmittance of one of the first modulation element 25 and the second modulation element 77 may also monotonically increase or decrease in one direction within the pupil plane or a plane conjugate to the pupil according to a linear function. As described above, the optical transmittance of at least one of the first modulation element 25 and the second modulation element 77 may also monotonically increase or decrease in one direction within the pupil plane or a plane conjugate to the pupil according to a continuous function, which may be any of a sine function, a cosine function, a quadratic function, and a Gaussian function. Even with this configuration, the three-dimensional refractive index distribution of the sample SA can be determined more accurately.

[0087] As described above, it is possible to select one of a plurality of first modulation elements 25 having different light transmittances, i.e., different light transmittance distributions, as the first modulation element 25 and place it at the illumination pupil position P1. In this case, a turret (not shown) holding the plurality of first modulation elements 25 may be provided, and the first modulation element 25 to be placed at the illumination pupil position P1 may be selected by rotating the turret. Note that the element selector that can select one of the plurality of first modulation elements 25 and place it at the illumination pupil position P1 is not limited to a turret, and an existing mechanism such as a slider may also be used. In this way, the control unit 90 controls the element selector to switch to one of the plurality of first modulation elements 25 and place it at the illumination pupil position P1, thereby changing the light transmittance distribution within the illumination pupil plane.

[0088] Furthermore, 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 placed at position P2 conjugate with the detection pupil. In this case, a turret (not shown) holding the plurality of second modulation elements 77 may be provided, and the second modulation element 77 to be placed at position P2 conjugate with the detection pupil may be selected by rotating the turret. Note that a means for selecting any one of the plurality of second modulation elements 77 and placing it at position P2 conjugate with the detection pupil (an element selection unit) similar to the means for selecting any one of the plurality of first modulation elements 25 and placing it at position P1 of the illumination pupil can be used. In this way, the control unit 90 controls the element selection unit to switch to any one of the plurality of second modulation elements 77 and place it at position P2 conjugate with the detection pupil, thereby changing the distribution of light transmittance in the plane conjugate with the detection pupil.

[0089] Next, a method for generating refractive index data in the microscope apparatus 1 according to the first embodiment will be described. FIG. 14 is a flowchart showing the data generating method according to the first embodiment. 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 in accordance with this control program.

[0090] 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 light detection signal (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 perpendicular to the first direction (step ST4). Next, the second detection optical system 75 receives the 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 light detection signal (step ST6). The processes of steps ST1 to ST6 are repeated to detect light from each cross-section of the sample SA corresponding to images of multiple cross-sections of the sample SA at different positions in the z direction (positions in the optical axis direction), i.e., z-stack images of the sample SA. For example, in steps ST1 to ST3, the stage 2 (sample SA) may be moved in the z direction by a stage driving unit (not shown), thereby detecting light from each cross-section of the sample SA by the first microscope unit 10 (first detector 40). 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, thereby detecting light from each cross-section of the sample SA by the second microscope unit 50 (second detector 80). Then, the image processing unit 91 generates a three-dimensional refractive index distribution in the sample SA (e.g., image data of the three-dimensional refractive index distribution of the sample SA) based on the light detection signals output from the first detector 40 and the light detection signals output from the second detector 80 (step ST7). In this case, for example, the image processing unit 91 may use the above formulas (10) and (11) to calculate the three-dimensional refractive index distribution of the sample SA based on the intensity of the image of the sample SA obtained by merging 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. This makes it possible to more accurately determine the three-dimensional refractive index distribution of the sample SA.

[0091] 14, the steps of irradiating the sample SA with first illumination light directed in a first direction and detecting light from the sample SA in response to the irradiation of the first illumination light (steps ST1 to ST3) by the first microscope unit 10 and detecting light from the sample SA in response to the irradiation of the first illumination light (steps ST4 to ST6) by the second microscope unit 50 are performed in this order, but this is not limiting. For example, the steps of the first microscope unit 10 (steps ST1 to ST3) and the steps of the second microscope unit 50 (steps ST4 to ST6) may be performed simultaneously. This allows the three-dimensional refractive index distribution of the sample SA to be determined in a short time.

[0092] In the first embodiment described above, the image processing unit 91 performs three-dimensional deconvolution for each cross section of the sample SA corresponding to the z-stack image of the sample SA based on the light detection signals detected by the first detector 40 and the light detection signals detected by the second detector 80 to obtain a three-dimensional refractive index distribution in the sample SA (e.g., image data of the three-dimensional refractive index distribution in the sample SA), but this is not limiting. For example, the image processing unit 91 may perform two-dimensional deconvolution (e.g., two-dimensional deconvolution based on the POTF at the cross section where Fz(Fx)=0 in the three-dimensional POTF distribution illustrated in FIG. 12) for each cross section of the sample SA corresponding to the z-stack image of the sample SA based on the light detection signals detected by the first detector 40 and the light detection signals detected by the second detector 80 to obtain a refractive index distribution in each cross section of the sample SA, thereby obtaining a three-dimensional refractive index distribution in the sample SA.

[0093] In the first embodiment described above, the light source unit 51 of the second microscope section 50 includes the first galvanometer mirror 54 and the second galvanometer mirror 57, but this 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 section 50a may include only one galvanometer mirror 64. In this case, a mirror 67 is disposed in the light source unit 51a 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 driving unit (not shown), thereby changing the focusing position of the second illumination light on the sample SA in the z direction. Furthermore, the galvanometer mirror 64 has the same configuration as the first galvanometer mirror 54, and changes the traveling direction of the second illumination light to change the focusing 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 focusing position of the second illumination light on the sample SA in three dimensions, thereby scanning the sample SA three-dimensionally. In this case, by moving the stage 2 (sample SA) in the z direction using the stage drive unit, the first microscope unit 10 (first detector 40) can detect light from each cross section of the sample SA, and simultaneously, the galvanometer mirror 64 and the liquid lens 59 can scan the sample SA, and the second microscope unit 50a (second detector 80) can detect light from each cross section of the sample SA. This allows the steps (steps ST1 to ST3) by the first microscope unit 10 and the steps (steps ST4 to ST6) by the second microscope unit 50a to be performed simultaneously.

[0094] Second Embodiment Next, a microscope apparatus 101 according to a second embodiment will be described with reference to FIG. 16. The microscope apparatus 101 according to the second embodiment has the same configuration as 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 descriptions thereof will be omitted. The microscope apparatus 101 according to the second embodiment has a first microscope unit 110 and a second microscope unit 150. Furthermore, the microscope apparatus 101 according to the second embodiment has a stage 2, a control unit 90, and an image processing unit 91.

[0095] The first microscope unit 110 has 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 has, 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. Furthermore, the first detection optical system 130 includes a 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.

[0096] The second microscope section 150 has 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.

[0097] The second illumination optical system 170 focuses and irradiates the second illumination light L2 emitted from the light source unit 51 (second light source 52) onto the sample SA in the -x direction (a second direction perpendicular to the first direction). As shown in FIGS. 16 and 17, the second illumination optical system 170 has a half mirror 172 and an illumination mirror 174. Furthermore, the second illumination optical system 170 includes the objective lens unit 31 (first objective lens 32) of the first microscope section 110 (first detection optical system 130).

[0098] 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 transmittance to reflectance of the half mirror 172 is set to, for example, 1:1. The half mirror 172 reflects a portion of the second illumination light L2 emitted from the light source unit 51 (second light source 52) toward the detection pupil (back focal plane) of the first objective lens 32. The first objective lens 32 collects the second illumination light L2 reflected by the half mirror 172. Note that the second illumination light L2 reflected by the half mirror 172 passes through the detection pupil (back focal plane) of the first objective lens 32 from a direction inclined with respect to the central axis of the first objective lens 32, and therefore, after passing through the first objective lens 32, it travels in the +z direction, offset from the central axis of the first objective lens 32. The illumination mirror 174 is disposed above the first objective lens 32 (and the stage 2) and faces the right side of the sample SA. The illumination mirror 174 reflects, toward the −x direction (second direction), the second illumination light L2 that passes through the first objective lens 32 and travels in the +z direction offset from the central axis of the first objective lens 32. An optical axis AX2 between the second illumination optical system 170 and the second detection optical system 175 in the second microscope unit 150 is perpendicular to an optical axis AX1 between the first illumination optical system 20 and the first detection optical system 130 in the first microscope unit 110.

[0099] The second detection optical system 175 receives light from the sample SA in response to the irradiation of the second illumination light from the side opposite the second illumination optical system 170 across the sample SA. As shown in FIGS. 16 and 17 , the second detection optical system 175 has, in order from the sample SA side, a second detection objective lens 176 and a second modulation element 177. The second detection objective lens 176 is provided on the side opposite the illumination mirror 174 across the sample SA. Light from the sample SA in response to the irradiation of the second illumination light is incident on the second detection objective lens 176. The second modulation element 177 is arranged in a plane conjugate with the detection pupil of the second detection objective lens 176 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 position P2 conjugate with the detection pupil). The second modulation element 177 has a configuration similar to that of the second modulation element 77 according to the first embodiment.

[0100] In the second embodiment, when bright-field observation of the sample SA is performed, 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 (first direction), as in the first embodiment.

[0101] The first detection light transmitted 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 130. The first detection light transmitted through the first objective lens 32 is incident on the half mirror 172. A portion of the first detection light incident on the half mirror 172 is transmitted through the half mirror 172 and incident on the imaging lens 36. The first detection light transmitted 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 light (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 (first detection light) output from the first detector 40 is sent to the image processing unit 91 via the control unit 90.

[0102] The second illumination light L2 emitted from the light source unit 51 (second light source 52) of the second microscope section 150 is incident on the half mirror 172 of the second illumination optical system 170. A portion 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, and passes through the detection pupil of the first objective lens 32 from a direction tilted 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 and collected by the illumination mirror 174, and is then irradiated onto the sample SA on the stage 2. As a result, the second illumination optical system 170 collects and irradiates the second illumination light L2 emitted from the light source unit 51 (second light source 52) onto the sample SA in the −x direction (a second direction perpendicular to the first direction).

[0103] The second detection light that has passed through the sample SA from the second illumination optical system 170 is incident on the second detection objective lens 176 of the second detection optical system 175. The second detection light that has passed through the second detection objective lens 176 passes through the second modulation element 177 and is incident on the second detector 80. 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 sent to the image processing unit 91 via the control unit 90.

[0104] Furthermore, when fluorescence observation of the sample SA is performed using the second microscope section 150, a cylindrical lens (not shown) of the light source unit 51 of the second microscope section 150 is inserted into the optical path between the first lens 53 and the first galvanometer mirror 54 in the light source unit 51. Furthermore, a fluorescence filter cube 173 is inserted into the optical path between the first objective lens 32 and the imaging lens 36 in place of the half mirror 172. The fluorescence filter cube 173 has a dichroic mirror 173a, an excitation filter 173b, and an absorption filter 173c.

[0105] 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 enters the dichroic mirror 173a. The excitation light that enters the dichroic mirror 173a of the fluorescence filter cube 173 is reflected by the dichroic mirror 173a and enters the first objective lens 32, and passes through the detection pupil of the first objective lens 32 from a direction tilted with respect to the central axis of the first objective lens 32. The excitation light that passes through the first objective lens 32 is reflected by the illumination mirror 174 and collected, and then irradiates the sample SA on the stage 2. In this way, 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).

[0106] Irradiation with the excitation light excites fluorescent substances contained in the sample SA, causing them to emit fluorescence. The fluorescence from the sample SA is incident on the first objective lens 32 of the first detection optical system 130. The fluorescence that passes through the first objective lens 32 is incident on the dichroic mirror 173a of the fluorescence filter cube 173. The fluorescence that passes through the dichroic mirror 173a of the fluorescence filter cube 173 is transmitted through the dichroic mirror 173a, passes through the absorption filter 173c, and is incident on the imaging lens 36. The fluorescence that passes 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 located. 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 fluorescence detection signal output from the first detector 40 is sent to the image processing unit 91 via the control unit 90.

[0107] In the second embodiment, 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) can be generated by a method similar to the refractive index data generation method according to the first embodiment. Therefore, according to the second embodiment, it is possible to obtain the same effects as those of the first embodiment.

[0108] In the second embodiment described above, the light source unit 51 of the second microscope section 150 has the first galvanometer mirror 54 and the second galvanometer mirror 57, but this is not limited to this. For example, as in a microscope apparatus 101a shown in Fig. 18, the light source unit 51a of the second microscope section 150a may be configured to have only one galvanometer mirror 64. In this case, similar to the microscope apparatus 1a according to the modified example of the first embodiment, a mirror 67 is arranged in place of the second galvanometer mirror 57 in the light source unit 51a.

[0109] Third Embodiment Next, a microscope apparatus 201 according to a third embodiment will be described with reference to FIG. 19. The microscope apparatus 201 according to the third embodiment has a configuration in which the main parts are common to 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 will be omitted. The microscope apparatus 201 according to the third embodiment has a first microscope unit 210 and a second microscope unit 250. Furthermore, the microscope apparatus 201 according to the third embodiment has a stage 2, a control unit 90, and an image processing unit 91.

[0110] The first microscope unit 210 has 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 has, in order from the sample SA side, an objective lens unit 31, an imaging lens 36, and a polarizing beam splitter 237. The first detection optical system 230 further includes a 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 polarizing beam splitter 237 reflects light (s-polarized light) from the sample SA in response to irradiation with the first illumination light and transmits light (p-polarized light) from the sample SA in response to irradiation with the second illumination light.

[0111] The second microscope section 250 has 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.

[0112] The second illumination optical system 270 focuses and irradiates the second illumination light L2 emitted from the light source unit 51 (second light source 52) onto the sample SA in the -x direction (a second direction perpendicular to the first direction). As shown in FIGS. 19 and 20, the second illumination optical system 270 has a half mirror 272 and an illumination mirror 274. Furthermore, the second illumination optical system 270 includes the objective lens unit 31 (first objective lens 32) of the first microscope section 210 (first detection optical system 230).

[0113] 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 transmittance to reflectance of the half mirror 272 is set to, for example, 1:1. The half mirror 272 reflects a portion of the second illumination light L2 emitted from the light source unit 51 (second light source 52) toward the detection pupil (back focal plane) of the first objective lens 32. The first objective lens 32 collects the second illumination light L2 reflected by the half mirror 272. Note that the second illumination light L2 reflected by the half mirror 272 passes through the detection pupil (back focal plane) of the first objective lens 32 from a direction inclined with respect to the central axis of the first objective lens 32, and therefore, after passing through the first objective lens 32, it travels in the +z direction, offset from the central axis of the first objective lens 32. The illumination mirror 274 is disposed above the first objective lens 32 (and the stage 2) and facing the right side of the sample SA. The illumination mirror 274 reflects, toward the −x direction (second direction), the second illumination light L2 that passes through the first objective lens 32 and travels in the +z direction offset from the central axis of the first objective lens 32. An optical axis AX2 between the second illumination optical system 270 and the second detection optical system 275 in the second microscope section 250 is perpendicular to an optical axis AX1 between the first illumination optical system 20 and the first detection optical system 230 in the first microscope section 210.

[0114] The second detection optical system 275 receives light from the sample SA in response to irradiation with the second illumination light from the side opposite the second illumination optical system 270 across the sample SA. As shown in Figures 19 and 20, the second detection optical system 275 has a detection mirror 276, a relay lens 278, and a second modulation element 277. Furthermore, the second detection optical system 275 includes the objective lens unit 31 (first objective lens 32) of the first microscope section 210 (first detection optical system 230), the imaging lens 36, the polarizing beam splitter 237, and the half mirror 272 of the second illumination optical system 270.

[0115] The detection mirror 276 is provided above the first objective lens 32 (and the stage 2) on the opposite side of the sample SA from the illumination mirror 274. The illumination mirror 274 reflects light from the sample SA irradiated with the second illumination light toward the −z direction (i.e., toward the first objective lens 32) that is offset from the central axis of the first objective lens 32. The light from the sample SA irradiated with the second illumination light is incident on the first objective lens 32 via the detection mirror 276. The relay lens 278 causes the light (p-polarized light) from the sample SA that has passed through the polarizing beam splitter 237 to be incident on the second modulation element 277. The second modulation element 277 is disposed in 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 (the second detection optical system 275) at 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.

[0116] In the third embodiment, when bright-field observation of the sample SA is performed, 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 (first direction), as in the first embodiment. Note that in the third embodiment, for example, a polarizing plate (not shown) is arranged in the optical path of 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 (first detection light) is incident on the polarizing beam splitter 237.

[0117] The first detection light (s-polarized light) transmitted 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 transmitted through the first objective lens 32 is incident on the half mirror 272. A portion of the first detection light incident on the half mirror 272 is transmitted through the half mirror 272 and enters the imaging lens 36. The first detection light (s-polarized light) transmitted through the imaging lens 36 is reflected by the polarizing 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 sent to an image processing unit 91 via the control unit 90.

[0118] The second illumination light L2 emitted from the light source unit 51 (second light source 52) of the second microscope section 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, which is linearly polarized, so that p-polarized light (second detection light) is incident on the polarizing beam splitter 237. A portion of the second illumination light L2 incident on the half mirror 272 is reflected by the half mirror 272 and enters the first objective lens 32, and passes through the detection pupil of the first objective lens 32 from a direction tilted 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 274 and condensed, and is irradiated onto the sample SA on the stage 2. As a result, the second illumination optical system 270 focuses and irradiates the second illumination light L2 emitted from the light source unit 51 (second light source 52) onto the sample SA in the -x direction (a second direction perpendicular to the first direction).

[0119] 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 portion 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 polarizing 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 from the sample SA (second detection light) 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 sent to the image processing unit 91 via the control unit 90.

[0120] In the third embodiment, 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) can be generated by a method similar to the refractive index data generation method according to the first embodiment. Therefore, according to the third embodiment, it is possible to obtain the same effects as those of the first embodiment.

[0121] In the third embodiment described above, the light source unit 51 of the second microscope section 250 has the first galvanometer mirror 54 and the second galvanometer mirror 57, but this is not limited to this. For example, as in a microscope apparatus 201a shown in Fig. 21, the light source unit 51a of the second microscope section 250a may be configured to have only one galvanometer mirror 64. In this case, similar to the microscope apparatus 1a according to the modified example of the first embodiment, a mirror 67 is arranged in place of the second galvanometer mirror 57 in the light source unit 51a.

[0122] <Fourth embodiment> Next, a microscope apparatus 301 according to a 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, 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 will be omitted. As shown in FIGS. 22 and 23, the microscope apparatus 301 according to the fourth embodiment has a first microscope unit 310 and a second microscope unit 350. Furthermore, as shown in FIG. 23, the microscope apparatus 301 according to the fourth embodiment has a stage 2, a control unit 90, and an image processing unit 91. Note that 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 coordinate axes perpendicular to the z-axis are defined as the x-axis and y-axis.

[0123] As shown in FIGS. 22 and 23, the first microscope unit 310 has 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 similarly to the first light source 11 according to the first embodiment. The first light source 311 generates first illumination light. The first light source 311 is also disposed at a position conjugate with the illumination pupil.

[0124] The first illumination optical system 320 irradiates the sample SA with first illumination light L1 emitted from the first light source 311 in the +x direction (first direction). In FIG. 23, the first illumination light L1 is indicated by a dashed line to easily distinguish it 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 similarly to 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 facing the left side of the stage 2. Furthermore, when a white light source is used as the first light source 311, an element (for example, a bandpass filter) that narrows the wavelength band of the first illumination light may be provided, similarly to the first embodiment.

[0125] The first detection optical system 330 receives light from the sample SA in response to irradiation with the first illumination light L1 from the side opposite the first illumination optical system 320 across the sample SA. As shown in FIGS. 23 and 24 , the first detection optical system 330 has, 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 the condenser lens 327 across the sample SA. Light from the sample SA in response to irradiation with 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 ).

[0126] A first detector 340 is disposed on the image plane I of the first detection optical system 330. The first detector 340 is configured similarly to 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.

[0127] As shown in FIGS. 22 and 23 , the second microscope section 350 has 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 has 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. The light source unit 351 also has a cylindrical lens (not shown) that is insertable into and removable from the optical path between the first lens 353 and the first galvanometer mirror 354.

[0128] 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. The first galvanometer mirror 354 changes the traveling direction of the second illumination light, thereby changing the focusing 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 focusing position of the second illumination light on the sample SA in the x direction.

[0129] The fourth lens 358 focuses the second illumination light reflected by the second galvanometer mirror 357 at a predetermined intermediate image plane IM and makes the light 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 focusing position of the second illumination light on the sample SA in three dimensions (x, y, and z directions), thereby enabling three-dimensional scanning of the sample SA.

[0130] The second illumination optical system 360 focuses and irradiates the second illumination light L2 emitted from the light source unit 351 (second light source 352) onto the sample SA in the +z direction (a second direction perpendicular to the first direction). As shown in FIGS. 22 and 23 , the second illumination optical system 360 has, 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.

[0131] The objective lens unit 366 has a plurality of second illumination objective lenses 367, a lens holder 368, and a unit driver 369. The second illumination objective lenses 367 are disposed below the stage 2 so as to face each other. The lens holder 368 holds a plurality of second illumination objective lenses 367 with different optical characteristics. The lens holder 368 is configured using, for example, a revolver or a turret. The unit driver 369 drives the lens holder 368 to select one of the plurality of second illumination objective lenses 367 and place it below the stage 2. The unit driver 369 may also move the lens holder 368 along the z-axis. In this case, the aforementioned stage driver may be used in combination, or the stage driver may not be used.

[0132] A second illumination objective lens 367 disposed below the stage 2 focuses the second illumination light L2 that has passed through the collimator lens 362 onto the sample SA. As also shown in Fig. 24 , an optical axis AX2 between the second illumination optical system 360 and the second detection optical system 370 in the second microscope unit 350 is perpendicular to an optical axis AX1 between the first illumination optical system 320 and the first detection optical system 330 in the first microscope unit 310.

[0133] The second detection optical system 370 receives light from the sample SA in response to irradiation with the second illumination light from the side opposite the second illumination optical system 360 across the sample SA. As shown in FIGS. 22 and 23 , the second detection optical system 370 has, in order from the sample SA side, a second detection objective lens 371, an aperture stop 372, a second modulation element 373, a condenser lens 374, a mirror 376, and a relay lens 377. The second detection objective lens 371 is provided on the side of the sample SA opposite the second illumination objective lens 367. As the second detection objective lens 371, any one of a plurality of second detection objective lenses 371 with different optical properties can be selected and placed above the stage 2. Light from the sample SA in response to irradiation with the second illumination light is incident on the second detection objective lens 371.

[0134] The aperture stop 372 and the second modulation element 373 are arranged on a plane conjugate with the detection pupil of the second detection objective lens 371 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 position P2A conjugate with the detection pupil). The second modulation element 373 is arranged adjacent to the aperture stop 372 (for example, above the aperture stop 372 as shown in FIG. 23). The second modulation element 373 has a configuration similar to that of the second modulation element 77 according to the first embodiment. The condenser lens 374 condenses light that has passed through the aperture stop 372 and the second modulation element 373. The mirror 376 reflects the light that has passed through the condenser lens 374 towards the relay lens 377. The relay lens 377 causes the light reflected by the mirror 376 to enter the second detector 380.

[0135] The second detector 380 is disposed at a position P2B conjugate with 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.

[0136] In the fourth embodiment, when bright-field observation of the sample SA is performed, 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 transmitted 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 transmitted through the relay lens 324 passes through a first modulation element 325 and an aperture stop (not shown), and is incident on the condenser lens 327. The first illumination light L1 transmitted through the condenser lens 327 becomes parallel light and is irradiated onto the sample SA on the stage 2. As a result, 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 (first direction).

[0137] The first detection light that has passed through the sample SA from the direction of 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 is imaged on a predetermined image plane I where the first detector 340 is disposed. The first detector 340 detects the light (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 (first detection light) output from the first detector 340 is sent to the image processing unit 91 via the control unit 90.

[0138] When bright-field observation of the sample SA is performed, the cylindrical lens (not shown) of the light source unit 351 of the second microscope section 350 is retracted from the optical path between the first lens 353 and the first galvanometer mirror 354 of 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 transmitted 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 is transmitted 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 is transmitted through the fourth lens 358 and is emitted to the outside of the light source unit 351. This causes the light source unit 351 to emit the second illumination light.

[0139] The second illumination light L2 emitted from the light source unit 351 (second light source 352) is reflected by a mirror 361 of the second illumination optical system 360 and enters a collimator lens 362. The second illumination light L2 that has passed through the collimator lens 362 becomes parallel light and enters a second illumination objective lens 367. The second illumination light L2 that has passed through the second illumination objective lens 367 is collected and irradiated onto the sample SA on the stage 2. As a result, the second illumination optical system 360 collects and irradiates the second illumination light L2 emitted from the light source unit 351 (second light source 352) onto the sample SA in the +z direction (a second direction perpendicular to the first direction).

[0140] The second detection light transmitted through the sample SA from the second illumination optical system 360 is incident on a second detection objective lens 371 of the second detection optical system 370. The second detection light transmitted through the second detection objective lens 371 passes through an aperture stop 372 and a second modulation element 373 and is incident on a condenser lens 374. The second detection light transmitted through the condenser lens 374 is reflected by a mirror 376 and is incident on a relay lens 377. The second detection light transmitted through the relay lens 377 is incident on a second detector 380. The second detector 380 detects the light from the sample SA (second detection light) via the second detection optical system 370 and outputs a detection signal of the light. The detection signal of the light (second detection light) output from the second detector 380 is transmitted to an image processing unit 91 via a control unit 90.

[0141] Furthermore, when fluorescent observation of the sample SA is performed using the second microscope section 350, the cylindrical lens (not shown) of the light source unit 351 of the second microscope section 350 is inserted into the optical path between the first lens 353 and the first galvanometer mirror 354 in the light source unit 351.

[0142] The excitation light emitted from the light source unit 351 (second light source 352) is reflected by a mirror 361 of the second illumination optical system 360 and enters a collimator lens 362. The excitation light that has passed through the collimator lens 362 becomes parallel light and enters a second illumination objective lens 367. The excitation light that has passed through the second illumination objective lens 367 is collected and irradiated onto the sample SA on the stage 2.

[0143] Irradiation with the excitation light excites fluorescent substances contained in the sample SA, causing them to emit fluorescence. The fluorescence from the sample SA is incident on 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 is incident on the imaging lens 336. The fluorescence that has passed through the imaging lens 336 is imaged on a predetermined image plane I where the first detector 340 is located. 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 fluorescence detection signal output from the first detector 340 is sent to the image processing unit 91 via the control unit 90.

[0144] In the fourth embodiment, 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) can be generated by a method similar to the refractive index data generation method according to the first embodiment. Therefore, according to the fourth embodiment, it is possible to obtain the same effects as those of the first embodiment.

[0145] Fifth Embodiment Next, a microscope apparatus 401 according to a fifth embodiment will be briefly described using FIG. 25. The microscope apparatus 401 according to the fifth embodiment has the same configuration as 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 will be omitted. The microscope apparatus 401 according to the fifth embodiment has a first microscope unit 410 and a second microscope unit 450. Furthermore, although not shown, the microscope apparatus 401 according to the fifth embodiment has a stage, a control unit, and an image processing unit. The stage, control unit, and image processing unit are configured similarly to the stage 2, control unit 90, and image processing unit 91 according to the first embodiment.

[0146] The first microscope unit 410 has 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 at a predetermined image plane IA.

[0147] The second microscope unit 450 has 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 similarly to the first light source 311 according to the fourth embodiment. The second illumination optical system 460 is configured similarly to the first illumination optical system 320 according to the fourth embodiment. The second modulation element 465 of the second illumination optical system 460 is configured similarly to the first modulation element 325 according to the fourth embodiment, and is disposed at the illumination pupil position P1B. The second detection optical system 470 is configured similarly to the first detection optical system 330 according to the fourth embodiment. The second detector 480 is configured similarly to the first detector 340 according to the fourth embodiment, and is disposed at a predetermined image plane IB.

[0148] In the fifth embodiment, 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) can be generated by a method similar to the refractive index data generation method according to the first embodiment. Therefore, according to the fifth embodiment, it is possible to obtain the same effects as those of the first embodiment.

[0149] Sixth Embodiment Next, a microscope apparatus 501 according to a sixth embodiment will be briefly described with reference to FIG. 26. The microscope apparatus 501 according to the sixth embodiment has the same configuration as the microscope apparatus 1 according to the first embodiment, except for the first 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 descriptions thereof will be omitted. The microscope apparatus 501 according to the sixth embodiment has a first microscope unit 510 and a second microscope unit 550. Furthermore, although not shown, the microscope apparatus 501 according to the sixth embodiment has a stage, a control unit, and an image processing unit. The stage, control unit, and image processing unit are configured similarly to the stage 2, control unit 90, and image processing unit 91 according to the first embodiment.

[0150] The first microscope section 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 similarly to the second light source unit 351 according to the fourth embodiment. The first illumination optical system 520 is configured similarly to the second illumination optical system 360 according to the fourth embodiment. The first detection optical system 530 is configured similarly to the second detection optical system 370 according to the fourth embodiment. The first modulation element 533 of the first detection optical system 530 is configured similarly to 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 similarly to the second detector 380 according to the fourth embodiment and is disposed at a position P2B conjugate with the detection pupil.

[0151] The second microscope section 550 has 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 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.

[0152] In the sixth embodiment, 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) can be generated by a method similar to the refractive index data generation method according to the first embodiment. Therefore, according to the sixth embodiment, it is possible to obtain the same effects as those of the first embodiment.

[0153] <Modification> In the first to third embodiments described above, the first detection optical system 30, 130, 230 of the first microscope unit 10, 110, 210 is provided separately from the first illumination optical system 20, but this is not limiting and the first microscope unit 610 may include part of the first illumination optical system. For example, as shown in Fig. 27, a first microscope unit 610 according to a modified example has 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.

[0154] The first illumination optical system 620 has, 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 (e.g., a bandpass filter) that narrows the wavelength band of the first illumination light may be provided, 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 has a configuration similar to that of the first modulation element 25 according to the first embodiment.

[0155] The half mirror 626 reflects a portion of the first illumination light from the first light source 11 toward the stage 2. The half mirror 626 transmits a portion of the light (first detection light) that has passed through the sample SA on the stage 2 toward an imaging lens 636 of the detection optical system 630. The ratio of transmittance to 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 holder 633, and a unit driver 634. The first objective lenses 632 are disposed below the stage 2 so as to face the stage 2. The lens holder 633 holds a plurality of first objective lenses 632 with different focal lengths. The lens holder 633 is configured using, for example, a revolver or a turret. The unit driver 634 can drive the lens holder 633 to select one of the plurality of first objective lenses 632 and place it below the stage 2. The illumination mirror 628 is disposed above the stage 2 so as to face the stage 2.

[0156] The first detection optical system 630 includes an objective lens unit 631 and a half mirror 626. Furthermore, the first detection optical system 630 has, in this 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.

[0157] The first illumination light emitted from the first light source 11 of the first microscope unit 610 is incident on a 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 is incident on a first relay lens 622. The first illumination light that has passed through the first relay lens 622 passes through a first modulation element 623 and is incident on a second relay lens 624. The first illumination light that has passed through the second relay lens 624 passes through a condenser lens 625 and is incident on a half mirror 626. A portion of the first illumination light that has passed through the half mirror 626 is reflected by the half mirror 626 and is incident on a 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 an illumination mirror 628. The first illumination light that has reflected by the illumination mirror 628 is irradiated onto the sample SA on the stage 2. As a result, 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 (first direction).

[0158] The first detection light reflected by the illumination mirror 628 and transmitted through the sample SA is incident on a first objective lens 632 serving as a first detection optical system 630. The first detection light transmitted through the first objective lens 632 is incident on a half mirror 626. A portion of the first detection light incident on the half mirror 626 is transmitted through the half mirror 626 and is incident on an imaging lens 636. The first detection light transmitted through the imaging lens 636 is reflected by a mirror 637 and forms an image on a predetermined image plane I where the first detector 40 is disposed. The first detector 40 detects light from the sample SA (first detection light) via the first detection optical system 630 and outputs a detection signal of the light.

[0159] In each of the above-described embodiments, the image processing unit 91 calculates the three-dimensional refractive index distribution of the sample SA based on the detection signal of light detected under one detection condition for light transmittance. However, this is not limiting. The image processing unit 91 may calculate the three-dimensional refractive index distribution of the sample SA based on the detection signal of light detected under multiple detection conditions for light transmittance. For example, the image processing unit 91 calculates the linear sum or difference of the POTF based on the detection signal of light detected under two detection conditions set by the user or the like. This makes it possible to obtain a high absolute value of the POTF over a wider frequency band than when the detection signal of light is detected under a single detection condition set by the user or the like. Therefore, it is possible to generate an image of the three-dimensional refractive index distribution of the sample SA with high contrast and resolution. The image processing unit 91 can calculate the three-dimensional refractive index distribution n(x, y, z) of the sample SA using the above equations (10) and (11), which include the POTF.

[0160] For example, in the microscope apparatus according to the first to fourth embodiments, under the first detection condition, a first modulation element (a first modulation element with a unidirectional change) whose light transmittance varies according to a linear function in the example shown in FIG. 4 is placed at the position of the illumination pupil. Under the second detection condition, a first modulation element (a first modulation element with a reversed change) whose light transmittance varies according to a linear function in the opposite direction to the example shown in FIG. 4 (the first detection condition) is placed at the position of the illumination pupil. In this modification, the difference between the POTF based on the detection signal of light detected under the first detection condition and the POTF based on the detection signal of light detected under the second detection condition is calculated. Since the POTF value under the first detection condition and the POTF value under the second detection condition have opposite signs, the width of the frequency band where the absolute value of the POTF does not become zero does not change significantly, but the absolute value of the POTF increases. Therefore, a three-dimensional refractive index distribution image of the sample SA with excellent contrast can be generated.

[0161] In the above-described embodiments, the first and second modulation elements are exemplified as elements whose light transmittance varies within the plane of the flat plate, and are formed by evaporating a film capable of reducing light transmittance onto a parallel flat plate such as a glass substrate. However, this is not limiting. For example, at least one of the first and second modulation elements may be a parallel flat plate such as a glass substrate on which a fine dot pattern capable of reducing light transmittance (having light-blocking properties) is formed. In this case, the light transmittance can be changed by forming a dot pattern with different densities on the parallel flat plate (glass substrate) using an existing lithography process or the like (the dense dot pattern area has lower transmittance than the sparse dot pattern area). At least one of the first and second modulation elements is not limited to the optical elements described above, and may be formed using an SLM (spatial light modulator) such as a transmissive liquid crystal element, a reflective liquid crystal element, or a DMD (digital mirror device). When an SLM is used, the SLM is positioned on the pupil (at least one of the illumination pupil and the detection pupil) or at a position conjugate to the pupil, as with the optical elements in the above-described embodiments. For example, if a transmissive liquid crystal element is used as the SLM, the desired light transmittance distribution can be set by controlling the transmittance of each pixel of the element.Also, if a DMD is used as the SLM, the desired light transmittance distribution can be set by controlling the angle of each mirror.

[0162] When the optical element (i.e., a light-transmitting flat plate) in each of the above-described embodiments is used as the first modulation element, the control unit 90 may change the light transmittance distribution in the plane of the illumination pupil by controlling the element selection unit to switch to one of the multiple first modulation elements and place it at the position of the illumination pupil. When the optical element (i.e., a light-transmitting flat plate) in each of the above-described embodiments is used as the second modulation element, the control unit 90 may change the light transmittance distribution in the plane conjugate to the detection pupil by controlling the element selection unit to switch to one of the multiple second modulation elements and place it at a position conjugate to the detection pupil. When an SLM is used as the first modulation element and the second modulation element, the control unit 90 controls the SLM to change the light transmittance distribution in the plane of the pupil or in a plane conjugate to the pupil. Therefore, there is no need to provide multiple elements or an element selection unit in order for the control unit 90 to change the light transmittance distribution.

[0163] In each of the above-described embodiments, for convenience of explanation, a "lens" such as the collector lens 21 is depicted as a single lens in each drawing, but this is not limited to this. For example, a "lens" such as the collector lens 21 may be composed of multiple lenses, or may be a combination of a lens and an existing optical element other than a lens. [Explanation of symbols]

[0164] 1 Microscope device (first embodiment) 2 Stage 10 First microscope section 50 Second microscope section 90 Control Unit 91 Image processing section (data processing section) 101 Microscope device (second embodiment) 110 First microscope section 150 Second microscope section 201 Microscope device (third embodiment) 210 First microscope section 250 Second microscope section 301 Microscope device (fourth embodiment) 310 First Microscope Section 350 Second Microscope Section 401 Microscope device (fifth embodiment) 410 First Microscope Section 450 Second Microscope Section 501 Microscope device (sixth embodiment) 510 First Microscope Section 550 Second Microscope Section

Claims

1. an illumination optical system including an illumination objective lens, which scans and illuminates a specimen with illumination light from a light source via a scanning unit within a plane intersecting an optical axis direction of the illumination objective lens; a detection optical system including a detection objective lens different from the illumination objective lens, and guiding light transmitted through the specimen to a detection unit; an image processing unit that generates an image based on a signal from the detection unit; Equipped with the detection optical system has an intensity modulation element that is disposed at a pupil of the detection objective lens or at a conjugate position thereof, and whose transmittance of the light changes within a plane of the pupil or within a plane of the conjugate position; The detection unit is disposed at a position conjugate with the pupil of the detection objective lens.

2. Further comprising a control unit, the control unit controls or inserts or removes the intensity modulation element to switch it with another intensity modulation element, thereby changing the state of transmittance of the light, so that the light guided by the detection optical system to the detection unit is in a different state from each other; The microscope according to claim 1 , wherein the detection unit receives the light in the different states.

3. 3. The microscope according to claim 1, wherein the transmittance of the intensity-modulating element varies according to a continuous function.

4. 4. The microscope according to claim 3, wherein the light transmittance of the intensity modulation element varies along one direction in the plane of the pupil or in the plane of a pre-conjugate position according to the continuous function.

5. 4. The microscope according to claim 3, wherein the light transmittance of the intensity modulation element monotonically increases or decreases in one direction in the plane of the pupil or the plane of the conjugate position according to the continuous function, and becomes zero at a portion of the outer periphery in the plane of the pupil or the plane of the conjugate position.

6. 4. The microscope according to claim 3, wherein the continuous function is any one of a linear function, a quadratic function, a Gaussian function, a sine function with a range smaller than one period, and a cosine function with a range smaller than one period.

7. 3. The microscope according to claim 2, wherein the change in the state of the light transmittance is a change from a state in which the transmittance monotonically increases in one direction according to a linear function to a state in which the transmittance monotonically decreases in the one direction according to a linear function.

8. The microscope according to claim 7 , wherein the image processing unit generates a differential image based on a plurality of images formed by a plurality of signals output from the detection unit.

9. 2. The microscope according to claim 1, wherein the front-illumination objective lens and the detection objective lens are arranged in a positional relationship facing each other across a stage on which the specimen is placed.

10. 2. The microscope according to claim 1, wherein the illumination objective lens and the detection objective lens are disposed in a positional relationship facing each other with respect to the specimen without a stage on which the specimen is placed.

11. 11. The microscope according to claim 1, wherein the numerical aperture of the illumination objective lens is the same as the numerical aperture of the detection objective lens.

12. The microscope according to claim 8 , wherein the image processing unit generates a three-dimensional refractive index distribution as the image based on the differential image.

Images