Reflective bright-field microscope, observation method, and program

Abstract

A reflective bright-field microscope 100 according to the present embodiment comprises: an illumination optical system 10, which has an objective lens 31 and an aperture pattern turret 16 capable of forming a plurality of annular illumination beams 10a having mutually different annular zone radii and which illuminates a sample S with the illumination beams 10a; a detection optical system 30, which focuses first reflected light from the sample S and second reflected light from an interface around the sample S to an imaging device 35, through the objective lens 31; and a control unit 50. Using each of the plurality of annular illumination beams 10ai formed by control of the aperture pattern turret 16 by the control unit 50, the imaging device 35 detects the first reflected light and the second reflected light at each of a plurality of positions for which the relative positions of the objective lens 31 and the sample S are different.

Description

Technical Field

[0001] The present invention relates to a reflection bright-field microscope, an observation method, and a program.

Background Art

[0002] Conventionally, a bright-field microscope is an optical device that magnifies and observes an illuminated sample using an objective lens, and has attracted attention as a quantitative phase microscope due to the technological development of two-dimensional detectors in recent years (see, for example, Non-Patent Document 1). The bright-field microscope is used not only for absorption objects but also for phase objects. In a reflection bright-field microscope, when a sample is illuminated by normal illumination such as Köhler illumination, reflected diffracted light from the sample that reflects the structure of the sample (hereinafter referred to as reflected light from the sample) interferes with reflected light from the interface around the sample that does not reflect the structure of the sample, for example, reflected light from a cover glass that supports the sample, and an object image is formed. However, since the phase of the reflected light from the cover glass shifts when the cover glass is driven in the optical axis direction together with the sample, pseudo-resolution occurs in the three-dimensional object image of the sample. Non-Patent Document 1 NATURE COMMUNICATIONS (2019) 10:4691 General Disclosure

[0003] (Item 1) The reflection bright-field microscope may include a first member capable of forming a plurality of annular illumination lights having different annular radii and an objective lens, and an illumination optical system that irradiates the sample with the illumination light. The reflection bright-field microscope may include a detection optical system that condenses the first reflected light from the sample and the second reflected light from the interface around the sample onto a detection unit via the objective lens. The reflection bright-field microscope may include a control unit. Using each of the plurality of annular illumination lights formed by the control unit controlling the first member, the detection unit may detect the first reflected light and the second reflected light at each of a plurality of positions where the relative position between the objective lens and the sample is different. (Item 2) The interface may be an interface between the sample and a second member in contact with the sample. (Item 3) The reflection bright-field microscope may include a processing unit that processes a plurality of detection results by the detection unit using parameters related to the plurality of annular illumination lights to generate a three-dimensional object image of the sample. (Item 4) The processing unit may generate a plurality of image frequencies in the frequency space from the plurality of detection results, process the plurality of image frequencies using the values of the parameters, and synthesize the resulting new plurality of image frequencies to generate a three-dimensional object image of the sample. (Item 5) The annular radius of the annular illumination pupil on the frequency plane corresponding to the two-dimensional plane orthogonal to the optical axis direction of the illumination optical system is defined by (NA ill / λ)(i - 1) / (M - 1), where M is the number of the annular illumination pupils, i is any one of 1 to M, NA ill is the numerical aperture of the illumination optical system, and λ is the wavelength of the illumination light. (Item 6) The processing unit: i) calculates each three-dimensional aperture B i (f) by shifting a three-dimensional aperture A i (f) determined from each annular illumination pupil of the illumination optical system and the imaging pupil of the detection optical system in a predetermined direction by the value of the parameter, where i is any one of 1 to M and M is the number of the annular illumination pupils; ii) calculates a positive-valued function using each three-dimensional aperture B i (f); and iii) may extract a region of the positive-valued function from the plurality of image frequencies calculated from the plurality of detection results. (Item 7) The processing unit may calculate the new plurality of image frequencies by shifting the extracted region in the reverse direction of the predetermined direction by the value of the parameter. (Item 8) The processing unit may synthesize the new plurality of image frequencies as a phase object or an absorption object. (Item 9) The processing unit may complement the plurality of image frequencies on the frequency axis corresponding to the optical axis direction of the objective lens using the plurality of image frequencies outside the frequency axis. (Item 10) The first member may be a spatial light modulator, an LED light source array, or a member on which a plurality of annular opening patterns having different annular radii are arranged.

[0004] (Item 11) The observation method may include a step of irradiating the sample with the illumination light through an illumination optical system having a first member capable of forming a plurality of annular illumination lights having different annular radii and an objective lens. The observation method may include a step of condensing, by the detection unit, the first reflected light from the sample and the second reflected light from the interface around the sample through the objective lens. The observation method may include a step of detecting, by the detection unit, the first reflected light and the second reflected light at each of a plurality of positions where the relative positions of the objective lens and the sample are different, using each of the plurality of annular illumination lights formed by controlling the first member.

[0005] (Item 12) The program may cause a computer to execute a procedure of irradiating a sample with the illumination light through an illumination optical system having a first member capable of forming a plurality of annular illumination lights having different annular radii and an objective lens. The program may cause a computer to execute a procedure of condensing, by the detection unit, the first reflected light from the sample and the second reflected light from the interface around the sample through the objective lens. The program may cause a computer to execute a procedure of detecting, by the detection unit, the first reflected light and the second reflected light at each of a plurality of positions where the relative positions of the objective lens and the sample are different, using each of the plurality of annular illumination lights formed by controlling the first member.

[0006] Note that the above summary of the invention does not list all the features of the present invention. Also, sub-combinations of these feature groups can also be inventions.

Brief Description of the Drawings

[0007]

Figure 1A

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Mode for Carrying Out the Invention

[0008] Hereinafter, the present invention will be described through embodiments of the invention. However, the following embodiments do not limit the invention according to the claims. Also, not all combinations of the features described in the embodiments are essential for the solution means of the invention.

[0009] Fig. 1A shows a schematic configuration of a reflection-type bright-field microscope (simply referred to as a microscope unless otherwise specified) 100 according to this embodiment. Fig. 1B shows that by illuminating the sample S, reflected light γ from the sample S and reflected light γ from the interface 22a of the periphery of the sample S (as an example, a cover glass 22) r are generated. The microscope 100 illuminates the sample S with illumination light 10a, and together with the reflected light γ from the sample S that reflects the structure of the sample S, reflected light γ from the interface of the periphery of the sample S that does not reflect the structure of the sample S, for example, the interface 22a of the cover glass 22 that supports the sample S rAn apparatus that receives them and generates a three-dimensional object image of the sample S in real space by detecting their interference, comprising an illumination optical system 10, a drive unit 20, a detection optical system 30, and a processing unit 40. Here, let the optical axis 10L of a part of the illumination optical system 10 and the optical axis 30L of the detection optical system 30 (objective lens). The sample S placed in the container 23 or on a slide glass (not shown) is supported by a support member such as a cover glass 22 and held on the stage 21.

[0010] The sample S is, for example, a cell section, a cell spheroid, an organoid, etc. A cell spheroid is a three-dimensional mass of three-dimensionally cultured cells, and an organoid is a small and simplified collection of cells having a part of the characteristics of an organ. An organoid can be produced three-dimensionally in vitro, for example, by using pluripotent stem cells such as iPS cells and ES cells as raw materials and differentiating the cells while controlling the cell culture conditions. In addition, the sample S also includes a two-dimensionally extended cell layer laminated over two or more layers (for example, a cell sheet). The cell sheet may be a single layer or a plurality of laminated layers.

[0011] The illumination optical system 10 is an optical system that generates a plurality of annular illumination lights 10a having different annular radii and irradiates the sample S with the illumination light 10a, and includes a light source 11, a collector lens 12, a field stop 13, a condenser lens 14, an aperture stop 15, an aperture pattern turret 16, a beam splitter 32 (for example, a half mirror), and an objective lens 31 arranged in order on the optical axis 10L.

[0012] The light source 11 generates, for example, incoherent illumination light 10a as the illumination light 10a. As the light source 11, an incoherent surface light source such as a halogen lamp or an LED is desirable.

[0013] The collector lens 12 is a lens element that shapes the illumination light 10a generated from each point of the light source 11 into parallel light.

[0014] The field stop 13 is an element that restricts the illumination light 10a and limits it to the observation range of the sample S.

[0015] The condenser lens 14 is a lens element that condenses the illumination light 10a passing through the field stop 13.

[0016] The aperture stop 15 is an element that restricts the illumination light 10a emitted from the condenser lens 14 and adjusts the numerical aperture of the illumination optical system 10. By adjusting the aperture stop 15, the brightness of the field of view can be changed. In the present embodiment, an aperture pattern turret 16 is disposed in the vicinity of the aperture stop 15.

[0017] FIG. 1C shows a schematic configuration of the aperture pattern turret 16. The aperture pattern turret 16 is configured such that a plurality of elements can be mounted thereon, and a plurality of elements (here, five elements 16a, 16b, 16c, 16d, 16e as an example) having annular aperture patterns (shown in white) with different annulus radii are mounted. The control unit 50 rotates the aperture pattern turret 16 to sequentially arrange elements having annular aperture patterns with different annulus radii on the optical axis 10L, and passes the illumination light 10a through the aperture pattern, thereby forming a plurality of annular illumination lights (i.e., annular illumination) 10a having different annulus radii. i to be shaped.

[0018] When the illumination light 10a is emitted from the light source 11, the illumination light 10a is shaped into parallel light by the collector lens 12, restricted by the field stop 13, then condensed by the condenser lens 14, and shaped into an annular shape with a size (radius and width) determined by being restricted by an element having an annular aperture pattern, and the annular illumination 10a i is formed. The illumination light 10a by the annular illumination 10a i is partially reflected by a beam splitter 32 disposed at the intersection of the optical axis 10L of a part (condenser lens 14) of the illumination optical system 10 and the optical axis 30L of the detection optical system 30 (objective lens 31), and is sent to the sample S through the objective lens 31. Thereby, the sample S is illuminated by the illumination light 10a.

[0019] FIG. 1D shows a cross-sectional shape of illumination light 10a generated by an element in which an annular opening pattern is formed. The illumination light 10a is shaped into annular illuminations 10a1 to 10a5 having a plurality (here, five as an example) of annular patterns. The annular illuminations 10a1 to 10a4 each have a different radius (the radius of the center of the annulus, which is referred to as the annular radius) and a width that spreads outward and inward about that radius. Here, the outer radius of the annular pattern is called the outer radius, and the inner radius is called the inner radius. The outer radius of the annular illumination 10a1 is equal to (or may be smaller than) the maximum radius of the effective light source (the light source image formed in the aperture stop 15). The outer radius of the annular illumination 10a2 is larger than (or may be equal to or slightly smaller than) the inner radius of the annular illumination 10a1. The outer radius of the annular illumination 10a3 is larger than (or may be equal to or slightly smaller than) the inner radius of the annular illumination 10a2. The outer radius of the annular illumination 10a4 is larger than (or may be equal to or slightly smaller than) the inner radius of the annular illumination 10a3. The annular illumination 10a5 is circular, and its radius is larger than (or may be equal to or slightly smaller than) the inner radius of the annular illumination 10a4. That is, by overlapping the annular illuminations 10a1 to 10a5, the effective light source that is most widely distributed is covered.

[0020] Note that although the annular illumination 10a5 is circular, by regarding the inner diameter radius as zero, it can be said to be an annular illumination with an inner diameter radius of zero. Since the inner diameter radius is regarded as zero, the annular radius is half of the outer radius.

[0021] Note that the number of annular illuminations, the annular radius, and the width may be such that the number (M) may be at least two as long as the approximate range of the effective light source can be covered, the annular radii may be approximately equally spaced including the maximum radius of the effective light source, and the width may be such that the adjacent annular illuminations inside and outside overlap each other, do not overlap and have a gap therebetween, or the inner annular is within the outer annular.

[0022] Note that in the microscope 100 according to the present embodiment, the control unit 50 sequentially switches a plurality of elements (16a to 16e) in which annular opening patterns having different annular radii are formed and arranged in the aperture pattern turret 16 to obtain the annular illumination 10ai Although it was decided to generate this, instead of this, a spatial modulation element (for example, a liquid crystal panel) is arranged at a position conjugate to the pupil of the objective lens 31 (near the aperture stop 15), and the voltage value applied to the spatial modulation element (liquid crystal panel) by the control unit 50 is controlled, thereby modulating the illumination light 10a to generate the annular illumination 10a i Alternatively, a micro LED light source array may be arranged as the light source 11 to directly generate the annular illumination 10a i may be generated.

[0023] The drive unit 20 is a unit that drives the sample S relative to the objective lens 31 in the direction of its optical axis 30L, and includes a stage 21 and a drive device 23.

[0024] The stage 21 holds the container 23 or the slide glass, and is configured to be able to move up and down at least along the optical axis 30L for the sample S disposed in the container 23 or on the slide glass and the cover glass (an example of a support member) 22 that supports it.

[0025] The drive device 23 drives the stage 21 at least in the direction of the optical axis 30L. As the drive device 23, for example, an electric motor or the like can be adopted. The drive device 23 is controlled by the control unit 50 to drive the stage 21 to the target position. Thereby, the sample S on the stage 21 moves along the optical axis 30L.

[0026] Instead of driving the stage 21 along the optical axis 30L by the drive device 23, the objective lens 31 may be driven along the optical axis 30L by the drive device 23 so that the sample S moves relative to the objective lens 31 along the optical axis 30L.

[0027] The detection optical system 30 is a unit that receives the reflected light γ from the sample S and images the sample S, and includes an objective lens 31, a beam splitter 32, an imaging lens 34, and an imaging device 35.

[0028] The objective lens 31 guides the illumination light 10a to illuminate the sample S on the stage 21, and also collects the reflected light γ from the sample S and the reflected light γ from the interface 22a around the sample S. r It is an optical system for condensing, and includes a plurality of lens elements in the lens barrel. In the present embodiment, the objective lens 31 is disposed directly above the stage 21. Note that the objective lens 31 may be configured to be movable along the optical axis 30L.

[0029] The beam splitter 32 is an optical element that reflects a part of the illumination light 10a toward the objective lens 31 and transmits a part of the reflected light γ from the sample S to send it to the imaging device 35.

[0030] The imaging lens 34 condenses the reflected light γ sent through the objective lens 31 onto the light receiving surface of the imaging device 35 and generates an object image of the sample S thereon.

[0031] The imaging device 35 detects the reflected light γ from the sample S through the objective lens 31 and the imaging lens 34, and images the image of the sample S. The imaging result is transmitted to the processing unit 40. As the imaging device 35, an imaging element such as a charge coupled device (CCD) or a CMOS sensor may be employed.

[0032] When the reflected light γ is emitted from the illuminated sample S, the reflected light γ is the reflected light γ from the interface 22a r and is condensed by the objective lens 31, transmitted through the beam splitter 32, condensed by the imaging lens 34, and detected by the imaging device 35. Thereby, the object image of the sample S by the reflected light γ is imaged.

[0033] In the microscope 100 according to the present embodiment, the illumination optical system 10 and the detection optical system 30 are disposed above the stage 21 and the sample S, but may be disposed below the stage 21 and the sample S. In that case, through the objective lens 31, the illumination light 10a illuminates the sample S disposed on the container 23 or the slide glass on the stage 21 from below, and through the objective lens 31, the reflected light γ from the sample S and the reflected light from the interface between the bottom surface of the container 23 in contact with the sample S or the slide are condensed.

[0034] The processing unit 40 uses each of the plurality of annular illuminations 10a i to process a plurality of imaging results obtained at each of a plurality of positions regarding the direction of the optical axis 30L of the sample S by the detection optical system 30, using parameters related to the plurality of annular illuminations 10a i to generate a three-dimensional object image of the sample S. Details of the processing of the imaging results will be described later.

[0035] The processing unit 40 is a computer device such as a personal computer, and is implemented by a device having at least a central processing unit (CPU). The CPU expresses a function of processing the imaging results in the processing unit 40 to generate an object image of the sample S by executing a dedicated program. The dedicated program is stored in, for example, a ROM and read by the CPU, or stored in a storage medium such as a DVD-ROM and read by the CPU using a reading device such as a DVD-ROM drive and expanded in a RAM to be started up. Note that a more detailed example of the hardware configuration of the computer device will be described later.

[0036] The control unit 50 controls the drive unit 20 (drive device 23) to drive the stage 21 (or the objective lens 31) at least in the direction of the optical axis 30L. In Z-stack imaging, the control unit 50 determines a target drive amount (Z-step amount) to the next Z-stack position. When the drive device 23 receives the target drive amount from the control unit 50, it drives the stage 21 (or the objective lens 31) by that target drive amount. Thereby, the sample S on the stage 21 sequentially moves along the optical axis 30L by the target drive amount, changing the observation surface in the sample S to the next Z-stack position. The control unit 50 controls the detection optical system 30 (imaging device 35) to image the sample at each Z-stack position. Thereby, a Z-stack image is obtained as an object image.

[0037] The control unit 50 is a computer device such as a personal computer, and is implemented by a device having at least a central processing unit (CPU). By executing a dedicated program, the CPU exhibits a function of controlling each component of the microscope 100 in the control unit 50. The dedicated program is stored in, for example, a ROM and read by the CPU, or stored in a storage medium such as a DVD-ROM and read by the CPU using a reading device such as a DVD-ROM drive and expanded in a RAM to be activated. Note that a more detailed example of the hardware configuration of the computer device will be described later. Further, the control unit 50 may be implemented by a single computer device together with the processing unit 40.

[0038] The cause of the pseudo-resolution of the three-dimensional image in the reflection bright-field microscope will be described.

[0039] FIG. 2A shows the three-dimensional shapes (pupil functions) of the imaging pupil P col (f) and the illumination pupil P ill (f) in the frequency space f{=(fx, fy, fz)}. The imaging pupil is the entrance pupil of the detection optical system 30, and the numerical aperture of the detection optical system 30 is limited by the objective lens 31. Further, the illumination pupil is the exit pupil of the illumination optical system 10, and the numerical aperture of the illumination optical system 10 is limited by the aperture stop 15. Here, for the three-dimensional variables fx, fy, fz in the frequency space f, fz is the spatial frequency with respect to the direction of the optical axis 10L (referred to as the Z direction), and fx, fy are the spatial frequencies with respect to the positions on the plane orthogonal to the optical axis 10L (the positions in the X direction and the Y direction). The convolution of the imaging pupil P col (f) and the illumination pupil P ill (f) gives the three-dimensional aperture A(f){=P col (f)·P ill (f)}. In this example, it is assumed that the numerical apertures (hereinafter referred to as NA) of the illumination optical system and the detection optical system are equal.

[0040] The imaging pupil P col (f) has a partial spherical shell shape cut by an NA that is axisymmetric with respect to the fz axis with the vertex facing the -fz direction. The illumination pupil P ill(f) has a partial spherical shell shape that is axisymmetric with respect to the fz-axis with its vertex pointing in the +fz direction. The spherical shell radius of the imaging pupil and the illumination pupil is f = n / λ, where n is the average refractive index inside the specimen (inside the sample S) and λ is the wavelength of the illumination light 10a.

[0041] Illumination pupil P ill By slicing (f) into M pieces perpendicular to the fz-axis (parallel to the fx-fy plane), a plurality (M) of annular pupils P max (=NA ill / λ) with different radii (referred to as zonal radii) fsinφ are obtained. Here, φ ill,i is the maximum angle that φ can take. The zonal radius of the i-th annular pupil P max can be given by (NA ill,i / λ)(i - 1) / (M - 1), where i is any value from 1 to M. The NA ill of the M-th annular pupil P ill,M is preferably equal to or close to (preferably 0.9 times or more) the NA of the imaging pupil, thereby obtaining high resolution. ill The zonal width Δf may be infinitesimal as long as it can approximately cover the illumination pupil P

[0042] (f) for all the annular pupils P ill,i and can also be a width that slightly overlaps or leaves a gap with the adjacent inner and outer annular pupils. For example, NA ill / 2λ ≤ MΔf ≤ NA ill / λ may be used. Thereby, the entire illumination pupil P ill (f) can be approximately covered with a small number (M) of zones, making it possible to obtain high resolution. ill (f)

[0043] In principle, the number (M) of zonal illuminations is preferably a large number so that all of the observable region is filled. As shown in Fig. 2D for the case of the number of zones M = 5, about five zones can approximately fill the ideal observable region shown in Fig. 2C.

[0044] Fig. 2B shows the imaging pupil P col (f) and the illumination pupil P ill(f), as an example, the annular pupil P ill,3 Shows the two-dimensional shape (pupil function) of fx - fz and fx - fy in (f). The annular pupil P ill,3 The annular radius of (f) is 0.4f. The annular pupil P in the fz direction ill,i and the imaging pupil P col The distance 2fcosφ from (f) i is defined as a parameter related to the annular illumination 10a i (also referred to as the annular parameter).

[0045] Figure 2C shows the convolution P col (f) of the imaging pupil P ill (f) and the illumination pupil P col (f)·P ill (f), which gives the three-dimensional aperture A(f). The three-dimensional aperture A(f) gives the observable object frequency region (i.e., the existence region of the image frequency). The three-dimensional aperture A(f) is a three-dimensional function that is distributed in the -fz region on the fx - fz plane as shown in the figure and is rotationally symmetric about the fz axis. By interfering the reflected light γ from the sample S with a reference light that travels on an optical path independent of the optical axes 10L and 30L, that is, a reference light whose phase does not change by driving the stage 21 that supports the sample S, an ideal image frequency can be obtained. However, when the reflected light γ from the sample S is interfered with the reflected light γ r from the interface around the sample S, for example, the interface 22a of the cover glass 22 that supports the sample S, when the stage 21 that supports the sample S is driven to obtain, for example, a Z-stack image, the phase of the reflected light γ r changes, resulting in pseudo-resolution in the three-dimensional object image.

[0046] Figure 2D shows the three-dimensional aperture Σ i A i (f) in the case where the number of annuli M = 5. It can be seen that it approximately fills the ideal observable region shown in Figure 2C.

[0047] Figure 3A is based on Figure 2B, showing the imaging pupil P col (f) and a plurality (M) of annular pupils P having different annular radii obtained by dividing the illumination pupil P ill (f) as described above ill,iAn example of a set with it is shown (from the right to the left of the drawing, i = 1 to 7). Here, M = 7 is taken as an example. The ring zone width Δf is a sufficiently small value, and the ring zone pupil P ill,i (i = 2 to 7) is distributed in a ring shape (two points on the fx - fz plane) within the frequency space. P ill,1 is distributed at one point on the fz axis.

[0048] Figure 3B shows the imaging pupil P shown in Figure 3A col (f) and the ring zone pupil P ill,i The three - dimensional aperture A i (f) of the ring zone illumination obtained from each set of (f) is shown (from the right to the left of the drawing, i = 1 to 7). The three - dimensional aperture A i (f) is located in the - fz region.

[0049] However, when the stage 21 that supports the sample S is driven, the phase of the reflected light γ from the interface around the sample S where the reflected light γ from the sample S interferes, for example, the reflected light γ from the interface 22a of the cover glass 22 that supports the sample S r changes. Therefore, as simulated in Figure 3C, the image frequency obtained by each ring zone illumination 10a i shifts in the + fz direction by the shift amount given by the ring zone parameter 2fcosφ i towards the origin. Note that the shift amount is different for each ring zone illumination 10a i .

[0050] Figure 3C shows the three - dimensional aperture A i of the ring zone illumination 10a shown in Figure 3B i (f) shifted in the + fz direction by the shift amount given by the ring zone parameter 2fcosφ i respectively, and shows the three - dimensional aperture B i (f) (including suspected defocus) of the ring zone illumination 10a obtained thereby. i (f)(including suspected defocus) is shown.

[0051] Figure 3D shows the ring zone illumination B shown in Figure 3C iIndicates the sum of (f) (i = 1 to 7). This sum gives the object frequency observable in a reflection bright-field microscope (i.e., the frequency distribution of the object image). It can be seen that it deviates significantly from the three-dimensional aperture A(f) shown in FIG. 2C. That is, in a reflection bright-field microscope, the sample S is illuminated by normal illumination, and the reflected light γ from the sample S that reflects the structure of the sample S interferes with the reflected light γ from the interface around the sample S that does not reflect the structure of the sample S, for example, the interface 22a of the cover glass 22 that supports the sample, and is received by the detection optical system 30. Here, the reflected light γ from the sample S interferes with the reflected light γ from the interface 22a that moves with the sample S, resulting in pseudo-resolution and forming a three-dimensional object image that does not correctly reflect the object structure of the sample S. r and is received by the detection optical system 30 through interference. Here, the reflected light γ from the sample S interferes with the reflected light γ from the interface 22a that moves with the sample S r to cause pseudo-resolution and form a three-dimensional object image that does not correctly reflect the object structure of the sample S.

[0052] An image processing method for generating a three-dimensional object image of the sample S performed by the microscope (reflection bright-field microscope) 100 according to this embodiment will be described.

[0053] The image processing method is executed by the processing unit 40. The processing unit 40 generates an object image (also called an image frequency) in the frequency space f from a plurality of imaging results obtained at each of a plurality of positions regarding the direction of the optical axis 30L of the sample S for each used annular illumination 10a (i = 1 to M), processes it using the annular parameter related to the annular radius of the used annular illumination 10a, synthesizes the obtained plurality of image frequencies, and performs inverse Fourier transform to generate a three-dimensional object image of the sample S in the real space. i (i = 1 to M), generates an object image (also called an image frequency) in the frequency space f from a plurality of imaging results obtained at each of a plurality of positions regarding the direction of the optical axis 30L of the sample S, and processes it using the annular parameter related to the annular radius of the used annular illumination 10a i to synthesize the obtained plurality of image frequencies and perform inverse Fourier transform to generate a three-dimensional object image of the sample S in the real space.

[0054] Note that the image frequency in the frequency space f of the sample S for each annular illumination 10a i is obtained by Fourier-transforming the object image of the sample S obtained using each annular illumination 10a into the frequency space f. Here, it is already assumed that the image frequency for each annular illumination 10a has been obtained. The procedure of the observation method for obtaining the image frequency of the sample S will be described later. i is obtained by Fourier-transforming the object image of the sample S obtained using each annular illumination 10a into the frequency space f. Here, it is already assumed that the image frequency for each annular illumination 10a i has been obtained. The procedure of the observation method for obtaining the image frequency of the sample S will be described later.

[0055] First, the processing unit 40 uses the illumination pupil P shown in FIGS. 2A and 2Bill Divide (f) into M parts to generate annular pupils P ill,i (i = 1 to M). Here, as an example, let M = 6. Thereby, as shown in FIG. 3A, the imaging pupil P col (f) and a plurality of annular pupils P having different annular radii ill,i are obtained as a set.

[0056] Next, the processing unit 40 calculates the convolution of each set of the imaging pupil P col (f) and the annular pupil P ill,i . Thereby, as shown in FIG. 3B, the three-dimensional aperture A i of each annular illumination 10a i (f) is obtained. (The sample S information is not included, and a theoretical value determined only by the optical system is obtained)

[0057] Next, the processing unit 40 shifts the three-dimensional aperture A i of the annular illumination 10a i (f) in the +fz direction by the shift amount given by the annular parameter 2fcosφ i respectively. Thereby, as shown in FIG. 3C, the three-dimensional aperture B i of each annular illumination 10a i (f) is obtained. (A part of the frequency region that can be obtained by the annular illumination calculated by theoretical calculation is obtained)

[0058] Next, the processing unit 40 uses the three-dimensional aperture B i of each annular illumination 10a i (f) to calculate the imaginary part of iB i (f) - iB i * (-f), and extracts only the positive value part (defined as α(f)). (Theoretical calculation) Thereby, as shown in FIG. 4, a positive value function (α(f)) is obtained in which only the extracted positive value part is 1 and the other parts are zero.

[0059] Next, the processing unit 40 uses the annular illumination 10a iFrom among the image frequencies (measured values) of the sample S generated each time, only the region of α(f) calculated above is extracted. (Only the region corresponding to the positive value part is extracted from the measured value.) Next, the processing unit 40 shifts the extracted part by the shift amount given by the annular parameter 2fcosφ in the -fz direction. The function obtained as a result of the shift is defined as the image frequency iA' i (f). When represented in a diagram, it is as shown in Fig. 5. However, for simplicity, it is illustrated without including the object frequency of the sample S. i (f). When represented in a diagram, it is as shown in Fig. 5. However, for simplicity, it is illustrated without including the object frequency of the sample S.

[0060] Next, the processing unit 40 uses the image frequency iA' i (f) obtained above to convert it to {iA' i (f) - iA' * i (-f)} and synthesizes the image frequencies for each annular illumination 10a, that is, calculates the sum Σ i {iA' i (f) - iA' i (f) - iA' * i (-f)}. Thereby, the distribution of the object frequencies of the observable phase object shown in Fig. 6 is obtained. This is equal to the ideal object frequency (Fig. 2C) obtained by interfering the reflected light γ from the sample S with a reference light that travels on an optical path independent of the optical axes 10L and 30L, that is, a reference light whose phase is not changed by driving the stage 21 that supports the sample S.

[0061] Here, the overlapping part of iB i (f) and -iB i (f) for each annular illumination 10a becomes a pure imaginary number by subtraction and the real part information is lost. Therefore, the processing unit 40 complements (or restores) the image frequency iA' i * (-f) on the fz axis using the value of the image frequency iA' i (fz = 0) outside the fz axis. Here, a complementation process such as linear complementation or spline complementation may be applied, or an estimation process such as Bayesian estimation may be applied. Furthermore, the processing unit 40 performs the image frequency iA' i (fz ≠ 0) outside the fz axis using the value of the image frequency iA' i(f) may be complemented (or restored) using the value of the image frequency outside the fx-fy plane. Thereby, iA' i (f) can be restored more accurately.

[0062] Finally, the processing unit 40 performs a Fourier transform on the object frequency of the phase object obtained above into real space. Thereby, a three-dimensional object image of the sample S in real space is obtained.

[0063] FIGS. 7A and 7B respectively show the object image reconstructed by the image processing method according to the present embodiment and the object image according to the comparative example. As the sample S, solid micron-sized polystyrene beads were used. According to the object image shown in FIG. 7A, it can be seen that the three-dimensional shape of the sample S is almost accurately reproduced. On the other hand, according to the object image shown in FIG. 7B, it can be seen that an artifact has occurred because the image processing method according to the present embodiment was not applied, and the three-dimensional shape of the sample S cannot be reproduced.

[0064] In the above-described image processing method, the image frequency iA' i (f) is used to convert to {iA' i (f)-iA' * i (-f)}, and the image frequencies for each annular illumination 10a i are synthesized to calculate the image frequency when the sample S is a phase object. Instead of this, the image frequency iA' i (f) can be used to convert to {-A' i (f)-A' * i (-f)}, and the image frequencies for each annular illumination 10a i are synthesized to calculate the image frequency when the sample S is an absorption object.

[0065] FIG. 8 shows the flow of an observation method for generating a three-dimensional object image of the sample S using the microscope 100 according to the present embodiment. It is assumed that the number M of annular illuminations, the number of Z-stack images, that is, the number N of times the stage 21 supporting the sample S is step-driven in the direction of the optical axis 30L and the driving amount thereof are determined in advance.

[0066] In step S102, the control unit 50 resets the index i (i = 0).

[0067] In step S104, the control unit 50 increments the index i (adds 1 to i).

[0068] In step S106, the control unit 50 generates illumination light 10a using a plurality of annular illuminations 10a having mutually different annular radii. Here, it is the i-th annular illumination 10a i and here, it generates illumination light 10a using the i-th annular illumination 10a i The method for generating the annular illumination 10a is as described above. i is as described above.

[0069] In step S108, the control unit 50 resets the index n (n = 0).

[0070] In step S110, the control unit 50 increments the index n (adds 1 to n).

[0071] In step S112, the control unit 50 images the sample S. First, the control unit 50 controls the illumination optical system 10 to generate the annular illumination 10a i and guides it through the objective lens 31 to illuminate the sample S supported on the stage 21. Next, the control unit 50 controls the detection optical system 30 to receive the reflected light γ from the sample S and the reflected light γ from the interface around the sample S r through the objective lens 31 and image the sample S. The imaging result (i, n) is transmitted to the processing unit 40.

[0072] In step S114, the control unit 50 determines whether n is equal to N. If they are equal, it proceeds to step S118. If they are not equal, it proceeds to step S116.

[0073] In step S116, the control unit 50 controls the drive unit 20 to step-drive the stage 21 that supports the sample S by a step amount defined in the direction of the optical axis 30L relative to the objective lens 31. Instead of driving the stage 21, the objective lens 31 may be step-driven in the direction of the optical axis 30L.

[0074] When step S116 is completed, the process returns to step S110. The imaging in step S112 and the step-driving in step S116 are repeated until the determination in step S114 is affirmed. Thereby, using the annular illumination 10a i the sample S is imaged at each of a plurality of positions in a direction parallel to the optical axis 30L, that is, a Z-stack image is obtained.

[0075] In step S118, the control unit 50 determines whether i is equal to M. If they are equal, the process proceeds to step S120. If they are not equal, the process returns to step S104.

[0076] Until the determination in step S118 is affirmed, the generation of the annular illumination 10a in step S106 i and the generation of the Z-stack images in steps S112 to S116 are repeated. Thereby, Z-stack images of the sample S are obtained using each of all the annular illuminations 10a i (i = 1 to M).

[0077] In step S120, the control unit 50 controls the processing unit 40 to execute the image processing method according to the present embodiment. The processing unit 40 performs Fourier transform on a plurality of imaging results (Z-stack images) obtained at each of a plurality of positions in the direction of the optical axis 30L of the sample S in step S112 using each of the plurality of annular illuminations 10a i to generate image frequencies in the frequency space. Then, the processing unit 40 processes the image frequencies for each of the plurality of annular illuminations 10a i using the annular parameters related to the plurality of annular illuminations 10a i and performs inverse Fourier transform to generate a three-dimensional object image of the sample S in the real space. The details of the image processing method are as described above.

[0078] In step S120, the control unit 50 controls the processing unit 40 to display the three-dimensional object image of the obtained sample S on the screen and / or record it in the storage device. Thereby, the flow is completed.

[0079] In addition, in the observation method according to the present embodiment, the annular illumination 10a i is generated, and while the stage 21 that supports the sample S is step-driven with respect to it, the sample S is imaged to generate a Z-stack image. Instead of this, the stage 21 that supports the sample S is step-driven and positioned in the direction of the optical axis 30L, and with respect to that, the annular illumination 10a i may be sequentially generated while imaging the sample S.

[0080] According to the microscope 100 according to the present embodiment, it has an aperture pattern turret 16 capable of forming a plurality of annular illumination lights 10a having different annular radii and an objective lens 31, an illumination optical system 10 that irradiates the sample S with the illumination light 10a, a detection optical system 30 that condenses the first reflected light from the sample S and the second reflected light from the interface around the sample S onto the imaging device 35 via the objective lens 31, and a control unit 50. The control unit 50 controls the aperture pattern turret 16 to form a plurality of annular illumination lights 10a i respectively, and at each of a plurality of positions where the relative position between the objective lens 31 and the sample S is different, the imaging device 35 detects the first reflected light and the second reflected light. According to this, a plurality of annular illumination lights 10a i each having a different annular radius are used to image the sample S at a plurality of positions regarding the optical axis direction by the imaging device 35, and for each used annular illumination 10a i every time, an image frequency in the frequency space (fx, fy, fz) is generated from a plurality of imaging results (XY images) obtained at each of a plurality of positions regarding the optical axis direction (Z direction) of the sample S, and using a parameter related to the annular radius of the used annular illumination 10a i to process and synthesize the obtained plurality of image frequencies, a three-dimensional object image of a sample without pseudo-resolution can be generated.

[0081] According to the observation method according to this embodiment, a step of irradiating the sample S with the illumination light 10a through the illumination optical system 10 having the aperture pattern turret 16 capable of forming a plurality of annular illumination lights 10a having different annular radii and the objective lens 31, a step of condensing the first reflected light from the sample and the second reflected light from the interface around the sample S onto the imaging device 35 through the objective lens 31, and a step of controlling the aperture pattern turret 16 to form each of the plurality of annular illumination lights 10a, and detecting the first reflected light and the second reflected light by the imaging device 35 at each of a plurality of positions where the relative positions of the objective lens 31 and the sample S are different. According to this, by using each of the plurality of annular illumination lights 10a having different annular radii i each, the imaging device 35 images the sample S at a plurality of positions in the optical axis direction, and for each of the used annular illuminations 10a i each, an image frequency in the frequency space (fx, fy, fz) is generated from a plurality of imaging results (XY images) obtained at each of a plurality of positions in the optical axis direction (Z direction) of the sample S, and is processed using a parameter related to the annular radius of the used annular illumination 10a i By synthesizing the obtained plurality of image frequencies, a three-dimensional object image of a sample without pseudo-resolution can be generated.

[0082] According to the program according to this embodiment, a procedure of irradiating the sample S with the illumination light 10a through the illumination optical system 10 having the aperture pattern turret 16 capable of forming a plurality of annular illumination lights 10a having different annular radii and the objective lens 31, a procedure of condensing the first reflected light from the sample and the second reflected light from the interface around the sample S onto the imaging device 35 through the objective lens 31, and a procedure of controlling the aperture pattern turret 16 to form each of the plurality of annular illumination lights 10a, and detecting the first reflected light and the second reflected light by the imaging device 35 at each of a plurality of positions where the relative positions of the objective lens 31 and the sample S are different are executed by the computer. According to this, by using each of the plurality of annular illumination lights 10a i each, the imaging device 35 images the sample S at a plurality of positions in the optical axis direction, and for each of the used annular illuminations 10a iEach time, image frequencies in the frequency space (fx, fy, fz) are generated from a plurality of imaging results (XY images) obtained at respective positions of a sample S with respect to the optical axis direction (Z direction), and are processed using parameters related to the ring zone radius of the used ring zone illumination 10a i By using the parameters related to the ring zone radius of i and synthesizing the obtained plurality of image frequencies, a three-dimensional object image of a sample without pseudo-resolution can be generated.

[0083] Various embodiments of the present invention may be described with reference to flowcharts and block diagrams, where the blocks may represent (1) stages of a process in which operations are performed or (2) sections of a device having the role of performing the operations. Specific stages and sections may be implemented by dedicated circuits, programmable circuits supplied with computer-readable instructions stored on a computer-readable medium, and / or processors supplied with computer-readable instructions stored on a computer-readable medium. The dedicated circuits may include digital and / or analog hardware circuits, and may include integrated circuits (ICs) and / or discrete circuits. The programmable circuits may include reconfigurable hardware circuits including memory elements such as logical AND, logical OR, logical XOR, logical NAND, logical NOR, and other logical operations, flip-flops, registers, field programmable gate arrays (FPGAs), programmable logic arrays (PLAs), etc.

[0084] A computer-readable medium may include any tangible device that can store instructions executable by a suitable device. As a result, a computer-readable medium having instructions stored therein will comprise a product that includes instructions that can be executed to create means for performing the operations specified in a flowchart or block diagram. Examples of computer-readable media may include electronic memory media, magnetic memory media, optical memory media, electromagnetic memory media, semiconductor memory media, and the like. More specific examples of computer-readable media may include floppy (registered trademark) disks, diskettes, hard disks, random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EPROM or flash memory), electrically erasable programmable read-only memory (EEPROM), static random access memory (SRAM), compact disc read-only memory (CD-ROM), digital versatile disc (DVD), Blu-ray (RTM) disc, memory stick, integrated circuit card, and the like.

[0085] Computer-readable instructions may include any combination of one or more programming languages, including assembly instructions, instruction set architecture (ISA) instructions, machine instructions, machine-dependent instructions, microcode, firmware instructions, state-setting data, or source code or object code written in any combination of object-oriented programming languages such as Smalltalk (registered trademark), JAVA (registered trademark), C++, and conventional procedural programming languages such as the "C" programming language or similar programming languages.

[0086] Computer-readable instructions may be provided locally or via a wide area network (WAN), such as a local area network (LAN), the Internet, etc., to a processor or programmable circuit of a general-purpose computer, a special-purpose computer, or other programmable data processing apparatus, and may be executed to create means for performing the operations specified in a flowchart or block diagram. Examples of processors include computer processors, processing units, microprocessors, digital signal processors, controllers, microcontrollers, and the like.

[0087] FIG. 9 shows an example of a computer 2200 in which multiple aspects of the present invention may be embodied, in whole or in part. Programs installed on the computer 2200 may cause the computer 2200 to function as an operation associated with an apparatus according to an embodiment of the present invention or as one or more sections of the apparatus, or to execute the operation or the one or more sections, and / or may cause the computer 2200 to execute a process according to an embodiment of the present invention or a stage of the process. Such a program may be executed by the CPU 2212 to cause the computer 2200 to perform certain operations associated with some or all of the blocks of the flowcharts and block diagrams described herein.

[0088] The computer 2200 according to this embodiment includes a CPU 2212, a RAM 2214, a graphic controller 2216, and a display device 2218, which are mutually connected by a host controller 2210. The computer 2200 also includes an input / output unit such as a communication interface 2222, a hard disk drive 2224, a DVD-ROM drive 2226, and an IC card drive, which are connected to the host controller 2210 via an input / output controller 2220. The computer also includes legacy input / output units such as a ROM 2230 and a keyboard 2242, which are connected to the input / output controller 2220 via an input / output chip 2240.

[0089] The CPU 2212 operates according to programs stored in the ROM 2230 and the RAM 2214, thereby controlling each unit. The graphic controller 2216 acquires image data generated by the CPU 2212 in a frame buffer or the like provided in the RAM 2214 or in itself, and causes the image data to be displayed on the display device 2218.

[0090] The communication interface 2222 communicates with other electronic devices via a network. The hard disk drive 2224 stores programs and data used by the CPU 2212 in the computer 2200. The DVD-ROM drive 2226 reads a program or data from the DVD-ROM 2201 and provides the program or data to the hard disk drive 2224 via the RAM 2214. The IC card drive reads programs and data from an IC card and / or writes programs and data to the IC card.

[0091] The ROM 2230 stores therein a boot program or the like executed by the computer 2200 upon activation and / or a program dependent on the hardware of the computer 2200. The input / output chip 2240 may also be connected to the input / output controller 2220 via various input / output units through a parallel port, a serial port, a keyboard port, a mouse port, etc.

[0092] The program is provided by a computer-readable medium such as a DVD-ROM 2201 or an IC card. The program is read from the computer-readable medium, installed in a hard disk drive 2224, a RAM 2214, or a ROM 2230, which are also examples of computer-readable media, and executed by the CPU 2212. The information processing described in these programs is read by the computer 2200, resulting in the cooperation between the programs and the various types of hardware resources described above. The apparatus or method may be configured by realizing the operation or processing of information according to the use of the computer 2200.

[0093] For example, when communication is executed between the computer 2200 and an external device, the CPU 2212 may execute a communication program loaded in the RAM 2214 and instruct the communication interface 2222 to perform communication processing based on the processing described in the communication program. Under the control of the CPU 2212, the communication interface 2222 reads the transmission data stored in a transmission buffer processing area provided in a recording medium such as the RAM 2214, the hard disk drive 2224, the DVD-ROM 2201, or the IC card, transmits the read transmission data to the network, or writes the received data received from the network to a reception buffer processing area or the like provided on the recording medium.

[0094] Further, the CPU 2212 may cause all or necessary portions of files or databases stored in external recording media such as a hard disk drive 2224, a DVD-ROM drive 2226 (DVD-ROM 2201), an IC card, etc. to be read into the RAM 2214, and execute various types of processing on the data on the RAM 2214. The CPU 2212 then writes back the processed data to the external recording media.

[0095] Various types of information such as various types of programs, data, tables, and databases may be stored in the recording media and may be subjected to information processing. The CPU 2212 may execute various types of processing on the data read from the RAM 2214, including various types of operations, information processing, condition determination, conditional branch, unconditional branch, information search / replacement, etc. described throughout this disclosure and specified by the instruction sequence of the program, and write back the results to the RAM 2214. Further, the CPU 2212 may search for information in files, databases, etc. within the recording media. For example, when a plurality of entries each having an attribute value of a first attribute associated with an attribute value of a second attribute are stored in the recording media, the CPU 2212 searches for an entry that matches the condition where the attribute value of the first attribute is specified from among the plurality of entries, reads the attribute value of the second attribute stored in the entry, and thereby may obtain the attribute value of the second attribute associated with the first attribute that satisfies a predetermined condition.

[0096] The programs or software modules described above may be stored in a computer-readable medium on or near the computer 2200. Also, a recording medium such as a hard disk or RAM provided in a server system connected to a dedicated communication network or the Internet can be used as a computer-readable medium, thereby providing the program to the computer 2200 via the network.

[0097] As described above, the present invention has been described using embodiments. However, the technical scope of the present invention is not limited to the scope described in the above embodiments. It is obvious to those skilled in the art that various changes or improvements can be made to the above embodiments. It is clear from the description of the claims that forms with such changes or improvements can also be included in the technical scope of the present invention.

[0098] It should be noted that the execution order of each process such as operations, procedures, steps, and stages in the devices, systems, programs, and methods shown in the claims, the specification, and the drawings is not explicitly indicated as "before" or "preceding" etc. in particular. Also, unless the output of the previous process is used in the subsequent process, it can be realized in any order. Regarding the operation flows in the claims, the specification, and the drawings, even if they are described using "first," "next," etc. for convenience, it does not mean that it is essential to implement them in this order.

Description of Reference Numerals

[0099] 10…Illumination optical system, 10L…Optical axis, 10a…Illumination light, 10a i ,10a1~10a5…Annular illumination, 11…Light source, 12…Collector lens, 13…Field stop, 14…Condenser lens, 15…Aperture stop, 16…Aperture pattern turret, 20…Drive unit, 21…Stage, 22…Cover glass, 22a…Interface, 23…Drive device, 30…Detection optical system, 30L…Optical axis, 31…Objective lens, 32…Beam splitter, 34…Imaging lens, 35…Imaging device, 40…Processing unit, 50…Control unit, 100…Reflecting bright-field microscope (microscope), 2200…Computer, 2201…DVD-ROM, 2210…Host controller, 2214…RAM, 2216…Graphic controller, 2218…Display device, 2220…Input / output controller, 2222…Communication interface, 2224…Hard disk drive, 2226…DVD-ROM drive, 2240…Input / output chip, 2242…Keyboard, S…Sample.

Claims

1. An illumination optical system having a first member capable of forming a plurality of annular illumination lights having different annular radii and an objective lens, and irradiating the sample with the illumination light; A detection optical system that condenses a first reflected light from the sample and a second reflected light from an interface around the sample onto a detection unit through the objective lens; A control unit; A processing unit, comprising: Using each of the plurality of annular illumination lights formed by controlling the first member by the control unit, at each of a plurality of positions where the relative position between the objective lens and the sample is different, the detection unit detects the first reflected light and the second reflected light, The processing unit generates a plurality of image frequencies in the frequency space from a plurality of detection results by the detection unit, processes the plurality of image frequencies using values of parameters related to the plurality of annular illumination lights, and synthesizes the resulting new plurality of image frequencies to generate a three-dimensional object image of the sample. A reflection bright-field microscope.

2. The reflection bright-field microscope according to claim 1, wherein the interface is an interface between the sample and a second member in contact with the sample.

3. The annular radius of the annular illumination pupil on the frequency plane corresponding to the two-dimensional plane orthogonal to the optical axis direction of the illumination optical system is defined by (NA ill / λ)(i - 1) / (M - 1), where M is the number of the annular illumination pupils, i is any one of 1 to M, NA ill is the numerical aperture of the illumination optical system, and λ is the wavelength of the illumination light. The reflection bright-field microscope according to claim 1 or 2.

4. The processing unit: i) Calculating each three-dimensional aperture B ｉ (f) by shifting a three-dimensional aperture A ｉ (f) determined from each annular illumination pupil of the illumination optical system and the imaging pupil of the detection optical system in a predetermined direction by the value of the parameter, where i is any one of 1 to M and M is the number of the annular illumination pupils, ii) Each of the three-dimensional apertures B ｉcalculating a positive-valued function using (f), and iii) extracting a region of the positive-valued function from the plurality of image frequencies calculated from the plurality of detection results; The reflection bright-field microscope according to claim 1 or 2.

5. The processing unit calculates the new plurality of image frequencies by shifting the extracted region in a direction opposite to the predetermined direction by the value of the parameter. The reflection bright-field microscope according to claim 4.

6. The processing unit synthesizes the new plurality of image frequencies as a phase object or an absorption object. The reflection bright-field microscope according to claim 1 or 2.

7. The processing unit complements the plurality of image frequencies on the frequency axis corresponding to the optical axis direction of the objective lens using the plurality of image frequencies outside the frequency axis. The reflection bright-field microscope according to claim 6.

8. The first member is a spatial light modulator, an LED light source array, or a member on which a plurality of annular opening patterns having different annular radii are arranged. The reflection bright-field microscope according to any one of claims 1 to 7.

9. irradiating the sample with the illumination light through an illumination optical system having a first member capable of forming a plurality of annular illumination lights having different annular radii and an objective lens; condensing, by the objective lens, the first reflected light from the sample and the second reflected light from the interface around the sample onto a detection unit; using each of the plurality of annular illumination lights formed by controlling the first member, the detection unit detecting the first reflected light and the second reflected light at each of a plurality of positions where the relative position between the objective lens and the sample is different; Generating a plurality of image frequencies in the frequency space from a plurality of detection results by the detection unit, processing the plurality of image frequencies using values of parameters related to the plurality of annular illumination lights, and synthesizing the resulting new plurality of image frequencies to generate a three-dimensional object image of the sample; An observation method comprising.

10. Irradiating the sample with the illumination light through an illumination optical system having a first member capable of forming a plurality of annular illumination lights having different annular radii and an objective lens; Collecting the first reflected light from the sample and the second reflected light from the interface around the sample by a detection unit through the objective lens; Using each of the plurality of annular illumination lights formed by controlling the first member, at each of a plurality of positions where the relative position between the objective lens and the sample is different, the detection unit detecting the first reflected light and the second reflected light; Generating a plurality of image frequencies in the frequency space from a plurality of detection results by the detection unit, processing the plurality of image frequencies using values of parameters related to the plurality of annular illumination lights, and synthesizing the resulting new plurality of image frequencies to generate a three-dimensional object image of the sample; A program for causing a computer to execute.

Images