Microscope, observation method, and program

Abstract

To provide a microscope that maintains a position of an illumination surface in a sample.SOLUTION: A microscope 100 has: a detection optical system 32 that has an objective lens 33 and receives detection light 30a from a sample 9; an illumination optical system 12 that irradiates the sample with illumination light 10a from a direction intersecting an optical axis direction of the objective lens to form an illumination surface 10S; a position detection unit 40 that detects information on a position in the optical axis direction of the sample; and a control unit 50. The control unit executes first control of moving at least one of the objective lens and a stage 21 holding the sample on the basis of a result of detection performed by the position detection unit in the optical axis direction of the objective lens to set a focal position of the objective lens in the detection optical system to a predetermined position of the sample, and second control of, when moving the objective lens in the optical axis direction, driving an optical element included in the illumination optical system to set an irradiation position of the illumination light to the predetermined position of the sample. The microscope can thus maintain the position of the illumination surface in the sample.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a microscope, an observation method, a refractive index determining method, and a program. [Background technology]

[0002] A light sheet microscope is known that generates an optical cross-sectional image of a sample by injecting a sheet-like excitation light (also called illumination light) into the sample from the side and detecting light (also called detection light) emitted upward or downward from the sample (see, for example, Non-Patent Document 1). In a light sheet microscope, the optical system that guides the illumination light to the sample and the optical system that guides the detection light to the detection device are independent, so it is necessary to control the illumination light and the detection light independently. Non-Patent Document 1 K. Greger, J. Swoger, EHK Stelzer, "Basic building units and properties of a fluorescence single plane illumination microscope," Rev. Sci. Instrum. 2007 Feb, 78(2), 023705 Summary of the Invention

[0003] In a first aspect of the present invention, a microscope is provided which has a first optical system having an objective lens and receiving a first light from a sample, a second optical system that irradiates a second light onto the sample from a direction intersecting the optical axis direction of the objective lens to form an illumination plane, a position detection unit that detects information about the position of the sample in the optical axis direction, and a control unit, wherein the control unit performs a first control to set the focal position of the objective lens in the first optical system to a predetermined position on the sample by moving at least one of the objective lens and a stage that holds the sample in the optical axis direction based on the detection result by the position detection unit, and when the objective lens is moved in the optical axis direction, further controls the second optical system to perform a second control to set the illumination plane at a predetermined position on the sample.

[0004] In a second aspect of the present invention, a microscope is provided which has a first optical system having an objective lens and receiving a first light from a sample, a second optical system that irradiates a second light onto the sample from a direction intersecting the optical axis direction of the objective lens to form an illumination plane, and a control unit, wherein when the relative positional relationship between the sample and the objective lens in the optical axis direction changes, the control unit controls the second optical system to adjust the position of the illumination plane in the optical axis direction based on the refractive index of the sample.

[0005] In a third aspect of the present invention, there is provided a microscope comprising: a first optical system having an objective lens and receiving a first light from a sample; a second optical system that irradiates a second light onto the sample from a direction intersecting the optical axis direction of the objective lens to form an illumination plane; a control unit that drives at least one of the objective lens and a stage that holds the sample in the optical axis direction and adjusts the position of the illumination plane in the optical axis direction based on the detection result of the first light from the sample; and a refractive index determination unit that determines the refractive index of the sample based on at least one of the driving amount of the objective lens and the driving amount of the stage and the adjustment amount of the illumination plane.

[0006] In a fourth aspect of the present invention, there is provided an observation method comprising the steps of receiving a first light from a sample through a first optical system having an objective lens; irradiating the sample with a second light through a second optical system from a direction intersecting the optical axis direction of the objective lens to form an illumination plane; detecting information regarding the position of the sample in the optical axis direction; performing a first control to set the focal position of the objective lens in the first optical system to a predetermined position on the sample by moving at least one of the objective lens and a stage holding the sample in the optical axis direction based on the detection result of the information regarding the position of the sample in the optical axis direction; and, when the objective lens is moved in the optical axis direction, further performing a second control to set the illumination plane at a predetermined position on the sample by controlling the second optical system.

[0007] In a fifth aspect of the present invention, there is provided a program for causing a computer to execute the following steps: receiving a first light from a sample through a first optical system having an objective lens; irradiating a second light onto the sample through a second optical system from a direction intersecting the optical axis direction of the objective lens to form an illumination plane; detecting information regarding the position of the sample in the optical axis direction; performing a first control to set the focal position of the objective lens in the first optical system to a predetermined position on the sample by moving at least one of the objective lens and a stage holding the sample in the optical axis direction based on the detection result of the information regarding the position of the sample in the optical axis direction; and, when the objective lens is moved in the optical axis direction, further controlling the second optical system to perform a second control to set the illumination plane to a predetermined position on the sample.

[0008] In a sixth aspect of the present invention, there is provided an observation method comprising the steps of receiving a first light from a sample via a first optical system having an objective lens, irradiating a second light onto the sample via a second optical system from a direction intersecting the optical axis direction of the objective lens to form an illumination plane, and, when the relative positional relationship between the sample and the objective lens in the optical axis direction changes, controlling the second optical system based on the refractive index of the sample to adjust the position of the illumination plane in the optical axis direction.

[0009] In a seventh aspect of the present invention, there is provided a program for causing a computer to execute the steps of receiving a first light from a sample via a first optical system having an objective lens, irradiating a second light onto the sample via a second optical system from a direction intersecting the optical axis direction of the objective lens to form an illumination plane, and, when the relative positional relationship between the sample and the objective lens in the optical axis direction changes, controlling the second optical system to adjust the position of the illumination plane in the optical axis direction based on the refractive index of the sample.

[0010] In an eighth aspect of the present invention, there is provided a refractive index determination method comprising the steps of receiving a first light from a sample via a first optical system having an objective lens; irradiating a second light onto the sample via a second optical system from a direction intersecting the optical axis direction of the objective lens to form an illumination plane; driving at least one of the objective lens and a stage holding the sample in the optical axis direction and adjusting the position of the illumination plane in the optical axis direction based on the detection result of the first light from the sample; and determining the refractive index of the sample based on at least one of the driving amount of the objective lens and the driving amount of the stage and the adjustment amount of the illumination plane.

[0011] In a ninth aspect of the present invention, there is provided a program for causing a computer to execute the following steps: receiving first light from a sample via a first optical system having an objective lens; irradiating second light onto the sample via a second optical system from a direction intersecting the optical axis direction of the objective lens to form an illumination plane; driving at least one of the objective lens and a stage holding the sample in the optical axis direction and adjusting the position of the illumination plane in the optical axis direction based on the detection result of the first light from the sample; and determining the refractive index of the sample based on at least one of the drive amount of the objective lens and the drive amount of the stage and the adjustment amount of the illumination plane.

[0012] The above summary of the invention does not list all of the features of the present invention. Also, subcombinations of these features may also be inventions. [Brief description of the drawings]

[0013] [Figure 1] 1 shows a schematic configuration of a microscope according to a first embodiment. [Diagram 2] The functional configuration of the control system of the microscope is shown. [Diagram 3] 1 shows the positional relationship between the sheet illumination and the objective lens. [Figure 4] 2 shows a schematic configuration of a position detection unit. [Diagram 5] 13 shows a schematic configuration of an illumination unit according to a modified example. [Figure 6]4 shows a procedure for correcting an illumination surface when a stage drift occurs according to the first embodiment. [Figure 7A] 1 shows one state (ideal state) of illumination surface correction when stage drift occurs. [Figure 7B] This shows one stage of illumination surface correction during stage drift (stage drift state). [Figure 7C] This shows one stage of illumination plane correction (state after detection plane correction) when the stage drifts. [Figure 7D] This shows one stage of illumination surface correction during stage drift (state after illumination surface correction). [Figure 8] 13 shows a procedure for correcting an illumination plane during Z-stack imaging according to the second embodiment. [Figure 9A] 1 shows one state (normal state) of illumination plane correction during Z-stack imaging. [Figure 9B] 1 shows one state of illumination plane correction during Z-stack imaging (state after objective lens is driven). [Figure 9C] FIG. 1 shows one state of illumination surface correction during Z-stack imaging (state after illumination light correction). [Figure 10] 13 shows a procedure for correcting an illumination plane during Z-stack imaging according to the third embodiment. [Figure 11A] 1 shows one state (normal state) of illumination plane correction during Z-stack imaging. [Figure 11B] FIG. 1 shows one state of illumination plane correction during Z-stack imaging (state after stage movement). [Figure 11C] FIG. 1 shows one state of illumination surface correction during Z-stack imaging (state after illumination light correction). [Figure 12] 1 illustrates an example of a configuration of a computer according to an embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention will be described below through embodiments of the invention, but the following embodiments do not limit the scope of the invention according to the claims. Furthermore, not all of the combinations of features described in the embodiments are necessarily essential to the solution of the invention.

[0015] First embodiment 1 and 2 respectively show the configuration of a light sheet microscope (also simply referred to as a microscope) 100 according to the first embodiment and the functional configuration of a control unit 50. The microscope 100 includes an illumination unit 10, a drive unit 20, a detection unit 30, a position detection unit 40, and a control unit 50. Here, in the microscope 100, the illumination unit 10 and the detection unit 30 each have an independent optical system, that is, an illumination optical system 12 and a detection optical system 32. The optical axis 30L of the detection optical system 32 intersects with the optical axis 10L of the illumination optical system 12 (orthogonal, as an example, in this embodiment). The direction parallel to the optical axis 10L is the Y-axis direction, the direction parallel to the optical axis 30L is the Z-axis direction, and the direction perpendicular to these is the X-axis direction.

[0016] The sample 9 is supported on the stage 21 via a support member (see FIG. 4) such as a sample container 3 that contains the sample 9. The sample 9 may be a biological cell, for example, and may be a stationary or non-stationary object. However, in this embodiment, the sample 9 is a stationary object. Also, a surface of the sample 9 that is to be observed is called an observation surface 9S.

[0017] The illumination unit 10 is a unit having a light source 11 and an illumination optical system 12, and illuminating light 10a is incident on the sample 9 via the illumination optical system 12 to define an illumination plane 10S (see FIGS. 3, 7A, etc.) within the sample 9.

[0018] The light source 11 may be a laser light source that generates, for example, coherent light as the illumination light 10a.

[0019] The illumination optical system 12 is an optical system that guides the illumination light 10 a to the sample 9 on the stage 21 , and includes, for example, an expander 13 , a reflecting element 14 , a relay lens 15 , and a cylindrical lens 16 .

[0020] The expander 13 has a plurality of lens elements and the like, and expands or reduces the beam diameter of the illumination light 10a. If there is no need to expand or reduce the beam diameter of the illumination light 10a, the expander 13 does not need to be provided in the illumination optical system 12.

[0021] The reflecting element 14 reflects the illumination light 10a and distributes it in the Z-axis direction. The reflecting element 14 includes, as an example, a mirror element such as a galvanometer mirror and a reflecting element driver 14a such as a motor that rotates the mirror surface around the X-axis. The reflecting element 14 may be an acousto-optical element (AOM), a spatial light modulator (SLM), or the like. Also, instead of the reflecting element 14, a transmitting element such as a transmission type phase modulation element may be used. The reflecting element 14 and the transmitting element are examples of members or optical elements that set or adjust the illumination surface 10S.

[0022] The relay lens 15 has a plurality of lens elements and the like, and guides the illumination light 10 a reflected by the reflecting element 14 to the cylindrical lens 16 .

[0023] The cylindrical lens 16 has refractive power only in the Z-axis direction, and focuses the illumination light 10a in the Z-axis direction to form it into a sheet-like shape, which is then introduced into the sample 9 from a direction intersecting the optical axis 30L of the detection optical system 32 (in this example, toward the Y-axis direction).

[0024] The illumination optical system 12 may include an objective lens on the sample 9 side of the cylindrical lens 16 .

[0025] 3, the illumination optical system 12 uses the above-mentioned optical element group to form an illumination plane 10S parallel to the XY plane within the sample 9, and irradiates illumination light 10a onto the illumination plane 10S along the Y-axis direction. The position of the illumination plane 10S can be adjusted in the Z-axis direction by controlling the reflection element driving device 14a to rotate the reflection element 14 constituting the illumination optical system 12 around the X-axis.

[0026] The driving unit 20 is a unit that drives a stage 21 that supports the sample 9 at least in the Z-axis direction, and includes the stage 21 and a driving device 22 .

[0027] The stage 21 is configured to be movable (i.e., raised and lowered) at least in the Z-axis direction while supporting the sample 9. The stage 21 has an opening (not shown) through which the detection light 30a passes, and supports the sample 9 above it.

[0028] The driving device 22 drives the stage 21 at least in the Z-axis direction. For example, an electric motor or the like can be used as the driving device 22. The driving device 22 is controlled by the control unit 50 (drive control unit 51) to drive the stage 21 to a target position. As a result, the sample 9 on the stage 21 moves along the optical axis 30L of the detection optical system 32.

[0029] The detection unit 30 is a unit that detects light (e.g., fluorescence emitted by the sample 9 upon excitation by illumination light) 30a emitted from a detection surface 30S (see FIGS. 3, 7A, etc.) defined in the sample 9 via a detection optical system 32. This light is also called detection light. The detection unit 30 has a detection device 31 and a detection optical system 32.

[0030] The detection device 31 detects the detection light 30a from the sample 9 via a detection optical system 32 to generate a detection image. As the detection device 31, an imaging element such as a charge-coupled device (CCD) or a CMOS sensor may be used.

[0031] The detection optical system 32 is an optical system that guides the detection light 30a from the sample 9 to the detection device 31, and includes, for example, an objective lens 33, an imaging lens 35, and a filter 36. The detection optical system 32 may be either an inverted system or an upright system.

[0032] The objective lens 33 is an optical element that forms an image of the sample 9 on the stage 21, and in this embodiment, it is arranged directly below the stage 21 so as to be movable in the Z-axis direction. Here, an immersion liquid such as oil or water is filled between the tip lens included in the objective lens 33 and the support member of the sample 9. It is not essential to fill the space between the tip lens included in the objective lens 33 and the support member of the sample 9 with immersion liquid, and air may be filled. The objective lens 33 includes a lens barrel that holds a group of lens elements therein, and can be moved by a lens driving device 33a such as an electric motor that drives the lens barrel in the Z-axis direction. Here, the detection plane 30S is defined parallel to the XY plane including the focal point of the objective lens 33, and can be moved in the Z-axis direction by controlling the lens driving device 33a to drive the objective lens 33 that constitutes the detection optical system 32 in the Z-axis direction.

[0033] The imaging lens 35 focuses the detection light 30 a sent via the objective lens 33 onto the light receiving surface of the detection device 31 , and generates a detection image of the sample 9 thereon.

[0034] The filter 36 transmits only light of a specific wavelength, for example, so that only the fluorescence emitted by the sample 9 is transmitted.

[0035] The detection optical system 32 defines a detection plane 30S parallel to the XY plane within the sample 9 by the above-mentioned optical element group, and sends detection light 30a emitted downward from there to the detection device 31. Here, the position of the detection plane 30S can be moved in the Z-axis direction by controlling the lens driving device 33a to drive the objective lens 33 constituting the detection optical system 32 in the Z-axis direction.

[0036] 1, the position detection unit 40 is a unit that detects information regarding the position of the sample 9 or the stage 21 in the Z-axis direction. The position detection unit 40 employs a configuration in which detection is performed particularly via at least one of the optical elements that configure the detection optical system 32, that is, the objective lens 33 in this embodiment.

[0037] 4 shows a schematic configuration of the position detection unit 40. The position detection unit 40 is a unit that detects the position of the bottom surface 3a of the sample container 3 that holds the sample 9, and includes an illumination optical system for focusing 40A, an imaging optical system for focusing 40B, a photoelectric converter 130, and a signal processing unit 41. The illumination optical system for focusing 40A and the imaging optical system for focusing 40B are collectively referred to as the optical system for focusing.

[0038] The focusing illumination optical system 40A is an optical system that irradiates probe light onto the bottom surface 3a of the sample container 3 on the stage 21, and includes, in order on its optical axis 40L, a light source 120, a first collector lens 121, a slit plate 122, a second collector lens 123, a first pupil limiting mask 124, a half mirror 125, an offset lens (also simply called a lens) 42, a visible light cut filter 43, and a dichroic mirror 34.

[0039] When a probe light (e.g., infrared light or near-infrared light) is emitted from the light source 120, the probe light is collected by the first collector lens 121 and enters the slit plate 122. The slit plate 122 has a rectangular slit aperture 122a at its center, and is disposed about the optical axis 40L such that the slit aperture 122a is at a position conjugate with the interface of the sample 9 and its longitudinal direction extends in a direction perpendicular to the paper surface in Fig. 4. The probe light is shaped into the shape of the slit aperture 122a by passing through the slit aperture 122a.

[0040] The probe light that has passed through slit aperture 122a is shaped into parallel light via second collector lens 123 and is irradiated onto first pupil limiting mask 124. First pupil limiting mask 124 is an optical element that blocks half of the pupil, and is disposed along the direction perpendicular to the paper surface in Fig. 4 with optical axis 40L as its center. Half of the probe light is blocked by first pupil limiting mask 124.

[0041] The probe light La that has passed through the first pupil limiting mask 124 passes through a half mirror 125. The half mirror 125 is an optical element that is disposed at a point where the optical axes of the focusing illumination optical system 40A and the focusing imaging optical system 40B intersect, reflects a part of the probe light, and transmits the rest, and is shared with the focusing imaging optical system 40B as described later.

[0042] The probe light La transmitted through the half mirror 125 undergoes a range factor change at the offset lens 42 (the function of the offset lens 42 will be described in detail later), the visible light component contained in the probe light La is removed through the visible light cut filter 43, and the probe light La is reflected by the dichroic mirror 34 and guided to the objective lens 33. The dichroic mirror 34 is disposed at the point where the optical axis 30L of the detection optical system 32 and the optical axis 40L of the focusing illumination optical system 40A intersect. Note that, by being disposed on the optical axis 30L of the detection optical system 32, the dichroic mirror 34 reflects the probe light La toward the objective lens 33 and transmits other light (particularly the detection light 30a).

[0043] The probe light Lb reflected by the dichroic mirror 34 is collected by the objective lens 33 and enters the bottom surface 3a of the sample container 3 on the stage 21 (see FIG. 1). Here, the imaging position in the Z-axis direction of the optical image based on the probe light Lb by the objective lens 33 is referred to as imaging position A. The probe light Lb is irradiated onto the bottom surface 3a of the sample container 3. In the following description, the bottom surface 3a is also referred to as a reflecting surface, an interface, or a boundary surface.

[0044] The focusing imaging optical system 40B is an optical system that receives the probe light that is irradiated onto the bottom surface 3a of the sample container 3 by the focusing illumination optical system 40A as described above and reflected by the bottom surface 3a. Here, the reflected probe light Ld passes through the objective lens 33, is reflected by the dichroic mirror 34, has visible light components removed via the visible light cut filter 43, enters the half mirror 125 via the offset lens 42, and is partially reflected and guided to the focusing imaging optical system 40B. The focusing imaging optical system 40B includes the half mirror 125, the autofocus objective lens 126, the autofocus relay lens 127a, the second pupil limiting mask 128, the autofocus relay lens 127b, and the cylindrical lens 129, which are arranged along the optical axis.

[0045] The probe light Ld reflected by the half mirror 125 is collected by the autofocus objective lens 126 and converted into imaging light, forming a slit image (light image). The slit image (the probe light Le forming the slit image) is relayed by the autofocus relay lenses 127a and 127b, and is re-imaged on the imaging surface of the photoelectric converter 130 via the cylindrical lens 129. Here, the second pupil limiting mask 128 is disposed between the autofocus relay lenses 127a and 127b so as to block half of the pupil. The light-blocked area corresponds to the area blocked by the first pupil limiting mask 124 described above. The cylindrical lens 129 is a lens that has a refracting effect only in a predetermined direction, and compresses the probe light Le in the direction perpendicular to the paper surface in FIG. 4 (the longitudinal direction of the slit image) to form an image on the imaging surface of the photoelectric converter 130.

[0046] The photoelectric converter 130 may be, for example, a line CCD sensor (charge-coupled device), which receives the probe light reflected by the bottom surface 3a (reflective surface) of the sample container 3, converts it into an electrical signal, and outputs the electrical signal to the signal processing unit 41.

[0047] The signal processing unit 41 performs signal processing on the electrical signal from the photoelectric converter 130 to generate a detection signal corresponding to the electrical signal, and outputs the generated detection signal to the control unit 50.

[0048] A driving unit that moves the position detection unit 40 together with the objective lens 33 in the Z-axis direction may be provided.

[0049] The function of the offset lens 42 included in the focusing illumination optical system 40A will be described in detail. The focusing illumination optical system 40A has a pair of convex and concave lenses (both not shown), and the lens driving unit 44 moves, for example, the concave lens relative to the convex lens in the optical axis 40L direction based on a control signal from the control unit 50, thereby moving the imaging position A of the optical image based on the probe light irradiated on the reflecting surface via the objective lens 33 in the Z-axis direction relative to the focal position F of the objective lens 33. By moving the imaging position A of the optical image in this manner, the offset value (the amount of deviation between the imaging position A and the focal position F) can be adjusted, thereby positioning the imaging position A on the reflecting surface and positioning the focal position F inside the sample 9 away from the reflecting surface by a certain amount in the Z-axis direction.

[0050] For example, if the stage 21 drifts so that the sample 9 (reference surface) gradually descends, a change in reflected light due to the descending reference surface is detected by the photoelectric converter 130. The control unit 50 gradually lowers the focal position F of the objective lens 33 via the lens driving device 33a based on the change in the detection signal output from the signal processing unit 42. This makes it possible to maintain a constant distance from the reference surface to the focal position F of the objective lens 33 (i.e., an offset value).

[0051] The control unit 50 is a computer device such as a personal computer, and has at least a central processing unit (CPU). The CPU executes a dedicated program to cause the control unit 50 to realize a function of controlling each component of the microscope 100. The dedicated program is, for example, stored in a ROM and read by the CPU, or stored in a storage medium such as a DVD-ROM and is started by the CPU reading the program using a reading device such as a DVD-ROM drive and expanding the program in a RAM. A more detailed example of the hardware configuration of the computer device will be described later. The control unit 50 includes a drive control unit 51, an illumination control unit 52, a detection control unit 53, an image processing unit 54, and a refractive index determination unit 55.

[0052] The drive control unit 51 controls the drive unit 20 (drive device 22) to drive the stage 21 at least in the Z-axis direction. When the drive device 22 receives the target drive amount from the drive control unit 51, it drives the stage 21 by the target drive amount. As a result, the sample 9 on the stage 21 moves along the optical axis 30L of the detection optical system 32 to the target position.

[0053] In observation of the sample 9, the drive control unit 51 determines a target drive amount to a target position, and thereby drives and controls the stage 21 or the objective lens 33 to maintain the position of the observation surface 9S in the sample 9 at the target position. In addition, in Z-stack imaging, the drive control unit 51 determines a target drive amount (Z step amount) to the next Z stack position, and thereby drives and controls the stage 21 or the objective lens 33 to change the observation surface 9S in the sample 9 to the next Z stack position.

[0054] The illumination control unit 52 controls the illumination optical system 12 to adjust the position of the illumination surface 10S in the Z-axis direction. In this embodiment, the illumination control unit 52 controls the reflective element driving device 14a based on the detection result of the drive amount of the objective lens 33 to rotate the reflective element 14 constituting the illumination optical system 12 around the X-axis. Thereby, for example, when the stage 21 supporting the sample 9 drifts in the Z-axis direction, the illumination control unit 52 controls the illumination optical system 12 to adjust the position of the illumination surface 10S in the optical axis direction, thereby making it possible to match the illumination surface 10S with the observation surface 9S within the sample 9.

[0055] In addition, the illumination control unit 52 can detect the position of the illumination surface 10S in the Z-axis direction based on the relationship between the inclination of the reflective element 14 and the reflection direction of the illumination light 10a by measuring the inclination of the reflective element 14 using a sensor such as an interferometer.

[0056] The detection control unit 53 controls the lens driving device 33a to drive the objective lens 33, thereby moving the imaging position (imaging plane) of the image formed by the probe light in the Z-axis direction so that the imaging plane coincides with the reflecting surface. Here, the position of the reflecting surface is identified by the position detection unit 40 based on the image detected by the probe light. Thereby, when the stage 21 supporting the sample 9 drifts in the Z-axis direction, the detection control unit 53 moves the objective lens 33 in the Z-axis direction based on the detection result by the position detection unit 40 so that the imaging plane coincides with the position of the reflecting surface, thereby making it possible to align the detection plane 30S of the objective lens to a position a certain distance away from the reflecting surface in the Z-axis direction. The amount of deviation between the imaging plane and the detection plane is the offset value.

[0057] The image processing unit 54 processes the detection result of the detection light 30a detected by the detection unit 30 to generate a detection image of the sample 9 on the observation surface 9S. The detection image may be displayed on a display device (not shown).

[0058] The refractive index determination unit 55 determines the refractive index of the sample 9. Here, due to a mismatch in the refractive index between the sample 9 and the container, cover glass, immersion liquid, etc. that contains the sample 9, when the stage 21 supporting the sample 9 is driven in the Z-axis direction, the detection surface 30S of the objective lens 33 in the sample 9 also moves slightly (the displacement (i.e., the optical distance) of the detection surface 30S in the sample 9 does not match the driving amount of the stage 21), and the detection surface 30S deviates from the illumination surface 10S. Conversely, by driving the stage 21 supporting the sample 9 in the Z-axis direction and adjusting the position of the illumination surface 10S so that it matches the detection surface 30S that has moved accordingly, the refractive index of the sample 9 can be determined from the relationship between the driving amount of the stage 21 and the adjustment amount of the illumination surface 10S (equal to the displacement of the detection surface 30S).

[0059] The refractive index determination unit 55 may hold refractive index data that associates the relationship between the driving amount of the stage 21 and the displacement of the detection surface 30S with the refractive index. The refractive index data may be in the form of a look-up table. The refractive index data is obtained by holding a sample having a known refractive index on the stage 21 using the microscope 100, driving the stage 21 in the Z-axis direction, adjusting the position of the illumination surface 10S so that it coincides with the detection surface 30S based on the detection light 30a (detection image) emitted by the detection unit 30 from the detection surface 30S in the sample 9, calculating the relationship (for example, ratio) between the driving amount of the stage 21 and the adjustment amount of the illumination surface 10S (equal to the displacement of the detection surface 30S), and storing the result in association with the refractive index of the sample. Similar measurements are performed on samples having different refractive indexes. Note that the refractive index data may be created by optical simulation instead of actual measurement using the microscope 100.

[0060] The refractive index determination unit 55 supports the sample 9 having an unknown refractive index on the stage 21, and adjusts the position of the illumination surface 10S in the Z-axis direction based on the detection light 30a (detection image) emitted from the detection surface 30S in the sample 9 by the detection unit 30 via the illumination control unit 52, and determines, by referring to the above-mentioned refractive index data, an estimated refractive index corresponding to the relationship (e.g., ratio) between the driving amount of the stage 21 and the adjustment amount of the illumination surface 10S as the refractive index of the sample 9.

[0061] In the above description, the refractive index determination unit 55 determines the refractive index of the sample 9 by driving the stage 21 in the Z-axis direction. However, the refractive index of the sample 9 can also be determined by a method similar to that described above when the objective lens 33 is driven in the Z-axis direction.

[0062] Instead of forming the illumination light 10a into a sheet shape, the illumination optical system 12 may be configured to scan the illumination light 10a in the X-axis direction.

[0063] 5 shows a schematic configuration of an illumination optical system 12d according to a modified example. The illumination optical system 12d has a reflecting element 17, an expander 13, a reflecting element 14, a relay lens 15, and a condenser lens 18. Here, the expander 13, the reflecting element 14, and the relay lens 15 are configured in the same manner as those described above.

[0064] The reflecting element 17 reflects the illumination light 10a and distributes it in the X-axis direction. The reflecting element 17 includes, for example, a mirror element such as a galvanometer mirror and a reflecting element driver 17a such as a motor that rotates the mirror surface around an axis tilted at 45 degrees with respect to the X-axis and Y-axis. The reflecting element 17 may be an acousto-optic element (AOM), a spatial light modulator (SLM), or the like. Also, a transmitting element such as a transmission type phase modulation element may be used instead of the reflecting element 14.

[0065] The condenser lens 18 condenses the illumination light 10 a transmitted via the relay lens 15 onto the sample 9 .

[0066] The illumination optical system 12d forms an illumination plane 10S parallel to the XY plane within the sample 9 by scanning the illumination light 10a in the X-axis direction using the reflecting element 17 while focusing the illumination light 10a within the sample 9 using the focusing lens 18. Note that in the illumination optical system 12d, the position of the illumination plane 10S can be adjusted in the Z-axis direction by controlling the reflecting element driving device 14a to rotate the reflecting element 14 around the X-axis.

[0067] In addition, the position of the illumination surface 10S may be adjusted in the Z-axis direction not only by rotating the reflective element 14 that constitutes the illumination optical system 12, but also by driving the entire illumination optical system 12 or some of the optical elements that constitute it, such as the reflective element 14, in the Z-axis direction.

[0068] 6 shows a procedure for correcting the illumination plane 10S when the stage drifts according to the first embodiment. It is assumed that the sample 9 is supported on the stage 21 via a support member (not shown) such as a container for containing the sample 9. Here, one surface of the sample 9 to be observed is assumed to be an observation surface 9S.

[0069] In step S102, as in the ideal state shown in Fig. 7A, the illumination unit 10 applies illumination light 10a to an illumination plane 10S in the sample 9 in the Y-axis direction via the illumination optical system 12 that forms the illumination plane 10S. The illumination plane 10S is assumed to coincide with the observation plane 9S by controlling the illumination optical system 12 by the illumination control unit 52. In addition, the detection plane 30S of the objective lens 33 is assumed to coincide with the observation plane 9S by controlling the lens driving device 33a by the detection control unit 53 to drive the objective lens 33.

[0070] 7B, assume that the stage 21 drifts in the Z-axis direction (-Z direction in this example) due to vibrations applied to the microscope 100, expansion or contraction of components due to heat, etc. Due to this stage drift, the sample 9 supported on the stage 21 also drifts in the -Z direction, and the illumination plane 10S and detection plane 30S shift from the observation plane 9S.

[0071] In step S104, information (light image based on the probe light) relating to the position of the sample 9 (or the stage 21) in the Z-axis direction is detected by the position detection unit 40. The detection result is transmitted to the control unit 50.

[0072] In step S106, as shown in FIG. 7C, the detection control unit 53 controls the detection optical system 32 based on the detection result (also simply referred to as the "detection result") of the information (optical image based on the probe light) on the position of the sample 9 (or the stage 21) in the Z-axis direction by the position detection unit 40 to drive the objective lens 33 in the Z-axis direction. As a result, the objective lens 33 is driven in the -Z direction, and the detection surface 30S is moved in the -Z direction accordingly to coincide with the observation surface 9S (the imaging position of the optical image based on the probe light coincides with the reflection surface). However, the illumination surface 10S remains shifted from the observation surface 9S. After driving the detection surface 30S, the detection control unit 53 may transmit the driving amount of the objective lens 33 to the illumination control unit 52.

[0073] 7D, in step S108, illumination control unit 52 controls illumination optical system 12 based on the detection result by position detection unit 40 to adjust the position of illumination surface 10S in the Z-axis direction. Thereby, reflection element driving device 14a is controlled to rotate reflection element 14 constituting illumination optical system 12 around the X-axis, thereby adjusting illumination surface 10S in the -Z direction to coincide with observation surface 9S.

[0074] In addition, steps S106 and S108 may be executed in the reverse order. In such a case, the illumination control unit 52 may transmit the adjustment amount of the illumination surface 10S to the detection control unit 53 after adjusting the illumination surface 10S.

[0075] In step S110, the detection unit 30 detects the detection light 30a emitted from the detection surface 30S in the sample 9 via the detection optical system 32 that defines the detection surface 30S. The detection result is transmitted to the control unit 50 and processed by the image processing unit 54 to generate a detection image.

[0076] The illumination control unit 52 may quickly roughly adjust the position of the illumination surface 10S based on the detection result by the position detection unit 40, and then precisely fine-tune the position of the illumination surface 10S by the detection unit 30 based on the detection light 30a (detection image) emitted from the detection surface 30S in the sample 9. This allows the position of the illumination surface 10S to be quickly and precisely adjusted.

[0077] Further, instead of controlling the detection optical system 32 based on the detection result by the position detector 40 to move the detection surface 30S in the Z-axis direction (the objective lens 33 is driven in the -Z direction, and the detection surface 30S is moved in the -Z direction accordingly to coincide with the observation surface 9S (the imaging position of the optical image based on the infrared light coincides with the reflecting surface)), the detection control unit 53 may control the stage 21 based on the detection result by the position detector 40 to move the detection surface 30S in the Z-axis direction (the stage 21 is driven in the +Z direction, and the detection surface 30S is moved in the -Z direction accordingly to coincide with the observation surface 9S (the imaging position of the optical image based on the probe light coincides with the reflecting surface)). When the stage 21 is controlled, it is not necessary to control the illumination optical system 12 by the illumination control unit 52 to adjust the position of the illumination surface 10S in the Z-axis direction.

[0078] According to the microscope 100 of the first embodiment, the microscope 100 has a detection optical system 32 having an objective lens 33 and receiving detection light 30a from a sample 9, an illumination optical system 12 that irradiates illumination light 10a onto the sample 9 from a direction intersecting the optical axis direction of the objective lens 33 to form an illumination plane 10S, a position detection unit 40 that detects information about the position of the sample 9 in the optical axis direction, and a control unit 50, and the control unit 50 performs a first control to set the focal position of the objective lens 33 in the detection optical system 32 to a predetermined position on the sample 9 by moving at least one of the objective lens 33 and the stage 21 that holds the sample 9 in the optical axis direction of the objective lens 33 based on the detection result by the position detection unit 40, and when the objective lens 33 has been moved in the optical axis direction, further controls the illumination optical system 12 to perform a second control to set the illumination plane 10S to a predetermined position on the sample 9. According to this, if the sample 9 or the stage 21 supporting it drifts in the Z-axis direction, the control unit 50 can control the illumination optical system 12 to set the illumination surface 10S to a predetermined position on the sample 9, thereby maintaining the position of the illumination surface 10S within the sample 9.

[0079] According to the observation method of the first embodiment, the steps include receiving detection light 30a from the sample 9 through a detection optical system 32 having an objective lens 33, irradiating illumination light 10a onto the sample 9 through an illumination optical system 12 from a direction intersecting the optical axis direction of the objective lens 33 to form an illumination plane 10S, detecting information about the position of the sample 9 in the optical axis direction, and moving at least one of the objective lens 33 and the stage 21 holding the sample 9 in the optical axis direction based on the detection result of the information about the position of the sample 9 in the optical axis direction to set the focal position of the objective lens 33 in the detection optical system 32 to a predetermined position on the sample 9, and when the objective lens 33 is moved in the optical axis direction, the illumination optical system 12 is further controlled to set the illumination plane 10S to the predetermined position on the sample 9. According to this, when the sample 9 or the stage 21 supporting it drifts in the Z-axis direction, the illumination optical system 12 is controlled to set the illumination plane 10S to the predetermined position on the sample 9, thereby making it possible to maintain the position of the illumination plane 10S in the sample 9.

[0080] Second Embodiment In order to perform Z-stack imaging, that is, to generate a Z-stack image by changing the depth of the observation plane 9S in the sample 9 and detecting an image at each depth, when the focal position (detection plane 30S) of the objective lens is moved by a Z interval, the detection plane 30S of the objective lens may also move slightly and become misaligned with the illumination plane 10S due to a mismatch (also called index mismatch) between the refractive index of the sample 9 and the container (also called sample container 3) that contains it, the cover glass, the immersion liquid, etc. In such a case, based on the refractive index of the sample 9 and the detection result by the position detection unit 40 (detection result of the optical image by the probe light), the illumination control unit 52 may determine a correction amount for the position of the illumination plane 10S, and based on the correction amount, control the illumination optical system 12 to adjust the position of the illumination plane 10S in the Z-axis direction, thereby correcting the misalignment and making the illumination plane 10S coincide with the detection plane 30S.

[0081] 8 shows a procedure for correcting the illumination plane 10S during Z-stack imaging according to the second embodiment. The microscope 100 is assumed to be configured as described above. The sample 9 is assumed to be supported on the stage 21 via a support member, such as a sample container 3, that accommodates the sample 9. Here, one surface within the sample 9 to be observed is assumed to be an observation surface 9S. In the Z-stack imaging, optical cross-sectional images of the sample 9 are assumed to be detected at N different depths.

[0082] In step S201, the control unit 50 sets a counter n to 1.

[0083] 9A, in step S202, the illumination unit 10 applies illumination light 10a to an illumination surface 10S in the sample 9 in the Y-axis direction via the illumination optical system 12 that forms the illumination surface 10S. The illumination surface 10S is made to coincide with the observation surface 9S by controlling the illumination optical system 12 by the illumination control unit 52. Furthermore, based on the detection result by the position detection unit 40, the detection surface 30S is made to coincide with the observation surface 9S by controlling the detection optical system 32 by the detection control unit 53 (the imaging position of the optical image based on the probe light coincides with the reflection surface).

[0084] 9B, in order to generate an image of the n-th observation surface 9S in the sample 9, the drive control unit 51 drives the objective lens 33 in the Z-axis direction (the +Z direction in this example) in order to generate an image of the n-th observation surface 9S in the sample 9. At this time, based on a change in the detection signal from the signal processing unit 41 accompanying a shift between the imaging position A of the optical image and the position of the reflecting surface due to the movement of the objective lens 33, the control unit 50 outputs a control signal to the lens driving unit 44 to move the offset lens 42 in the direction of the optical axis 40L so as to eliminate the shift between the imaging position A of the optical image and the position of the reflecting surface caused by the movement of the objective lens 33 (the distance from the reflecting surface to the detection surface 30S is the offset value OSn). Also, at this time, due to index mismatch, by driving the objective lens 33 in the Z-axis direction, the detection surface 30S of the objective lens 33 in the sample 9 also moves slightly (the displacement of the detection surface 30S in the sample 9 (i.e., the optical distance) does not match the driving amount of the objective lens 33), and the detection surface 30S of the objective lens 33 deviates from the illumination surface 10S.

[0085] In step S208, the control unit 50 checks whether or not the value of the refractive index of the sample 9 is held. If the value of the refractive index is not held, the process proceeds to step S210, and if the value is held, the process proceeds to step S212.

[0086] In step S210, the refractive index of the sample 9 is determined. The refractive index determination unit 55 drives the stage 21 in the Z-axis direction by a predetermined amount via the drive unit 20, adjusts the position of the illumination surface 10S in the Z-axis direction based on the detection light 30a (detection image) emitted from the detection surface 30S in the sample 9 by the detection unit 30 via the illumination control unit 52, and determines the refractive index of the sample 9 to be a refractive index corresponding to the relationship (for example, ratio) between the drive amount of the stage 21 and the adjustment amount of the illumination surface 10S by referring to the refractive index data. The determined refractive index is transmitted to the illumination control unit 52 and held therein.

[0087] Once the refractive index has been determined, the drive control unit 51 drives the stage 21 to return it to a position for generating an image of the sample 9 on the n-th observation plane 9S in step S204.

[0088] In step S212, when the relative positional relationship between the sample 9 and the objective lens 33 in the optical axis direction has changed as described above, as shown in FIG. 9C, the illumination control unit 52 refers to the refractive index data and determines a correction amount for the position of the illumination plane 10S based on the refractive index of the sample 9 and the change in the offset value (or the movement amount of the offset lens 42), and controls the illumination optical system 12 based on the correction amount to adjust the position of the illumination plane 10S in the Z axis direction, thereby correcting the deviation and making the illumination plane 10S coincident with the detection plane 30S.

[0089] In step S214, the detection unit 30 detects the detection light 30a emitted from the detection surface 30S in the sample 9 via the detection optical system 32 that defines the detection surface 30S. The detection result is transmitted to the control unit 50 and processed by the image processing unit 54 to generate a detection image.

[0090] In step S216, the control unit 50 judges whether the counter n is smaller than N. If n is smaller than N, the process proceeds to step S218, where the counter n is incremented by 1, and the process returns to step S202. If n is equal to N, the flow ends. As a result, cross-sectional images of the sample 9 at N different depths, i.e., Z-stack images, are obtained.

[0091] The microscope 100 according to the second embodiment includes a detection optical system 32 having an objective lens and receiving detection light 30a from a sample 9, an illumination optical system 12 irradiating illumination light 10a onto the sample 9 from a direction intersecting the optical axis 30L direction of the objective lens 33 to form an illumination plane 10S, a position detection unit 40 detecting information regarding the position of the sample 9 in the optical axis 30L direction, and a control unit 50. The control unit 50 moves the objective lens 33 in the optical axis 30L direction to set the focal position of the objective lens 33 in the detection optical system 32 to a predetermined position on the sample 9, and then moves the offset lens 42 of the position detection unit 40 in the optical axis 40L direction to match the imaging position A of the optical image based on the probe light with the position of the reflecting surface, and then drives an optical element included in the illumination optical system 12 to set the irradiation position of the illumination light 10a to the predetermined position on the sample 9. According to this, when the objective lens 33 is driven, the control unit 50 drives the optical elements included in the illumination optical system 12 to set the irradiation position of the illumination light 10a to a predetermined position on the sample 9, thereby maintaining the position of the illumination surface 10S within the sample 9.

[0092] According to the observation method of the second embodiment, the steps include the steps of receiving detection light 30a from sample 9 via detection optical system 32 having an objective lens, irradiating illumination light 10a onto sample 9 via illumination optical system 12 from a direction intersecting with the optical axis 30L direction of objective lens 33 to form an illumination plane 10S, detecting information about the position of sample 9 in the optical axis 30L direction, setting the focal position of objective lens 33 in detection optical system 32 to a predetermined position on sample 9 by moving objective lens 33 in the optical axis 30L direction, and then matching imaging position A of the optical image based on the probe light with the position of the reflecting surface by moving an optical element included in illumination optical system 12. According to this, when the objective lens 33 is driven, the control unit 50 drives the optical elements included in the illumination optical system 12 to set the irradiation position of the illumination light 10a to a predetermined position on the sample 9, thereby maintaining the position of the illumination surface 10S within the sample 9.

[0093] Third embodiment In order to perform Z-stack imaging, i.e., to generate a Z-stack image by changing the depth of the observation plane 9S in the sample 9 and detecting an image at each depth, when the stage 21 supporting the sample 9 or the objective lens 33 is driven in the Z-axis direction, the detection plane 30S of the objective lens may also move slightly and become misaligned from the illumination plane 10S due to a mismatch (also called index mismatch) in the refractive index between the sample 9 and the container (also called sample container 3) that contains it, the cover glass, the immersion liquid, etc. In such a case, the illumination control unit 52 may determine a correction amount for the position of the illumination plane 10S based on the refractive index of the sample 9 and the driving amount of the stage 21 or the objective lens 33, and may control the illumination optical system based on the correction amount to adjust the position of the illumination plane 10S in the Z-axis direction, thereby correcting the misalignment and making the illumination plane 10S coincide with the detection plane 30S.

[0094] 10 shows a procedure for correcting the illumination plane 10S during Z-stack imaging according to the third embodiment. The microscope 100 is assumed to be configured as described above. The sample 9 is assumed to be supported on the stage 21 via a support member, such as a sample container 3, that accommodates the sample 9. Here, one surface within the sample 9 to be observed is assumed to be an observation surface 9S. In the Z-stack imaging, optical cross-sectional images of the sample 9 are assumed to be detected at N different depths.

[0095] In step S301, the control unit 50 sets a counter n to 1.

[0096] 11A, the illumination unit 10 applies illumination light 10a to an illumination surface 10S in the sample 9 in the Y-axis direction via an illumination optical system 12 that forms the illumination surface 10S. The illumination surface 10S is assumed to coincide with the observation surface 9S by controlling the illumination optical system 12 by the illumination control unit 52.

[0097] In step S306, as shown by a white arrow in Fig. 11B, in order to generate an image of the n-th observation surface 9S in the sample 9, the drive control unit 51 drives the stage 21 by a predetermined amount in the Z-axis direction (-Z direction in this example). Alternatively, as shown in Fig. 9B, the objective lens 33 may be driven by a predetermined amount in the Z-axis direction. At this time, due to index mismatch, by driving the stage 21 supporting the sample 9 or the objective lens 33 in the Z-axis direction, the detection surface 30S of the objective lens 33 in the sample 9 also moves slightly (the displacement (i.e., the optical distance) of the detection surface 30S in the sample 9 does not match the driving amount of the stage 21), and the detection surface 30S of the objective lens 33 is shifted from the illumination surface 10S.

[0098] In step S308, the control unit 50 checks whether or not the value of the refractive index of the sample 9 is held. If the value of the refractive index is not held, the process proceeds to step S310, and if the value is held, the process proceeds to step S312.

[0099] In step S310, the refractive index of the sample 9 is determined. The refractive index determination unit 55 drives the stage 21 in the Z-axis direction by a predetermined amount via the drive unit 20, adjusts the position of the illumination surface 10S in the Z-axis direction based on the detection light 30a (detection image) emitted from the detection surface 30S in the sample 9 by the detection unit 30 via the illumination control unit 52, and determines the refractive index of the sample 9 to be a refractive index corresponding to the relationship (for example, ratio) between the drive amount of the stage 21 and the adjustment amount of the illumination surface 10S by referring to the refractive index data. The determined refractive index is transmitted to the illumination control unit 52 and held therein.

[0100] Once the refractive index has been determined, the drive control unit 51 drives the stage 21 to return it to a position for generating an image of the sample 9 on the n-th observation plane 9S in step S306.

[0101] In step S312, when the relative positional relationship between the sample 9 and the objective lens 33 in the optical axis direction has changed as described above, as shown in FIG. 11C, the illumination control unit 52 refers to the refractive index data to determine a correction amount for the position of the illumination plane 10S based on the refractive index of the sample 9 and the driving amount of the stage 21 or the objective lens 33, and controls the illumination optical system 12 based on the correction amount to adjust the position of the illumination plane 10S in the Z axis direction, thereby correcting the deviation and making the illumination plane 10S coincident with the detection plane 30S.

[0102] If the correction amount is small, step S312 may be skipped, or step S312 may be executed every several times, such as executing step S312 only when n is an even number.

[0103] In step S314, the detection unit 30 detects the detection light 30a emitted from the detection surface 30S in the sample 9 via the detection optical system 32 that defines the detection surface 30S. The detection result is transmitted to the control unit 50 and processed by the image processing unit 54 to generate a detection image.

[0104] In step S316, the control unit 50 determines whether the counter n is smaller than N. If n is smaller than N, the process proceeds to step S318, where the counter n is incremented by 1, and the process returns to step S302. If n is equal to N, the flow ends. As a result, cross-sectional images of the sample 9 at N different depths, i.e., Z-stack images, are obtained.

[0105] The microscope 100 according to the third embodiment includes a detection optical system 32 having an objective lens 33 and receiving detection light 30a from a sample 9, an illumination optical system 12 irradiating illumination light 10a onto the sample 9 from a direction intersecting the optical axis direction of the objective lens 33 to form an illumination plane 10S, and a control unit 50. When the relative positional relationship between the sample 9 and the objective lens 33 in the optical axis direction changes, the control unit 50 controls the illumination optical system 12 to adjust the position of the illumination plane 10S in the optical axis direction based on the refractive index of the sample 9. According to this, when the stage 21 supporting the sample 9 or the objective lens 33 is driven in the Z axis direction to move the detection plane 30S within the sample 9, the illumination control unit 52 controls the illumination optical system 12 based on the refractive index of the sample 9 to adjust the position of the illumination plane 10S in the Z axis direction, thereby correcting the deviation between the drive amount of the stage 21 or the objective lens 33 and the movement amount of the detection plane 30S due to index mismatch, and the illumination plane 10S can be made to coincide with the detection plane 30S. At this time, based on the refractive index of the sample 9, a correction amount corresponding to the deviation between the driving amount of the stage 21 or the objective lens 33 due to index mismatch and the movement amount of the observation surface 9S is determined, and by using this, the illumination surface 10S can be made to coincide with the observation surface 9S.

[0106] The observation method according to the third embodiment includes a step of receiving detection light 30a from a sample 9 via a detection optical system 32 having an objective lens 33, a step of irradiating illumination light 10a onto the sample 9 via an illumination optical system 12 from a direction intersecting the optical axis direction of the objective lens 33 to form an illumination plane 10S, and a step of controlling the illumination optical system 12 to adjust the position of the illumination plane 10S in the optical axis direction based on the refractive index of the sample when the relative positional relationship between the sample 9 and the objective lens 33 in the optical axis direction changes. According to this, when the stage 21 supporting the sample 9 or the objective lens 33 is driven in the Z axis direction to move the detection plane 30S within the sample 9, the illumination optical system 12 is controlled based on the refractive index of the sample 9 to adjust the position of the illumination plane 10S in the Z axis direction, thereby correcting the deviation between the drive amount of the stage 21 or the objective lens 33 and the movement amount of the detection plane 30S due to index mismatch, and the illumination plane 10S can be made to coincide with the detection plane 30S. At this time, based on the refractive index of the sample 9, a correction amount corresponding to the deviation between the driving amount of the stage 21 or the objective lens 33 due to index mismatch and the movement amount of the observation surface 9S is determined, and by using this, the illumination surface 10S can be made to coincide with the observation surface 9S.

[0107] The microscope 100 may be a refractive index determining device for determining the refractive index of the sample 9, or the steps S302 to S306 and S310 may be a refractive index determining method for determining the refractive index of the sample 9.

[0108] In the microscope 100 and observation method according to the first to third embodiments, the illumination light 10a is directed to the sample 9 in the +Y direction via the illumination optical system 12. However, it is also possible to provide two illumination optical systems 12, or to split the illumination light 10a guided from the illumination optical system 12 into two and direct them to the sample 9 from both the +Y direction and the -Y direction.

[0109] Various embodiments of the present invention may be described with reference to flow charts and block diagrams, where the blocks may represent (1) stages of a process in which operations are performed or (2) sections of an apparatus responsible for performing the operations. Particular stages and sections may be implemented by dedicated circuitry, programmable circuitry provided with computer readable instructions stored on a computer readable medium, and / or a processor provided with computer readable instructions stored on a computer readable medium. Dedicated circuitry may include digital and / or analog hardware circuitry and may include integrated circuits (ICs) and / or discrete circuits. Programmable circuitry may include reconfigurable hardware circuitry including logical AND, logical OR, logical XOR, logical NAND, logical NOR, and other logical operations, memory elements such as flip-flops, registers, field programmable gate arrays (FPGAs), programmable logic arrays (PLAs), and the like.

[0110] A computer-readable medium may include any tangible device capable of storing instructions that are executed by a suitable device, such that the computer-readable medium having instructions stored thereon comprises an article of manufacture that includes instructions that can be executed to create means for performing the operations specified in the flowcharts or block diagrams. Examples of computer-readable media may include electronic storage media, magnetic storage media, optical storage media, electromagnetic storage media, semiconductor storage media, and the like. More specific examples of computer-readable media may include floppy disks, diskettes, hard disks, random access memories (RAMs), read-only memories (ROMs), erasable programmable read-only memories (EPROMs or flash memories), electrically erasable programmable read-only memories (EEPROMs), static random access memories (SRAMs), compact disk read-only memories (CD-ROMs), digital versatile disks (DVDs), Blu-ray (RTM) disks, memory sticks, integrated circuit cards, and the like.

[0111] The computer readable instructions may include either assembler instructions, Instruction Set Architecture (ISA) instructions, machine instructions, machine-dependent instructions, microcode, firmware instructions, state setting data, or source or object code written in any combination of one or more programming languages, including object-oriented programming languages ​​such as Smalltalk®, JAVA®, C++, etc., and conventional procedural programming languages ​​such as the “C” programming language or similar programming languages.

[0112] Computer readable instructions may be provided to a processor or programmable circuitry of a general purpose computer, special purpose computer, or other programmable data processing apparatus, either locally or over a local area network (LAN), a wide area network (WAN), such as the Internet, etc., to execute the computer readable instructions to create means for performing the operations specified in the flowcharts or block diagrams. Examples of processors include computer processors, processing units, microprocessors, digital signal processors, controllers, microcontrollers, etc.

[0113] 12 illustrates an example of a computer 2200 in which 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 or perform operations associated with an apparatus or one or more sections of the apparatus according to an embodiment of the present invention and / or to perform a process or steps of a process according to an embodiment of the present invention. Such programs may be executed by the CPU 2212 to cause the computer 2200 to perform specific operations associated with some or all of the blocks of the flowcharts and block diagrams described herein.

[0114] 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 connected to each other by a host controller 2210. The computer 2200 also includes input / output units 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.

[0115] The CPU 2212 operates according to a program stored in the ROM 2230 and the RAM 2214, thereby controlling each unit. The graphic controller 2216 retrieves image data generated by the CPU 2212 into a frame buffer or the like provided in the RAM 2214 or into the graphic controller 2216 itself, and causes the image data to be displayed on the display device 2218.

[0116] 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 programs or data from the DVD-ROM 2201 and provides the programs 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 an IC card.

[0117] The ROM 2230 stores therein a boot program or the like executed by the computer 2200 upon activation, and / or a program that depends on the hardware of the computer 2200. The input / output chip 2240 may also connect various input / output units to the input / output controller 2220 via a parallel port, a serial port, a keyboard port, a mouse port, and the like.

[0118] The programs are provided by a computer-readable medium such as a DVD-ROM 2201 or an IC card. The programs are read from the computer-readable medium, installed in the hard disk drive 2224, the RAM 2214, or the ROM 2230, which are also examples of computer-readable media, and executed by the CPU 2212. Information processing described in these programs is read by the computer 2200, and brings about cooperation between the programs and the various types of hardware resources described above. An apparatus or method may be constructed by realizing information manipulation or processing according to the use of the computer 2200.

[0119] For example, when communication is performed 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 transmission data stored in a transmission buffer processing area provided in the RAM 2214, the hard disk drive 2224, the DVD-ROM 2201, or a recording medium such as an IC card, and transmits the read transmission data to the network, or writes reception data received from the network to a reception buffer processing area or the like provided on the recording medium.

[0120] Furthermore, the CPU 2212 may cause all or a necessary portion of a file or database stored in an external recording medium such as the hard disk drive 2224, the DVD-ROM drive 2226 (DVD-ROM 2201), an IC card, etc. to be read into the RAM 2214, and perform various types of processing on the data on the RAM 2214. The CPU 2212 then writes back the processed data to the external recording medium.

[0121] Various types of information, such as various types of programs, data, tables, and databases, may be stored in the recording medium and undergo information processing. The CPU 2212 may perform various types of processing on the data read from the RAM 2214, including various types of operations, information processing, conditional judgment, conditional branching, unconditional branching, information search / replacement, etc., as described throughout this disclosure and specified by the instruction sequence of the program, and write back the results to the RAM 2214. The CPU 2212 may also search for information in a file, database, etc. in the recording medium. 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 medium, the CPU 2212 may search for an entry that matches a condition, in which an attribute value of the first attribute is specified, from among the plurality of entries, read the attribute value of the second attribute stored in the entry, and thereby obtain the attribute value of the second attribute associated with the first attribute that satisfies a predetermined condition.

[0122] The above-described programs or software modules may be stored in a computer-readable medium on the computer 2200 or in the vicinity of the computer 2200. Also, a recording medium such as a hard disk or a 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 programs to the computer 2200 via the network.

[0123] Although the present invention has been described above using the embodiments, the technical scope of the present invention is not limited to the scope described in the above embodiments. It is clear to those skilled in the art that various modifications and improvements can be made to the above embodiments. It is clear from the description of the claims that such modifications and improvements can also be included in the technical scope of the present invention.

[0124] It should be noted that the order of execution of each process, such as operations, procedures, steps, and stages, in the devices, systems, programs, and methods shown in the claims, specifications, and drawings is not specifically stated as "before," "prior to," etc., and may be realized in any order unless the output of a previous process is used in a later process. Even if the operational flow in the claims, specifications, and drawings is explained using "first," "next," etc. for convenience, it does not mean that it is essential to perform the process in this order. [Explanation of symbols]

[0125] 3...sample container, 3a...bottom surface, 9...sample, 9S...observation surface, 10...illumination unit, 10L...optical axis, 10S...illumination surface, 10a...illumination light, 11...light source, 12...illumination optical system, 12d...illumination optical system, 13...expander, 14...reflecting element, 14a...reflecting element driving device, 15...relay lens, 16...cylindrical lens, 17...reflecting element, 17a...reflecting element driving device, 18...condensing lens, 20...driving unit, 21...stage, 22...driving device, 30...detection output portion, 30L...optical axis, 30S...detection surface, 30a...detection light, 31...detection device, 32...detection optical system, 33...objective lens, 33a...lens driving device, 34...dichroic mirror, 35...imaging lens, 36...filter, 40...position detection portion, 40A...illumination optical system for focusing, 40B...imaging optical system for focusing, 40L...optical axis, 41...signal processing portion, 42...offset lens, 42...signal processing portion, 43...visible light cut filter, 44...lens lens drive unit, 50...control unit, 51...drive control unit, 52...illumination control unit, 53...detection control unit, 54...image processing unit, 55...refractive index determination unit, 100...microscope, 120...light source, 122...slit plate, 122a...slit aperture, 125...half mirror, 126...objective lens for autofocus, 127a, 127b...relay lens for autofocus, 129...cylindrical lens, 130...photoelectric converter, 2200...computer, 2201...DVD-ROM, 2210...host controller, 2214...RAM, 2216...graphics 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, La...probe light, Lb...probe light, Ld...probe light, Le...probe light.

Claims

1. a first optical system having an objective lens and receiving a first light from the sample; a second optical system that irradiates the sample with a second light from a direction intersecting with an optical axis direction of the objective lens to form an illumination plane; a position detection unit that detects information regarding a position of the sample in the optical axis direction; A control unit; having The control unit is execute a first control for setting a focal position of the objective lens in the first optical system to a predetermined position of the sample by moving at least one of the objective lens and a stage for holding the sample along the optical axis based on a detection result by the position detection unit; and when the objective lens is moved in the optical axis direction, a second control is executed to set the illumination plane at a predetermined position on the sample by further controlling the second optical system. microscope.

2. a first optical system having an objective lens and receiving a first light from the sample; a second optical system that irradiates the sample with a second light from a direction intersecting with an optical axis direction of the objective lens to form an illumination plane; A control unit; having The control unit is when a relative positional relationship between the sample and the objective lens in the optical axis direction changes, the second optical system is controlled based on a refractive index of the sample to adjust a position of the illumination surface in the optical axis direction. microscope.

3. 3. The microscope according to claim 2, wherein the change in the relative positional relationship is achieved by the control unit driving at least one of the objective lens and a stage that holds the sample in the optical axis direction, and a correction amount for the position of the illumination surface is determined based on at least one of the driving amount of the objective lens and the driving amount of the stage and the refractive index of the sample, and the second optical system is controlled based on the correction amount.

4. a position detection unit that detects information regarding a position of the sample in the optical axis direction, The microscope according to claim 2 , wherein the control unit determines a correction amount for the position of the illumination plane based on the detection result of the position detection unit and the refractive index of the sample, and controls the second optical system based on the correction amount.

5. the control unit drives at least one of the objective lens and a stage for holding the sample in the optical axis direction, and adjusts a position of the illumination surface in the optical axis direction based on a detection result of the first light from the sample; 5. The microscope according to claim 2, further comprising a refractive index determination unit that determines the refractive index of the sample based on at least one of a drive amount of the objective lens and a drive amount of the stage and an adjustment amount of the illumination surface.

6. 5. The microscope according to claim 1, wherein the position detection unit includes a third optical system that includes an offset optical system and focuses a third light at a predetermined position on a support member that supports the sample, and a detection unit that detects the third light reflected at the predetermined position on the support member.

7. The microscope according to claim 1 , wherein the second optical system has a member that sets or adjusts the illumination surface under the control of the control unit.

8. By moving the offset optical system, a focal position of the objective lens is moved to a position different from a predetermined position of the sample; and executing control to set the illumination surface at the different position by driving an optical element included in the second optical system.

7. The microscope according to claim 6.

9. a first optical system having an objective lens and receiving a first light from the sample; a second optical system that irradiates the sample with a second light from a direction intersecting with an optical axis direction of the objective lens to form an illumination plane; a control unit that drives at least one of the objective lens and a stage that holds the sample in the optical axis direction, and adjusts a position of the illumination surface in the optical axis direction based on a detection result of the first light from the sample; a refractive index determining unit that determines a refractive index of the sample based on at least one of a driving amount of the objective lens and a driving amount of the stage and an adjustment amount of the illumination surface; A microscope equipped with

10. receiving a first light from a sample via a first optical system having an objective lens; irradiating the sample with second light from a direction intersecting with an optical axis direction of the objective lens via a second optical system to form an illumination plane; detecting information regarding a position of the sample along the optical axis; a step of executing a first control for setting a focal position of the objective lens in the first optical system to a predetermined position of the sample by moving at least one of the objective lens and a stage for holding the sample in the optical axis direction based on a detection result of information regarding a position of the sample in the optical axis direction; a step of executing a second control for setting the illumination plane at a predetermined position on the sample by further controlling the second optical system when the objective lens is moved in the optical axis direction; An observation method comprising:

11. On the computer, receiving a first light from a sample via a first optical system having an objective lens; irradiating the sample with second light via a second optical system from a direction intersecting with an optical axis direction of the objective lens to form an illumination plane; detecting information regarding a position of the sample in the optical axis direction; a step of executing a first control for setting a focal position of the objective lens in the first optical system to a predetermined position of the sample by moving at least one of the objective lens and a stage for holding the sample in the optical axis direction based on a detection result of information regarding a position of the sample in the optical axis direction; a step of executing a second control for setting the illumination plane at a predetermined position on the sample by further controlling the second optical system when the objective lens is moved in the optical axis direction; A program that executes the following.

12. receiving a first light from a sample via a first optical system having an objective lens; irradiating the sample with second light from a direction intersecting with an optical axis direction of the objective lens via a second optical system to form an illumination plane; when a relative positional relationship between the sample and the objective lens in the optical axis direction changes, adjusting a position of the illumination surface in the optical axis direction by controlling the second optical system based on a refractive index of the sample; A microscope equipped with

13. On the computer, receiving a first light from a sample via a first optical system having an objective lens; irradiating the sample with second light via a second optical system from a direction intersecting with an optical axis direction of the objective lens to form an illumination plane; a step of controlling the second optical system to adjust a position of the illumination surface in the optical axis direction based on a refractive index of the sample when a relative positional relationship between the sample and the objective lens in the optical axis direction changes; A program that executes the following.

14. receiving a first light from a sample through a first optical system having an objective lens; irradiating the sample with second light from a direction intersecting with an optical axis direction of the objective lens via a second optical system to form an illumination plane; driving at least one of the objective lens and a stage for holding the sample in the optical axis direction, and adjusting a position of the illumination surface in the optical axis direction based on a detection result of the first light from the sample; determining a refractive index of the sample based on at least one of a driving amount of the objective lens and a driving amount of the stage and an adjustment amount of the illumination surface; A method for determining a refractive index comprising:

15. On the computer, receiving a first light from a sample via a first optical system having an objective lens; irradiating the sample with second light via a second optical system from a direction intersecting with an optical axis direction of the objective lens to form an illumination plane; driving at least one of the objective lens and a stage for holding the sample in the optical axis direction, and adjusting a position of the illumination surface in the optical axis direction based on a detection result of the first light from the sample; determining a refractive index of the sample based on at least one of a driving amount of the objective lens and a driving amount of the stage and an adjustment amount of the illumination surface; A program that executes the following.

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