microscope

Abstract

This invention provides a microscope that maintains the position of the illumination surface within the sample. [Solution] The microscope 100 has an objective lens 33 and a detection optical system 32 that 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 the optical axis direction of the objective lens to form an illumination surface 10S, a position detection unit 40 that detects information regarding the position of the sample in the optical axis direction, and a control unit 50. The control unit performs a first control to set the focal position of the objective lens in the detection optical system to a predetermined position on the sample by moving at least one of the objective lens and the stage 21 that holds the sample in the optical axis direction of the objective lens based on the detection result by the position detection unit. When the objective lens is moved in the optical axis direction, it further performs a second control to set the irradiation position of the illumination light to a predetermined position on the sample by driving the optical elements included in the illumination optical system. This makes it possible to maintain the position of the illumination surface within the sample.

Description

Technical Field

[0001] The present invention relates to a microscope.

Background Art

[0002] A light sheet microscope that generates an optical cross-sectional image of a sample by introducing sheet-like excitation light (also referred to as illumination light) into the sample from the side and detecting light (also referred to as detection light) emitted from the sample upward or downward is known (see, for example, Non-Patent Document 1). In a light sheet microscope, since the optical system for guiding illumination light to the sample and the optical system for guiding detection light to the detection device are independent, it is necessary to independently control the illumination light and the detection light. Non-Patent Document 1 K. Greger, J. Swoger, E.H.K. 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, there is provided a microscope including: a first optical system having an objective lens and receiving first light from a sample; a second optical system that irradiates the sample with second light from a direction intersecting the optical axis direction of the objective lens to form an illumination surface; a position detection unit that detects information regarding the position of the sample in the optical axis direction; and a control unit. The control unit executes a first control for setting the focal position of the objective lens in the first optical system at a predetermined position of the sample by moving at least one of the objective lens and a stage holding the sample in the optical axis direction based on a detection result by the position detection unit. When the objective lens is moved in the optical axis direction, the control unit further controls the second optical system to execute a second control for setting the illumination surface at a predetermined position of the sample.

[0004] A second embodiment of the present invention provides a microscope comprising: a first optical system having an objective lens and receiving first light from a sample; a second optical system irradiating the sample with second light from a direction intersecting the optical axis direction of the objective lens to form an illumination surface; and a control unit, wherein the control unit controls the second optical system to adjust the position of the illumination surface in the optical axis direction based on the refractive index of the sample when the relative positional relationship between the sample and the objective lens in the optical axis direction changes.

[0005] A third embodiment of the present invention provides a microscope comprising: a first optical system having an objective lens and receiving first light from a sample; a second optical system irradiating the sample with second light from a direction intersecting the optical axis direction of the objective lens to form an illumination surface; a control unit that drives at least one of the objective lens and a stage holding the sample in the optical axis direction and adjusts the position of the illumination surface 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 amount of drive of the objective lens and the amount of drive of the stage and the adjustment amount of the illumination surface.

[0006] A fourth aspect of the present invention provides an observation method comprising: receiving first light from a sample through a first optical system having an objective lens; irradiating the sample with second light through a second optical system from a direction intersecting the optical axis direction of the objective lens to form an illumination surface; 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 has been moved in the optical axis direction, performing a second control to set the illumination surface to a predetermined position on the sample by further controlling the second optical system.

[0007] In a fifth aspect of the present invention, a program is provided that causes a computer to perform the following steps: receiving 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 the optical axis direction of the objective lens to form an illumination surface; 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 the 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 has been moved in the optical axis direction, performing a second control to set the illumination surface to a predetermined position on the sample by further controlling the second optical system.

[0008] A sixth aspect of the present invention provides an observation method comprising: receiving first light from a sample through a first optical system having an objective lens; irradiating the sample with second light through a second optical system from a direction intersecting the optical axis direction of the objective lens to form an illumination surface; 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 surface in the optical axis direction.

[0009] In a seventh aspect of the present invention, a program is provided that causes a computer to perform the following steps: receiving first light from a sample through a first optical system having an objective lens; irradiating the sample with second light through a second optical system from a direction intersecting the optical axis direction of the objective lens to form an illumination surface; and, if 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 surface in the optical axis direction.

[0010] An eighth aspect of the present invention provides a refractive index determination method comprising: receiving first light from a sample through a first optical system having an objective lens; irradiating the sample with second light through a second optical system from a direction intersecting the optical axis direction of the objective lens to form an illumination surface; 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 surface 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 amount of drive of the objective lens and the amount of drive of the stage and the amount of adjustment of the illumination surface.

[0011] In a ninth aspect of the present invention, a program is provided that causes a computer to perform the following steps: receiving first light from a sample through a first optical system having an objective lens; irradiating the sample with second light through a second optical system from a direction intersecting the optical axis direction of the objective lens to form an illumination surface; 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 surface 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 amount of drive of the objective lens and the amount of drive of the stage and the adjustment amount of the illumination surface.

[0012] It should be noted that the above summary of the invention does not enumerate all of its features. Furthermore, subcombinations of these features may also constitute an invention. [Brief explanation of the drawing]

[0013] [Figure 1] A schematic configuration of the microscope according to the first embodiment is shown. [Figure 2] This shows the functional configuration of the microscope's control system. [Figure 3] This shows the positional relationship between the sheet light and the objective lens. [Figure 4] The schematic configuration of the position detection unit is shown. [Figure 5] The schematic configuration of the lighting unit in the modified example is shown. [Figure 6]Shows the correction procedure of the illumination surface during stage drift according to the first embodiment. [Figure 7A] Shows a state (ideal state) of the correction of the illumination surface during stage drift. [Figure 7B] Shows a stage (stage drift state) of the correction of the illumination surface during stage drift. [Figure 7C] Shows a stage (state after detecting surface correction) of the correction of the illumination surface during stage drift. [Figure 7D] Shows a stage (state after illumination surface correction) of the correction of the illumination surface during stage drift. [Figure 8] Shows the correction procedure of the illumination surface during Z-stack imaging according to the second embodiment. [Figure 9A] Shows a state (normal state) of the correction of the illumination surface during Z-stack imaging. [Figure 9B] Shows a state (state after objective lens drive) of the correction of the illumination surface during Z-stack imaging. [Figure 9C] Shows a state (state after illumination light correction) of the correction of the illumination surface during Z-stack imaging. [Figure 10] Shows the correction procedure of the illumination surface during Z-stack imaging according to the third embodiment. [Figure 11A] Shows a state (normal state) of the correction of the illumination surface during Z-stack imaging. [Figure 11B] Shows a state (state after stage drive) of the correction of the illumination surface during Z-stack imaging. [Figure 11C] Shows a state (state after illumination light correction) of the correction of the illumination surface during Z-stack imaging. [Figure 12] Shows an example of the configuration of a computer according to an embodiment.

Mode for Carrying Out the Invention

[0014] 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 features described in the embodiments are essential for the solution of the invention.

[0015] <First Embodiment> FIG. 1 and FIG. 2 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, respectively. 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 (orthogonal as an example in this embodiment) the optical axis 10L of the illumination optical system 12. A direction parallel to the optical axis .....

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

[0017] The illumination unit 10 is a unit having a light source 11 and an illumination optical system 12, and puts illumination light 10a into the sample 9 through the illumination optical system 12, and defines an illumination surface 10S (see FIGS. 3, 7A, etc.) in 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 10a to the sample 9 on the stage 21, and has, as an example, an expander 13, a reflection element 14, a relay lens 15, and a cylindrical lens 16.

[0020] The expander 13 has multiple lens elements and the like, and expands or contracts the beam diameter of the illumination light 10a. However, if it is not necessary to expand or contract 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 reflective element 14 reflects the illumination light 10a and distributes it in the Z-axis direction. The reflective element 14 includes, for example, a mirror element such as a galvanometer mirror and a reflective element drive device 14a such as a motor that rotates its mirror surface around the X-axis. The reflective element 14 may also be an acousto-optic element (AOM), a spatial light modulator (SLM), etc. Alternatively, a transmissive element such as a transmissive phase modulation element may be used instead of the reflective element 14. These reflective elements 14 and transmissive elements are examples of members or optical elements that set or adjust the illumination surface 10S.

[0022] The relay lens 15 has multiple lens elements and guides the illumination light 10a 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 a sheet, which is then inserted into the sample 9 from a direction intersecting the optical axis 30L of the detection optical system 32 (towards the Y-axis direction in this example).

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

[0025] As shown in Figure 3, the illumination optical system 12 forms an illumination surface 10S parallel to the XY plane within the sample 9 using the above-mentioned optical elements, and illuminates it with illumination light 10a along the Y axis. Here, the position of the illumination surface 10S can be adjusted in the Z axis direction by controlling the reflector element driving device 14a to rotate the reflector element 14 constituting the illumination optical system 12 around the X axis.

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

[0027] Stage 21 is configured to support the sample 9 and be movable (i.e., vertically) at least in the Z-axis direction. Stage 21 has an aperture (not shown) through which detection light 30a passes, and supports the sample 9 above it.

[0028] The drive device 22 drives the stage 21 at least in the Z-axis direction. For example, an electric motor can be used as the drive device 22. The drive device 22 is controlled by the control unit 50 (drive control unit 51) to drive the stage 21 to the 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 (for example, fluorescence emitted by the sample 9 when excited by illumination light) 30a emitted from a detection surface 30S (see Figures 3, 7A, etc.) defined within the sample 9 via a detection optical system 32. This light is also called detection light. The detection unit 30 includes a detection device 31 and a detection optical system 32.

[0030] The detection device 31 detects detection light 30a from the sample 9 via the detection optical system 32 and generates a detection image. The detection device 31 may employ an image sensor such as a charge-coupled device (CCD) or a CMOS sensor.

[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 for example, it includes an objective lens 33, an imaging lens 35, and a filter 36. The detection optical system 32 may be either an inverted or 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 positioned 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. Note that 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 an immersion liquid; it may be filled with air instead. The objective lens 33 includes a lens barrel that holds the lens element group inside, and can be moved by a lens drive device 33a such as an electric motor that drives it in the Z-axis direction. Here, the detection surface 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 drive 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 30a sent through the objective lens 33 onto the light-receiving surface of the detection device 31, and generates a detection image of the sample 9 on it.

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

[0036] As shown in Figure 1, the position detection unit 40 is a unit that detects information regarding the position of the sample 9 or stage 21 in the Z-axis direction. In particular, the position detection unit 40 employs a configuration in which it detects through at least one of the optical elements constituting the detection optical system 32, which in this embodiment is the objective lens 33.

[0037] Figure 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 a focusing illumination optical system 40A, a focusing imaging optical system 40B, a photoelectric converter 130, and a signal processing unit 41. The focusing illumination optical system 40A and the focusing imaging optical system 40B are collectively referred to as the focusing optical system.

[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 along 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 called simply a lens) 42, a visible light cut filter 43, and a dichroic mirror 34.

[0039] When probe light (for example, infrared or near-infrared light) is emitted from the light source 120, the probe light is focused by the first collector lens 121 and incident on the slit plate 122. The slit plate 122 has a rectangular slit opening 122a in its center, and the slit opening 122a is positioned at a conjugate position with the interface of the sample 9, with its longitudinal direction extending perpendicular to the plane of the paper in Figure 4, centered on the optical axis 40L. The probe light passes through the slit opening 122a and is shaped to the shape of the opening.

[0040] The probe light passing through the slit opening 122a is shaped into parallel light via the second collector lens 123 and irradiated onto the first pupil limiting mask 124. The first pupil limiting mask 124 is an optical element that blocks half of the pupil and is arranged along the direction perpendicular to the plane of the paper in Figure 4, centered on the optical axis 40L. Half of the probe light is blocked by the first pupil limiting mask 124.

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

[0042] The probe light La that has passed through the half mirror 125 undergoes a distance ratio change in the offset lens 42 (details of the function of the offset lens 42 will be described later), the visible light component contained in the probe light La is removed via the visible light cut filter 43, and the light is reflected by the dichroic mirror 34 and guided to the objective lens 33. The dichroic mirror 34 is positioned 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. The dichroic mirror 34 is positioned on the optical axis 30L of the detection optical system 32 so as to reflect the probe light La toward the objective lens 33 and transmit other light (especially the detection light 30a).

[0043] The probe light Lb reflected by the dichroic mirror 34 is focused by the objective lens 33 and incident on the bottom surface 3a of the sample container 3 on the stage 21 (see Figure 1). Here, the imaging position of the optical image based on the probe light Lb by the objective lens 33 in the Z-axis direction is called the 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 will also be referred to as the reflective surface, interface, or boundary surface.

[0044] The focusing imaging optical system 40B is an optical system that receives 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 its visible light component removed via the visible light cut filter 43, enters the half mirror 125 via the offset lens 42, and a portion of it is reflected and guided to the focusing imaging optical system 40B. The focusing imaging optical system 40B includes a half mirror 125, an autofocus objective lens 126, an autofocus relay lens 127a, a second pupil limiting mask 128, an autofocus relay lens 127b, and a cylindrical lens 129 arranged along its optical axis.

[0045] The probe light Ld reflected by the half mirror 125 is focused by the autofocus objective lens 126 and converted into imaging light, forming a slit image (optical image). The slit image (the probe light Le that images it) is relayed by the autofocus relay lenses 127a and 127b and re-imaged to the imaging surface of the photoelectric converter 130 via the cylindrical lens 129. Here, the second pupil limiting mask 128 is positioned between the autofocus relay lenses 127a and 127b to block half of the pupil. The blocked area corresponds to the area blocked by the first pupil limiting mask 124 described above. The cylindrical lens 129 is a lens that refracts only in a predetermined direction, compressing the probe light Le in the direction perpendicular to the plane of the paper (the longitudinal direction of the slit image) in Figure 4, and imaging it to the imaging surface of the photoelectric converter 130.

[0046] The photoelectric converter 130 may be, for example, a line CCD sensor (charge-coupled element), which receives probe light reflected from 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 generates a detection signal corresponding to the electrical signal by performing signal processing on the electrical signal from the photoelectric converter 130, and outputs the generated detection signal to the control unit 50.

[0048] Furthermore, a drive unit may be provided to move the position detection unit 40 together with the objective lens 33 in the Z-axis direction.

[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 (neither of which are shown), and the lens drive unit 44 moves the concave lens relative to the convex lens in the direction of the optical axis 40L based on a control signal from the control unit 50, for example, thereby moving the imaging position A of the optical image based on the probe light irradiated onto the reflective 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 way, 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 reflective surface and positioning the focal position F inside the sample 9, which is a certain distance away from the reflective surface in the Z-axis direction.

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

[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 enable the control unit 50 to perform functions to control each component of the microscope 100. The dedicated program is, for example, stored in 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 loaded into RAM to be activated. 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 a target drive amount from the drive control unit 51, it drives the stage 21 by that 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 the observation of the sample 9, the drive control unit 51 determines a target drive amount to the target position, and drives the stage 21 or objective lens 33 to maintain the position of the observation surface 9S within the sample 9 at the target position. In Z-stack imaging, the drive control unit 51 determines a target drive amount (Z-step amount) to the next Z-stack position, and drives the stage 21 or objective lens 33 to change the position of the observation surface 9S within the sample 9 at 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 drive device 14a based on the detection result of the amount of drive of the objective lens 33 to rotate the reflective element 14 constituting the illumination optical system 12 around the X-axis. As a result, for example, if 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 aligning the illumination surface 10S with the observation surface 9S within the sample 9.

[0055] Furthermore, the lighting control unit 52 can measure the inclination of the reflecting element 14 using a sensor such as an interferometer, and based on the relationship between the inclination of the reflecting element 14 and the reflection direction of the illumination light 10a, it can detect the position of the illumination surface 10S in the Z-axis direction.

[0056] The detection control unit 53 controls the lens drive device 33a to drive the objective lens 33, thereby moving the imaging position (imaging plane) of the probe light in the Z-axis direction so that the imaging plane coincides with the reflective surface. Here, the position of the reflective surface is determined by the position detection unit 40 based on the image detected by the probe light. As a result, 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 from the position detection unit 40 so that the imaging plane is at the position of the reflective surface, thereby aligning the detection surface 30S of the objective lens to a position a certain distance away from the reflective surface in the Z-axis direction. The amount of deviation between the imaging plane and the detection surface 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. This 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 refractive index between the sample 9 and the container, cover glass, immersion liquid, etc. that contain it, when the stage 21 supporting the sample 9 is driven in the Z-axis direction, the detection surface 30S of the objective lens 33 inside the sample 9 also moves slightly (the displacement of the detection surface 30S inside the sample 9 (i.e., optical distance) does not match the amount of drive of the stage 21), and the detection surface 30S shifts 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 to match the detection surface 30S that has moved as a result, the refractive index of the sample 9 can be determined from the relationship between the amount of drive of the stage 21 and the amount of adjustment of the illumination surface 10S (equal to the displacement of the detection surface 30S).

[0059] The refractive index determination unit 55 may store refractive index data that associates the relationship between the drive amount of the stage 21 and the displacement of the detection surface 30S with the refractive index. The refractive index data may be in lookup table format. The refractive index data is obtained by using the microscope 100 to hold a sample with a known refractive index on the stage 21, 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 from the detection surface 30S in the sample 9 by the detection unit 30, calculating the relationship (e.g., ratio) between the drive 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 for samples with different refractive indices. Alternatively, 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 a sample 9 having an unknown refractive index on the stage 21, and via the illumination control unit 52, 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, and determines the estimated refractive index of the sample 9 as the refractive index of the sample 9, by referring to the above refractive index data, and corresponding to the relationship (e.g., ratio) between the drive amount of the stage 21 and the adjustment amount of the illumination surface 10S.

[0061] In the description above, the refractive index determination unit 55 was described in the case where the stage 21 is driven in the Z-axis direction to determine the refractive index of the sample 9. However, even when the objective lens 33 is driven in the Z-axis direction, the refractive index of the sample 9 can be determined by the same method as described above.

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

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

[0064] The reflective element 17 reflects the illumination light 10a and distributes it in the X-axis direction. The reflective element 17 includes, for example, a mirror element such as a galvanometer mirror and a reflective element drive device 17a such as a motor that rotates its mirror surface around an axis tilted 45 degrees with respect to the X and Y axes. The reflective element 17 may also be an acousto-optic element (AOM), a spatial light modulator (SLM), etc. Alternatively, a transmissive element such as a transmissive phase modulation element may be used instead of the reflective element 14.

[0065] The focusing lens 18 focuses the illumination light 10a, which is sent via the relay lens 15, into the sample 9.

[0066] The illumination optical system 12d focuses the illumination light 10a into the sample 9 using the focusing lens 18, and scans the illumination light 10a in the X-axis direction using the reflecting element 17, thereby forming an illumination surface 10S parallel to the XY plane within the sample 9. In the illumination optical system 12d, the position of the illumination surface 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] Furthermore, the position of the illumination surface 10S is not limited to adjusting the position of the illumination surface 10S in the Z-axis direction by rotating the reflective element 14 that constitutes the illumination optical system 12. The position of the illumination surface 10S may also be adjusted in the Z-axis direction by driving the entire illumination optical system 12 or some of its optical elements, such as the reflective element 14, in the Z-axis direction.

[0068] Figure 6 shows the correction procedure for the illumination surface 10S during stage drift according to the first embodiment. The sample 9 is assumed to be supported on the stage 21 via a support member (not shown), such as a container that houses it. Here, one surface within the sample 9 to be observed is referred to as the observation surface 9S.

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

[0070] As shown by the white arrows in Figure 7B, assume that the stage 21 drifts in the Z-axis direction (in this example, the -Z direction) due to vibrations applied to the microscope 100, expansion or contraction of components due to heat, etc. This stage drift causes the sample 9 supported on the stage 21 to also drift in the -Z direction, resulting in a shift of the illumination surface 10S and the detection surface 30S from the observation surface 9S.

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

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

[0073] In step S108, as shown in Figure 7D, the illumination control unit 52 controls the illumination optical system 12 based on the detection result from the position detection unit 40 to adjust the position of the illumination surface 10S in the Z-axis direction. By controlling the reflective element drive device 14a and rotating the reflective elements 14 that make up the illumination optical system 12 around the X-axis, the illumination surface 10S is adjusted in the -Z direction to coincide with the observation surface 9S.

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

[0075] In step S110, the detection unit 30 detects the detection light 30a emitted from the detection surface 30S within 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] Furthermore, the illumination control unit 52 may quickly and roughly adjust the position of the illumination surface 10S based on the detection result from the position detection unit 40, and the detection unit 30 may precisely fine-tune the position of the illumination surface 10S based on the detection light 30a (detection image) emitted from the detection surface 30S within the sample 9. This allows for quick and precise adjustment of the position of the illumination surface 10S.

[0077] Alternatively, instead of the detection control unit 53 controlling the detection optical system 32 based on the detection result from the position detection unit 40 to move the detection surface 30S in the Z-axis direction (the objective lens 33 is driven in the -Z direction, and consequently the detection surface 30S is moved in the -Z direction to coincide with the observation surface 9S (the imaging position of the light image based on infrared light coincides with the reflective surface)), the detection control unit 53 may control the stage 21 based on the detection result from the position detection unit 40 to move the detection surface 30S in the Z-axis direction (the stage 21 is driven in the +Z direction, and consequently the detection surface 30S is moved in the -Z direction to coincide with the observation surface 9S (the imaging position of the light image based on probe light coincides with the reflective surface)). If the stage 21 is controlled, it is not necessary to control the illumination optical system 12 with 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, it includes a detection optical system 32 having an objective lens 33 that receives detection light 30a from a sample 9, an illumination optical system 12 that irradiates the sample 9 with illumination light 10a from a direction intersecting the optical axis direction of the objective lens 33 to form an illumination surface 10S, a position detection unit 40 that detects information regarding the position of the sample 9 in the optical axis direction, and a control unit 50. 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 of the position detection unit 40. When the objective lens 33 is moved in the optical axis direction, the control unit 50 further controls the illumination optical system 12 to set the illumination surface 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 controls 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 are: receiving detection light 30a from the sample 9 via a detection optical system 32 having an objective lens 33; irradiating the sample 9 with illumination light 10a via an illumination optical system 12 from a direction intersecting the optical axis direction of the objective lens 33 to form an illumination surface 10S; detecting information regarding the position of the sample 9 in the optical axis direction; and setting 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 holding the sample 9 in the optical axis direction based on the detection result regarding the position of the sample 9 in the optical axis direction, and further controlling the illumination optical system 12 to set the illumination surface 10S to a predetermined position on the sample 9 when the objective lens 33 is moved in the optical axis direction.

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

[0081] Figure 8 shows the correction procedure for the illumination surface 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 supported on the stage 21 via a support member such as the sample container 3 that houses it. Here, the surface within the sample 9 that is to be observed is referred to as the observation surface 9S. In Z-stack imaging, optical cross-sectional images of the sample 9 are detected at N different depths.

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

[0083] In step S202, as shown in the normal state in Figure 9A, the illumination unit 10 directs illumination light 10a onto the illumination surface 10S within the sample 9 via the illumination optical system 12 that forms the illumination surface 10S, in the Y-axis direction. The illumination surface 10S coincides with the observation surface 9S by controlling the illumination optical system 12 with the illumination control unit 52. Furthermore, based on the detection result from the position detection unit 40, the detection surface 30S coincides with the observation surface 9S by controlling the detection optical system 32 with the detection control unit 53 (the imaging position of the light image based on the probe light coincides with the reflection surface).

[0084] In step S204, as shown by the white arrow in Figure 9B, the drive control unit 51 drives the objective lens 33 in the Z-axis direction (in this example, the +Z direction) to generate an image of the nth observation surface 9S within the sample 9. At this time, based on the change in the detection signal from the signal processing unit 41 due to the shift between the image formation position A and the position of the reflective surface caused by the movement of the objective lens 33, the control unit 50 outputs a control signal to the lens drive unit 44, thereby moving the offset lens 42 in the direction of the optical axis 40L so that the shift between the image formation position A and the position of the reflective surface caused by the movement of the objective lens 33 is eliminated (the distance from the reflective surface to the detection surface 30S is the offset value OSn). Furthermore, due to the index mismatch, driving the objective lens 33 in the Z-axis direction causes the detection surface 30S of the objective lens 33 within the sample 9 to move slightly (the displacement of the detection surface 30S within the sample 9 (i.e., the optical distance) does not match the amount of drive of the objective lens 33), causing the detection surface 30S of the objective lens 33 to shift away from the illumination surface 10S.

[0085] In step S208, the control unit 50 checks whether it has retained the refractive index value of the sample 9. If it does not have retained the refractive index value, it proceeds to step S210; if it does have retained the value, it 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 by a predetermined amount in the Z-axis direction via the drive unit 20, and via the illumination control unit 52 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. The unit then refers to the refractive index data and determines the refractive index corresponding to the relationship (e.g., ratio) between the amount of drive of the stage 21 and the amount of adjustment of the illumination surface 10S as the refractive index of the sample 9. The determined refractive index is transmitted to the illumination control unit 52 and held therein.

[0087] Once the refractive index is determined, the drive control unit 51 drives the stage 21 back to the position for generating an image of the sample 9 on the nth observation surface 9S in step S204.

[0088] In step S212, if the relative positional relationship between the sample 9 and the objective lens 33 in the optical axis direction changes as described above, the illumination control unit 52, as shown in Figure 9C, refers to the refractive index data and determines the amount of correction for the position of the illumination surface 10S based on the displacement of the refractive index and offset value of the sample 9 (or the amount of movement of the offset lens 42). Based on the correction amount, the illumination optical system 12 is controlled to adjust the position of the illumination surface 10S in the Z axis direction, thereby correcting the misalignment and aligning the illumination surface 10S with the detection surface 30S.

[0089] In step S214, the detection unit 30 detects the detection light 30a emitted from the detection surface 30S within 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 determines whether the counter n is less than N. If n is less than N, the process proceeds to step S218, incrementing the counter n by 1, and returning to step S202. If n is equal to N, the process ends. This yields cross-sectional images of the sample 9 at N different depths, i.e., Z-stack images.

[0091] According to the microscope 100 of the second embodiment, it includes a detection optical system 32 having an objective lens and receiving detection light 30a from a sample 9, an illumination optical system 12 that irradiates the sample 9 with illumination light 10a from a direction intersecting the optical axis 30L direction of the objective lens 33 to form an illumination surface 10S, a position detection unit 40 that detects information regarding the position of the sample 9 in the optical axis 30L direction, and a control unit 50. The control unit 50 sets the focal position of the objective lens 33 in the detection optical system 32 to a predetermined position on the sample 9 by moving the objective lens 33 in the optical axis 30L direction, 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 light image based on the probe light to the position of the reflective surface, and then 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. 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, thereby setting the irradiation position of the illumination light 10a to a predetermined position on the sample 9, and thus maintaining the position of the illumination surface 10S within the sample 9.

[0092] According to the observation method of the second embodiment, the steps are: receiving detection light 30a from the sample 9 via a detection optical system 32 having an objective lens; irradiating the sample 9 with illumination light 10a via an illumination optical system 12 from a direction intersecting the optical axis 30L direction of the objective lens 33 to form an illumination surface 10S; detecting information regarding the position of the sample 9 in the optical axis 30L direction; setting the focal position of the objective lens 33 in the detection optical system 32 to a predetermined position on the sample 9 by moving the objective lens 33 in the optical axis 30L direction; then moving the offset lens 42 of the position detection unit 40 in the optical axis 40L direction to align the imaging position A of the light image based on the probe light with the position of the reflective surface; and finally, driving 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. 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, thereby setting the irradiation position of the illumination light 10a to a predetermined position on the sample 9, and thus maintaining the position of the illumination surface 10S within the sample 9.

[0093] <Third Embodiment> In Z-stack imaging, that is, generating a Z-stack image by changing the depth of the observation surface 9S within the sample 9 and detecting an image at each depth, when the stage 21 or objective lens 33 supporting the sample 9 is driven in the Z-axis direction, the detection surface 30S of the objective lens may also move slightly and shift away from the illumination surface 10S due to a refractive index mismatch (also called index mismatch) between the sample 9 and the container containing it (also called the sample container 3), cover glass, immersion liquid, etc. In such cases, the illumination control unit 52 may determine a correction amount for the position of the illumination surface 10S based on the refractive index of the sample 9 and the drive amount of the stage 21 or objective lens 33, and then control the illumination optical system based on the correction amount to adjust the position of the illumination surface 10S in the Z-axis direction, thereby correcting the shift and aligning the illumination surface 10S with the detection surface 30S.

[0094] Figure 10 shows the correction procedure for the illumination surface 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 supported on the stage 21 via a support member such as the sample container 3 that houses it. Here, the surface within the sample 9 that is to be observed is referred to as the observation surface 9S. In Z-stack imaging, optical cross-sectional images of the sample 9 are detected at N different depths.

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

[0096] In step S302, as shown in Figure 11A, the illumination unit 10 directs illumination light 10a onto the illumination surface 10S within the sample 9 via the illumination optical system 12 that forms the illumination surface 10S, in the Y-axis direction. The illumination surface 10S is assumed to coincide with the observation surface 9S by controlling the illumination optical system 12 with the illumination control unit 52.

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

[0098] In step S308, the control unit 50 checks whether it has retained the refractive index value of the sample 9. If it does not have retained the refractive index value, it proceeds to step S310; if it does have retained the value, it 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 by a predetermined amount in the Z-axis direction via the drive unit 20, and via the illumination control unit 52 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. The unit then refers to the refractive index data and determines the refractive index corresponding to the relationship (e.g., ratio) between the amount of drive of the stage 21 and the amount of adjustment of the illumination surface 10S as the refractive index of the sample 9. The determined refractive index is transmitted to the illumination control unit 52 and held therein.

[0100] Once the refractive index is determined, the drive control unit 51 drives the stage 21 back to the position for generating an image of the sample 9 on the nth observation surface 9S in step S306.

[0101] In step S312, if the relative positional relationship between the sample 9 and the objective lens 33 in the optical axis direction changes as described above, the illumination control unit 52, as shown in Figure 11C, refers to the refractive index data and determines the correction amount for the position of the illumination surface 10S based on the refractive index of the sample 9 and the drive amount of the stage 21 or the objective lens 33. Based on the correction amount, the illumination optical system 12 is controlled to adjust the position of the illumination surface 10S in the Z axis direction, thereby correcting the misalignment and aligning the illumination surface 10S with the detection surface 30S.

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

[0103] In step S314, the detection unit 30 detects the detection light 30a emitted from the detection surface 30S within 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 less than N. If n is less than N, the process proceeds to step S318, incrementing the counter n by 1, and returning to step S302. If n is equal to N, the process ends. This yields cross-sectional images of the sample 9 at N different depths, i.e., Z-stack images.

[0105] According to the microscope 100 of the third embodiment, it has a detection optical system 32 having an objective lens 33 that receives detection light 30a from a sample 9, an illumination optical system 12 that irradiates the sample 9 with illumination light 10a from a direction intersecting the optical axis direction of the objective lens 33 to form an illumination surface 10S, and a control unit 50. The control unit 50 controls the illumination optical system 12 based on the refractive index of the sample 9 to adjust the position of the illumination surface 10S in the optical axis direction when the relative positional relationship between the sample 9 and the objective lens 33 in the optical axis direction changes. As a result, when the stage 21 supporting the sample 9 or the objective lens 33 is driven in the Z axis direction to move the detection surface 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 surface 10S in the Z axis direction, thereby correcting the discrepancy between the amount of drive of the stage 21 or objective lens 33 and the amount of movement of the detection surface 30S due to index mismatch, and making the illumination surface 10S align with the detection surface 30S. At this time, based on the refractive index of the sample 9, a correction amount corresponding to the discrepancy between the drive amount of the stage 21 or 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 aligned with the observation surface 9S.

[0106] According to the observation method of the third embodiment, the method includes the steps of: receiving detection light 30a from a sample 9 via a detection optical system 32 having an objective lens 33; irradiating the sample 9 with illumination light 10a via an illumination optical system 12 from a direction intersecting the optical axis direction of the objective lens 33 to form an illumination surface 10S; and, when the relative positional relationship between the sample 9 and the objective lens 33 in the optical axis direction changes, controlling the illumination optical system 12 based on the refractive index of the sample to adjust the position of the illumination surface 10S in the optical axis direction. With 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 surface 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 surface 10S in the Z-axis direction, thereby correcting the discrepancy between the amount of drive of the stage 21 or objective lens 33 and the amount of movement of the detection surface 30S due to index mismatch, and making the illumination surface 10S coincide with the detection surface 30S. At this time, based on the refractive index of the sample 9, a correction amount corresponding to the discrepancy between the drive amount of the stage 21 or 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 aligned with the observation surface 9S.

[0107] Furthermore, the microscope 100 may also be used as a refractive index determination device to determine the refractive index of the sample 9, or the refractive index determination method from steps S302 to S306 and S310 may be used to determine the refractive index of the sample 9.

[0108] In the microscope 100 and observation method according to the first to third embodiments, illumination light 10a is introduced into the sample 9 in the +Y direction via the illumination optical system 12. However, two illumination optical systems 12 may be provided, or the illumination light 10a guided from the illumination optical system 12 may be separated into two and introduced into the sample 9 from both the +Y and -Y directions.

[0109] Various embodiments of the present invention may be described with reference to flowcharts and block diagrams, where a block may represent (1) a stage in a process in which an operation is performed or (2) a section of a device having the role of performing the operation. 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. Dedicated circuits may include digital and / or analog hardware circuits, and may include integrated circuits (ICs) and / or discrete circuits. Programmable circuits may include reconfigurable hardware circuits, including logical AND, logical OR, logical XOR, logical NAND, logical NOR, and other logic operations, flip-flops, registers, memory elements such as field-programmable gate arrays (FPGAs), programmable logic arrays (PLAs), etc.

[0110] Computer-readable media may include any tangible device capable of storing instructions to be executed by a suitable device, and as a result, computer-readable media having instructions stored therein will comprise a product containing instructions that can be executed to create means for performing operations specified in a flowchart or block diagram. Examples of computer-readable media may include electronic storage media, magnetic storage media, optical storage media, electromagnetic storage media, semiconductor storage media, etc. More specific examples of computer-readable media may include floppy 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 disk read-only memory (CD-ROM), digital versatile disk (DVD), Blu-ray (RTM) disk, memory stick, integrated circuit card, etc.

[0111] Computer-readable instructions may include assembler 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 one or more programming languages, including object-oriented programming languages ​​such as Smalltalk®, Java®, C++, and traditional procedural programming languages ​​such as the C programming language or similar programming languages.

[0112] Computer-readable instructions may be provided locally or via a wide area network (WAN), such as a local area network (LAN) or the internet, to a processor or programmable circuit of a general-purpose computer, a special-purpose computer, or other programmable data processing device, and these instructions may be executed to create means for performing 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.

[0113] Figure 12 shows an example of a computer 2200 in which multiple aspects of the present invention may be embodied in whole or in part. A program installed on the computer 2200 can cause the computer 2200 to function as an operation or one or more sections of an apparatus according to an embodiment of the present invention, or to execute such operation or one or more sections, and / or to cause the computer 2200 to execute a process or a stage of such process according to an embodiment of the present invention. Such a program may be executed by the CPU 2212 to cause the computer 2200 to perform a particular operation associated with some or all of the blocks in the flowcharts and block diagrams described herein.

[0114] The computer 2200 according to this embodiment includes a CPU 2212, RAM 2214, a graphics controller 2216, and a display device 2218, which are interconnected 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 programs stored in the ROM 2230 and RAM 2214, thereby controlling each unit. The graphics controller 2216 retrieves image data generated by the CPU 2212 from a frame buffer provided in RAM 2214 or from itself, and displays the image data 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 them to the hard disk drive 2224 via the RAM 2214. The IC card drive reads programs and data from the IC card and / or writes programs and data to the IC card.

[0117] The ROM 2230 stores boot programs and / or programs that depend on the computer 2200's hardware, which are executed by the computer 2200 when activated. The input / output chip 2240 may also connect various input / output units to the input / output controller 2220 via parallel ports, serial ports, keyboard ports, mouse ports, etc.

[0118] The program is provided on a computer-readable medium such as a DVD-ROM 2201 or an IC card. The program is read from the computer-readable medium and installed on a hard disk drive 2224, RAM 2214, or ROM 2230, which are also examples of computer-readable medium, and executed by the CPU 2212. The information processing described within these programs is read by the computer 2200, resulting in coordination between the program and the various types of hardware resources described above. The apparatus or method may be configured to realize the manipulation or processing of information in accordance with the use of the computer 2200.

[0119] For example, when communication is performed between a computer 2200 and an external device, the CPU 2212 may execute a communication program loaded into RAM 2214 and, based on the processing described in the communication program, instruct the communication interface 2222 to perform communication processing. Under the control of the CPU 2212, the communication interface 2222 reads transmission data stored in a transmission buffer processing area provided in a recording medium such as RAM 2214, a hard disk drive 2224, a DVD-ROM 2201, or an IC card, transmits the read transmission data to the network, or writes received data received from the network to a reception buffer processing area provided on the recording medium.

[0120] Furthermore, the CPU 2212 may read all or necessary parts of files or databases stored on external storage media such as the hard disk drive 2224, DVD-ROM drive 2226 (DVD-ROM 2201), or IC card into the RAM 2214, and perform various types of processing on the data in the RAM 2214. The CPU 2212 then writes the processed data back to the external storage media.

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

[0122] The programs or software modules described above may be stored on or near computer 2200 on a computer-readable medium. Alternatively, recording media such as hard disks or RAM provided within a server system connected to a dedicated communication network or the Internet can be used as computer-readable media, thereby providing programs to computer 2200 via the network.

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

[0124] It should be noted that the execution order of operations, procedures, steps, and stages in the apparatus, systems, programs, and methods shown in the claims, specifications, and drawings is not explicitly stated as "before," "prior to," etc., and that these can be implemented in any order unless the output of a previous process is used in a later process. Even if the operation flow in the claims, specifications, and drawings is described using phrases such as "first," "next," etc. for convenience, it does not mean that it is essential to perform the operations in that 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 optics, 12d...Illumination optics, 13...Expander, 14...Reflector, 14a...Reflector drive unit, 15...Relay lens, 16...Cylindrical lens, 17...Reflector, 17a...Reflector drive unit, 18...Focusing lens, 20...Drive unit, 21...Stage, 22...Drive unit, 30...Detection Outlet, 30L...Optical axis, 30S...Detection surface, 30a...Detection light, 31...Detection device, 32...Detection optical system, 33...Objective lens, 33a...Lens drive device, 34...Dichroic mirror, 35...Imaging lens, 36...Filter, 40...Position detection unit, 40A...Illumination optical system for focusing, 40B...Imaging optical system for focusing, 40L...Optical axis, 41...Signal processing unit, 42...Offset lens, 42...Signal processing unit, 43...Visible light cut filter, 44...Lens 50...Drive 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 opening, 125...Half mirror, 126...Autofocus objective lens, 127a,127b...Autofocus relay lens, 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

[Claim 1] A first optical system having an objective lens and receiving first light from a sample, A second optical system that irradiates the sample with a second light from a direction intersecting the optical axis direction of the objective lens to form an illumination surface, A position detection unit for detecting information regarding the position of the sample in the optical axis direction, It has a control unit and The control unit, when the position detection unit detects that the sample has moved at least in the optical axis direction from a preset position due to drift of the stage on which the sample is placed, and the focal position of the objective lens and the illumination surface have been distorted from their state set at a predetermined position on the sample, moves at least the focal position of the objective lens in the optical axis direction and controls the second optical system to return the focal position of the objective lens and the illumination surface to their state set at the predetermined position on the sample.

Images