Optical observation device and imaging method used in the optical observation device

Abstract

This invention provides an optical observation device and an imaging method using the optical observation device that suppress the generation of shadows caused by the unevenness of a sample when observing a sample containing cells using ultraviolet light. [Solution] The optical observation apparatus disclosed herein includes a placement unit on which a sample containing cells can be placed, A light source 12 capable of projecting ultraviolet light, An optical unit that reflects ultraviolet light (UV) incident from light source 12 onto sample S, irradiating sample S with ultraviolet light (UV), and transmits observation light emitted from sample S by ultraviolet light (UV), An imaging optical system that forms an image of the aforementioned observation light as an observation image, It includes an imaging unit that captures the formed observation image using an image sensor, The ultraviolet light reflected by the optical unit is incident on the sample S from a direction substantially perpendicular to the surface of the sample S.

Description

Technical Field

[0001] The present disclosure relates to an optical observation device and an imaging method used in the optical observation device.

Background Art

[0002] As a method for observing biological tissues, a method (ultraviolet surface excitation method, MUSE method) has been developed that uses ultraviolet light to excite and observe the surface of a sample. As shown in FIG. 16(A), in the MUSE method, the sample S is stained with a fluorescent dye that can be excited by ultraviolet light UV, and the obtained sample S is irradiated with ultraviolet light UV at an oblique angle. Then, the fluorescence FL emitted from the sample S is observed by a camera 95 installed in a direction orthogonal (perpendicular) to the placement (mounting) surface of the sample S (Patent Document 1). Since the ultraviolet light UV has a short wavelength, it is known that in the MUSE method, the surface layer of the sample S can be observed by irradiating the sample S at an oblique angle.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] In the MUSE method, the sample surface can be observed by staining it with a fluorescent dye. Therefore, compared with a general method for observing biological tissues in which tissue staining is performed by HE staining or the like and the stained biological tissue is observed, the MUSE method is expected to enable diagnosis of pathological tissues and the like in a short time.

[0005] However, as shown in FIG. 16(B), there are irregularities on the surface of the sample. Therefore, the inventors of the present invention have found that in the MUSE method, in the obtained observation image G, due to the irregularities on the surface of the sample S (tissue surface or cell surface), in addition to the observation image G of the cells, a shadow G C is also present. SShadow G S We found that this method results in a lack of information regarding the tissue or cells in that portion. Furthermore, due to the above problem, the observed image G obtained by the MUSE method differs from the observed image obtained by general methods of observing living tissue, which may make it impossible to apply existing tissue diagnostic techniques.

[0006] Therefore, the present disclosure aims to provide an optical observation device and an imaging method using the optical observation device in which the generation of shadows caused by the unevenness of a sample is suppressed when observing a sample containing cells using ultraviolet light. [Means for solving the problem]

[0007] To achieve the aforementioned objective, the optical observation apparatus of this disclosure (hereinafter also referred to as the "first optical observation apparatus") includes a placement unit on which a sample containing cells can be placed, A light source capable of projecting ultraviolet light, An optical unit that reflects ultraviolet light incident from the light source onto the sample, irradiating the sample with ultraviolet light, and transmits observation light emitted from the sample by the ultraviolet light, An imaging optical system that forms an image of the aforementioned observation light as an observation image, It includes an imaging unit that captures the formed observation image using an image sensor, The ultraviolet light reflected by the optical unit is incident on the sample from a direction substantially perpendicular to the surface of the sample.

[0008] The optical observation apparatus of this disclosure (hereinafter also referred to as the "second optical observation apparatus") includes a placement unit on which a sample containing cells can be placed, A light source capable of projecting ultraviolet light, An optical unit that reflects ultraviolet light incident from the light source onto the sample, irradiating the sample with ultraviolet light, and transmits observation light emitted from the sample by the ultraviolet light, An imaging optical system that forms an image of the aforementioned observation light as an observation image, An imaging unit that captures the formed observation image using an image sensor, A drive unit that can move the position of the imaging optical system relative to the sample, Control unit, including, The control unit is The drive unit is controlled to drive the imaging optical system so that it is positioned at a first position relative to the sample and at a second position relative to the sample. Furthermore, the imaging unit is controlled to capture a first observation image formed when the imaging optical system is located in the first position, and to capture a second observation image formed when the imaging optical system is located in the second position, thereby correcting the first observation image with the second observation image and clarifying the image of the cells contained in the sample.

[0009] The optical observation apparatus of this disclosure (hereinafter also referred to as the "third optical observation apparatus") includes a placement unit on which a sample containing cells can be placed, A light source capable of projecting ultraviolet light, A focusing optical system for focusing the ultraviolet light onto the sample, An optical unit that reflects the ultraviolet light onto the sample, irradiates the sample with ultraviolet light, and transmits the observation light emitted from the sample by the ultraviolet light, An imaging optical system that forms an image of the aforementioned observation light as an observation image, An imaging unit that captures the formed observation image using an image sensor, The light-gathering optical system is provided with a light-gathering optical system drive unit that can move along the ultraviolet light path, Control unit, including, The control unit is The light-gathering optical system drive unit is driven and controlled so that the light-gathering optical system is positioned at a first position on the optical path and a second position on the optical path, where the positional relationship of the ultraviolet light on the optical path is different. Furthermore, the focusing optical system drive unit is controlled to capture a first observation image formed when the focusing optical system is in the first position, and to capture a second observation image formed when the focusing optical system is in the second position, thereby correcting the first observation image with the second observation image and clarifying the image of the cells contained in the sample.

[0010] The optical observation device of the present disclosure (hereinafter, also referred to as the "fourth optical observation device") includes an arrangement unit on which a sample containing cells can be arranged, a light source capable of projecting ultraviolet light, an illumination optical system that guides the ultraviolet light projected from the light source to the sample, an imaging optical system that forms the observation light into an observation image, an imaging unit that images the formed observation image with an imaging element, and includes: the illumination optical system includes a collimating optical system that converts the ultraviolet light into parallel light, and irradiates the sample with the parallel light from an oblique direction with respect to the surface of the sample.

[0011] The imaging method of the present disclosure (hereinafter, also referred to as the "first imaging method") is an imaging method used in an optical observation device including an arrangement unit, a light source, an optical unit, an imaging optical system, and an imaging unit, and includes: an irradiation step of irradiating a sample containing cells arranged in the arrangement unit with ultraviolet light projected from the light source, which is reflected by the optical unit and incident on the sample from a direction substantially orthogonal to the surface of the sample; an imaging step of forming an observation image by the imaging optical system through which the observation light emitted from the sample by the ultraviolet light passes; an imaging step of imaging the observation image with the imaging element of the imaging unit. and includes:

[0012] The imaging method of the present disclosure (hereinafter, also referred to as the "second imaging method") is an imaging method used in an optical observation device including an arrangement unit, a light source, an optical unit, an imaging optical system, an imaging unit, and a drive unit, and includes: an irradiation step of irradiating a sample containing cells arranged in the arrangement unit with ultraviolet light projected from the light source, which is reflected by the optical unit; An imaging step in which the observation light emitted from the sample by the ultraviolet light passes through the optical unit and is imaged as an observation image by the imaging optical system; An imaging step in which the driving unit is driven to control the position of the imaging optical system, and the imaging optical system is in a state of being positioned at a first position and a second position having different positional relationships with respect to the sample, and the imaging element of the imaging unit captures the observation image as a first observation image and a second observation image, respectively; A correction step of correcting the first observation image corresponding to the first position with the second observation image corresponding to the second position to clarify the image of the cells contained in the sample.

[0013] The imaging method of the present disclosure (hereinafter, also referred to as "third imaging method") is an imaging method used in an optical observation apparatus including a placement unit, a light source, a condensing optical system, an optical unit, an imaging optical system, an imaging unit, and a condensing optical system driving unit, An irradiation step of irradiating the sample containing cells arranged in the placement unit with ultraviolet light projected from the light source, which is reflected by the optical unit after passing through the condensing optical system; An imaging step in which the observation light emitted from the sample by the ultraviolet light passes through the optical unit and is imaged as an observation image by the imaging optical system; An imaging step in which the driving unit is driven to control the position of the condensing optical system, and the condensing optical system is in a state of being positioned at a first position on the optical path and a second position on the optical path having different positional relationships on the optical path of the ultraviolet light, and the imaging element of the imaging unit captures the observation image as a first observation image and a second observation image, respectively; A correction step of correcting the first observation image corresponding to the first position on the optical path with the second observation image corresponding to the second position on the optical path to clarify the image of the cells contained in the sample.

[0014] The imaging method of the present disclosure (hereinafter, also referred to as "fourth imaging method") is an imaging method used in an optical observation apparatus including a placement unit, a light source, an illumination optical system, an imaging optical system, and an imaging unit, The illumination optical system includes a collimating optical system that converts the ultraviolet light into parallel light, For a sample containing cells arranged in the aforementioned arrangement unit, the irradiation step involves converting ultraviolet light projected from the light source into parallel light using the collimating optical system, and then irradiating the sample with the parallel light from an oblique direction onto the surface layer of the sample. The imaging step involves the imaging process in which the observation light emitted from the sample by the ultraviolet light is formed as an observation image by the imaging optical system, The imaging step involves capturing the observation image using the image sensor of the imaging unit, Includes. [Effects of the Invention]

[0015] According to this disclosure, an optical observation device and an imaging method using the optical observation device can be provided, which suppress the generation of shadows caused by the unevenness of a sample when observing a sample containing cells using ultraviolet light. [Brief explanation of the drawing]

[0016] [Figure 1] Figure 1 is a schematic cross-sectional view showing an example of the optical observation apparatus of Embodiment 1. [Figure 2] Figure 2 is a flowchart showing an example of the imaging method in Embodiment 1. [Figure 3] Figure 3 is a schematic cross-sectional view showing an example of the optical observation device of Modification 1. [Figure 4] Figure 4 is a schematic cross-sectional view showing an example of an optical observation device according to Modification 2. [Figure 5] Figure 5 is a schematic cross-sectional view showing an example of the optical observation device of Modification 3. [Figure 6] Figure 6 is a schematic cross-sectional view showing an example of the optical observation apparatus of Embodiment 2. [Figure 7] Figure 7 is a block diagram showing an example of a controller for the optical observation device of Embodiment 2. [Figure 8] Figure 8 is a flowchart showing an example of the imaging method in Embodiment 2. [Figure 9]Figure 9 is a schematic diagram showing the imaging position and the observed image at the imaging position in the optical observation device of Embodiment 2. [Figure 10] Figure 10 is a photograph showing the uncorrected observation image G1 and the corrected observation image G3 in Embodiment 2. [Figure 11] Figure 11 is a schematic cross-sectional view showing an example of the optical observation apparatus of Embodiment 3. [Figure 12] Figure 12 is a schematic cross-sectional view showing an example of an optical observation device according to Modification 4. [Figure 13] Figure 13 is a schematic cross-sectional view showing an example of an optical observation device according to Modification 5. [Figure 14] Figure 14 is a schematic cross-sectional view showing an example of the optical observation apparatus of Embodiment 4. [Figure 15] Figure 15 is a schematic cross-sectional view showing an example of an optical observation device according to Modification 6. [Figure 16] Figure 16 is a block diagram showing an example of an observation apparatus used in the MUSE method. [Modes for carrying out the invention]

[0017] In this specification, "optical axis direction" means the direction of the optical axis (axis of symmetry) in the imaging optical system, and is also referred to as the "Z-axis direction." The optical axis direction can also be defined as, for example, the direction orthogonal (perpendicular) to the surface on which the sample is placed (mounted). Furthermore, in this disclosure, "X-axis direction" means one direction in the plane (XY plane) orthogonal to the optical axis direction, and "Y-axis direction" means the direction in the XY plane that is orthogonal (perpendicular) to the X-axis direction.

[0018] In this specification, "observation" means observation of a sample, which may include, for example, observation accompanied by imaging, or observation without imaging.

[0019] In this specification, “cell” means a cell or a component containing cells. The cell may be, for example, a cell, a cell mass composed of cells, a tissue, an organ, etc. The cell may be, for example, a cultured cell or a cell isolated from a living organism. The origin of the cell may be, for example, an animal such as a human or a non-human animal. Examples of non-human animals include monkeys, horses, pigs, cattle, sheep, dogs, cats, rats, mice, etc. Examples of a sample containing the cell may be an organ, tissue, or part thereof surgically removed or excised from an animal. Examples of a sample containing the cell may be lymph fluid, blood, plasma, serum, saliva, tears, gastric juice, sputum, urine, pleural fluid, ascites, biopsy samples, fine-needle aspiration cell samples (for fine-needle aspiration cytology), etc. Examples of the aforementioned tissues or organs include the esophagus, stomach, small intestine, large intestine, duodenum, rectum, liver, pancreas, gallbladder, bladder, kidneys, prostate, uterus, ovaries, breasts, lungs, bronchi, thyroid gland, parathyroid gland, adrenal gland, skin, brain, spinal cord, bones, muscles, soft tissues such as smooth muscle, bone marrow, lymph nodes, peritoneum, diaphragm, etc.

[0020] The sample containing the aforementioned cells may also be a sample that has undergone fixation treatment with, for example, formalin, paraformaldehyde, and / or membrane permeabilization treatment of the cell membrane using a surfactant such as saponin.

[0021] The sample containing the cells may be contained in, for example, a cell culture apparatus (e.g., a dish, plate, flask (cell culture flask), etc.), or it may be placed on a substrate such as glass, plastic, or a slide.

[0022] In this specification, "ultraviolet light" means light with a shorter wavelength than visible light. Specifically, ultraviolet light is, for example, 200-300 nm, 240-300 nm, or about 280 nm.

[0023] In this specification, "fluorescent dye" means, for example, a dye that becomes excited by excitation light and emits fluorescence when returning to the ground state. Examples of such fluorescent dyes include fluorescent dyes that are excited by ultraviolet light, that is, fluorescent dyes that have an absorption region in the ultraviolet wavelength range. Specific examples of fluorescent dyes excited by ultraviolet light include, for example, eosin dyes such as eosin B, toluidine blue O, methylene blue, DAPI, acridine orange, DRAQ5, Hoechst 33342 and Hoechst 33528, calcein-AM, propidium iodide, Nile blue, Nile red, oil red O, Congo red, fast green FCF, DiI, DiO, DiD, TOTO® dyes, YO-PRO® dyes, neutral red, nuclear fast red, pyronin Y, acid fuchsin, astrazon dyes, MitoTracker, mitochondrial dyes, LysoTracker dyes, lysosome dyes, safranin dyes, thioflavin dyes, fluorescent phalloidin, terbium such as terbium chloride (TbCl3), etc. The fluorescent dyes may also be bioactive molecules that exhibit autofluorescence. Specific examples of biomolecules exhibiting autofluorescence include, for instance, nucleic acid bases, aromatic amines, dopamine, serotonin, and the like.

[0024] The optical observation apparatus of this disclosure will be described in detail below with reference to the drawings. However, the present invention is not limited to the following description. In Figures 1 to 16 below, the same parts are denoted by the same reference numerals, and their descriptions may be omitted. Also, in the drawings, for the sake of explanation, the structure of each part may be shown in a simplified manner, and the dimensional ratios of each part may be shown schematically and may differ from the actual dimensions. Furthermore, unless otherwise specified, each embodiment is combinable, and the descriptions of each embodiment can be used interchangeably.

[0025] (Embodiment 1) Embodiment 1 relates to a first optical observation apparatus and a first imaging method according to the present disclosure.

[0026] This embodiment is an example of a first optical observation apparatus. Figure 1 is a schematic diagram showing the configuration of the optical observation apparatus 100 of Embodiment 1. As shown in Figure 1, the optical observation apparatus 100 of Embodiment 1 mainly comprises a stage 11 which is a placement unit, a light source 12, a dichroic mirror 13 which is an optical unit, an objective lens 14 which is an imaging optical system, a camera 15 which is an imaging unit including an image sensor, and a bandpass filter 16 which is a filter unit. As shown in Figure 1, the objective lens 14, the dichroic mirror 13, the bandpass filter 16, and the camera 15 are arranged in this order on the optical axis from the stage 11 side. In Figure 1, the dashed line shows the optical path of observation light including ultraviolet light UV projected from the light source 12 and fluorescence FL emitted from the sample S.

[0027] Stage 11 contains a sample S, which is an external component of the optical observation device 100 of Embodiment 1, including cells. The sample S is stained with a fluorescent dye capable of staining cells, as described later. Stage 11 may be any configuration capable of arranging the sample S. Specifically, the arrangement unit can utilize the configuration of the arrangement unit in an optical observation device. Examples of the optical observation device include bright-field microscopes, stereomicroscopes, phase-contrast microscopes, differential interference microscopes, polarizing microscopes, fluorescence microscopes, confocal laser microscopes, total internal reflection fluorescence microscopes, Raman microscopes, etc., and is preferably a phase-contrast microscope. In Stage 11, the arrangement area for the sample S is configured so that the sample S can be observed from an objective lens 14 located below Stage 11. The arrangement area for the sample S may be formed of a translucent material such as glass, quartz, plastic, or resin, and through holes may be formed in a part thereof.

[0028] The light source 12 irradiates the sample S, which is placed on the stage 11, with ultraviolet light (UV). In the optical observation apparatus 100 of Embodiment 1, the ultraviolet light (UV) projected from the light source 12 is irradiated onto the sample S via a dichroic mirror 13 and an objective lens 14. However, the optical observation apparatus 100 of Embodiment 1 of this disclosure may, in addition to these configurations, include an illumination optical system that guides the ultraviolet light (UV) from the light source 12 onto the sample S. The light source 12 can be, for example, an LED (Light Emitting Diode) (any wavelength between 200 and 400 nm), a laser light source (any wavelength between 200 and 400 nm), a high-pressure mercury UV lamp (main wavelength 365 nm), a metal halide UV lamp (continuous wavelength between 200 and 400 nm), a low-pressure mercury UV lamp (254 nm), an ozone lamp (185 nm and 254 nm), a xenon light source (continuous wavelength between 200 and 400 nm), a heavy water lamp (continuous wavelength between 200 and 400 nm), etc. The illumination optical system can, for example, adopt the configuration of the illumination optical system in the optical observation device described above.

[0029] The dichroic mirror 13 irradiates the sample S with ultraviolet (UV) light by reflecting the UV light projected from the light source 12 onto the sample S. At this time, the dichroic mirror 13 reflects the UV light so that the optical axis of the UV light and the surface on which the stage 11 is positioned are approximately perpendicular, that is, the incident angle θ is approximately 90° (for example, 88~92° or 89~91°). As a result, when the optical observation device 100 of Embodiment 1 irradiates the surface of the sample S, which is in contact with the surface on which the stage 11 is positioned, the UV light can also irradiate the recesses on the surface of the sample S, thereby suppressing the occurrence of shadows due to the unevenness of the surface of the sample S. Furthermore, by setting the incident angle θ to approximately 90°, preferably 90°, in the center of the observation field of view of the objective lens 14, the occurrence of shadows due to the unevenness of the surface of the sample S can be suppressed even more effectively. Furthermore, the dichroic mirror 13 transmits observation light, which includes fluorescence FL, generated from the sample S by ultraviolet light (UV) and transmitted (passed through) the objective lens 14.

[0030] The optical properties of the dichroic mirror 13 can be set according to the wavelength range of ultraviolet light (UV) and the fluorescence wavelength range of the fluorescent dye used to stain the sample S, or the fluorescence wavelength range of the fluorescent dye (FL). Specifically, the wavelength range reflected by the dichroic mirror 13 may include the ultraviolet light (UV) wavelength range, or it may include other wavelength ranges. Also, the wavelength range transmitted by the dichroic mirror 13 includes the fluorescence wavelength range of the fluorescent dye (FL) but does not include the ultraviolet light (UV) wavelength range. As a specific example, when using Hoechst® 33342 or terbium as the fluorescent dye, a dichroic mirror 13 that reflects wavelengths of 400 nm or less and transmits wavelengths of 400 nm or more (a cut-on wavelength of 400 nm) can be used.

[0031] The objective lens 14 transmits ultraviolet light (UV) reflected from the dichroic mirror 13, thereby irradiating the sample S with ultraviolet light (UV). The objective lens 14 also forms an observation image of the observation light, including the fluorescence FL of the sample S, onto the camera 15, which is the image sensor. More specifically, the objective lens 14 forms an observation image of the observation light of the cells in the sample S onto the camera 15, and more specifically, onto the image sensor of the camera 15. This enables the optical observation device 100 of Embodiment 1 to observe and image the cells in the sample S. In the optical observation device 100 of Embodiment 1, the imaging optical system is configured as the objective lens 14, but it is sufficient if it is capable of forming an observation image of the sample S. The imaging optical system can, for example, adopt the configuration of the imaging optical system in the optical observation device described above.

[0032] In the optical observation apparatus 100 of Embodiment 1, there is one objective lens 14, but there may be multiple. In this case, the magnification of each objective lens 14 may be the same or different. In the MUSE method described above, ultraviolet light is incident on the sample S at an oblique angle. In this case, the observation light (fluorescence FL) generated from the sample S has an asymmetrical ultraviolet light (UV) irradiation distribution on the surface of the sample S. That is, the side of the objective lens 14 onto which ultraviolet light (UV) from the light source 12 is projected (the side of the light source 92 in Figure 16(A)) becomes brighter, while the opposite side becomes darker. Also, since the field of view differs depending on the magnification of the objective lens 14, the optimal illumination position needs to be adjusted according to each field of view. For this reason, when the objective lens 14 is replaced, there is a problem that the positional relationship between the light source 12 and the objective lens 14 needs to be adjusted. On the other hand, in the optical observation apparatus 100 of Embodiment 1, ultraviolet light (UV) is incident on the surface of the sample S from a direction approximately perpendicular to it, so the irradiation distribution of ultraviolet light (UV) on the surface of the sample S is symmetrical. Therefore, when the objective lens 14 is replaced, it is not necessary to adjust the positional relationship between the light source 12 and the objective lens 14, and thus the first optical observation device of this disclosure can be equipped with multiple objective lenses 14.

[0033] In the optical observation apparatus 100 of Embodiment 1, the objective lens 14 is positioned below the sample S, but it may also be positioned above the sample S. In this case, the light source 12, dichroic mirror 13, camera 15, and bandpass filter 16 in the optical observation apparatus 100 of Embodiment 1 are also positioned above the sample S.

[0034] The camera 15 is capable of capturing an observation image of the sample S, and more specifically, is configured to capture an observation image of the cells within the sample S. In the optical observation apparatus 100 of Embodiment 1, the imaging unit uses a camera 15 equipped with an image sensor, but a configuration capable of capturing an observation image of the sample S can be adopted. For example, known image sensors can be used as the image sensor, and specific examples include elements such as Charge-Coupled Devices (CCDs) and Complementary Metal Oxide Semiconductors (CMOS). Therefore, the imaging unit can be, for example, an imaging device such as a camera equipped with these image sensors.

[0035] Camera 15 is configured to capture an image of the sample S upon receiving an imaging trigger signal input by the user, for example. The imaging time (exposure time) of Camera 15 can be set appropriately according to the brightness of the sample S, for example.

[0036] The bandpass filter 16 alters the wavelength range of the observation light containing the fluorescent FL transmitted through the dichroic mirror 13. Specifically, the bandpass filter 16 has optical properties that allow it to extract the wavelength range of the fluorescent FL of the fluorescent dye used to stain the sample S in the observation light. That is, the bandpass filter 16 transmits the wavelength range of the fluorescent FL and does not transmit or attenuates the wavelength range of noise other than the fluorescent FL. The optical observation apparatus 100 of Embodiment 1, by including the bandpass filter 16, can reduce noise in the observation light, and thus can capture an observation image with reduced noise. The wavelength range of the observation light changes to, for example, the fluorescent dye used to stain the sample S, and is, for example, 400 to 600 nm. The wavelength range of the fluorescent FL changes to, for example, the fluorescent dye used to stain the sample S. If the fluorescent dye is Hoechst® 33342, the wavelength range of the fluorescent FL is, for example, 450 to 480 nm. Furthermore, if the fluorescent dye is terbium, the wavelength range of the fluorescence FL is, for example, 520 to 550 nm. In the optical observation apparatus 100 of Embodiment 1, the bandpass filter 16 can be of any configuration and may or may not be present. In addition, the optical observation apparatus of this disclosure may use a long-wavelength transmission filter, a short-wavelength transmission filter, a superconducting transition edge sensor, etc., instead of the bandpass filter 16 as the filter unit.

[0037] In the optical observation device 100 of Embodiment 1, after the observation image is formed on the camera 15, the camera 15 may capture the observation image, and the obtained image may be displayed on an external display device or the like. In addition to displaying on the display device, the optical observation device 100 of Embodiment 1 may also relay the image obtained by the objective lens 14 (primary image) to the eyepiece used by the user of the optical observation device 100 of Embodiment 1. In this case, the optical observation device 100 of Embodiment 1 includes, for example, a relay optical system and an eyepiece that relay the primary image to the eyepiece. The relay optical system and the eyepiece can, for example, adopt the configuration of the relay optical system and eyepiece in the optical observation device described above. Specific examples of the display device will be described later.

[0038] Furthermore, the imaging optical system, camera 15, may transmit the captured image to a computing device such as a computer. In this case, it is preferable for the camera 15 to associate the image with the image acquisition position (for example, coordinates such as XYZ coordinates) and transmit it to the computing device.

[0039] Next, the imaging method of Embodiment 1 using the optical observation device 100 of Embodiment 1 will be described.

[0040] Figure 2 is a flowchart of the imaging method of Embodiment 1. As shown in Figure 2, the imaging method of Embodiment 1 includes step S1 (irradiation), step S2 (imaging), and step S3 (imaging).

[0041] First, prior to step S1, the sample S to be placed on stage 11 is prepared. Specifically, the cells to be detected by the optical observation device 100 of Embodiment 1 are pre-stained with a fluorescent dye in the sample S. The fluorescent staining of the cells can be carried out by known staining methods and can be carried out appropriately depending on the type of cells to be detected and the type of molecules in the cells. The fluorescent staining may be performed specifically using nucleic acid molecules such as antibodies, aptamers, ligands, or receptors labeled with the fluorescent dye, for example. Then, the sample S stained with the fluorescent dye is placed on stage 11 of the optical observation device 100 of Embodiment 1.

[0042] Next, in step S1, ultraviolet light (UV) projected from the light source 12 is irradiated onto the sample S placed on the stage 11. Specifically, in step S1, the ultraviolet light (UV) projected from the light source 12 is reflected by the dichroic mirror 13 and irradiated onto the sample S containing cells placed on the stage 11. At this time, in step S1, ultraviolet light (UV) is incident on the sample S from a direction approximately perpendicular to the plane direction of the stage 11, that is, on the surface of the sample S that is in contact with the placement surface of the stage 11. When the sample S is irradiated with ultraviolet light (UV), the fluorescent dye on the sample S is excited by the ultraviolet light and enters an excited state, and fluorescence FL is emitted when the fluorescent dye returns to its ground state.

[0043] In step S2, the observation light containing fluorescence FL emitted from the sample S by ultraviolet light UV in step S1 is imaged onto the image sensor of the camera 15 by the objective lens 14. Specifically, in step S2, the observation light in the direction of the optical axis of the observation light containing fluorescence FL is transmitted through the objective lens 14. At this time, the objective lens 14 images the observation light so as to form an observation image on the image sensor of the camera 15. The observation light transmitted through the objective lens 14 comes into contact with the dichroic mirror 13, and the observation light in the wavelength range that can be transmitted through the dichroic mirror 13 is transmitted through the dichroic mirror 13. The observation light transmitted through the dichroic mirror 13 comes into contact with the bandpass filter 16, where the wavelength range of fluorescence FL is transmitted and the wavelength range other than the wavelength range of fluorescence FL is attenuated. The observation light transmitted through the dichroic mirror 13 is imaged onto the image sensor of the camera 15.

[0044] In step S3, the image sensor of camera 15 captures the formed observation image. This allows for the acquisition of an observation image of sample S.

[0045] This concludes the imaging method of Embodiment 1.

[0046] In the optical observation apparatus 100 of Embodiment 1, the dichroic mirror 13 is configured to receive ultraviolet light (UV) projected from the light source 12 onto the surface of the sample S, which is in contact with the placement surface of the stage 11, in a direction substantially perpendicular to the surface direction of the stage 11. Therefore, in the optical observation apparatus 100 of Embodiment 1, compared to the MUSE method, ultraviolet light (UV) can be irradiated onto a wider area of ​​the depressions on the surface of the sample S, thereby allowing observation light including fluorescence (FL) to be emitted from cells in the depressions. Consequently, the optical observation apparatus 100 of Embodiment 1 can suppress the generation of shadows caused by the unevenness of the sample S.

[0047] (Variation 1) The optical observation apparatus of this disclosure is not limited to the form of Embodiment 1, and various modifications are possible. For example, in the optical observation apparatus 100 of Embodiment 1, an example is given in which an objective lens is used as the imaging optical system, but the invention is not limited thereto, and a telecentric optical system may be used as the imaging optical system. Figure 3 shows another example of an optical observation apparatus in which the telecentric lens, which is the telecentric optical system, is used as the imaging optical system.

[0048] Figure 3 is a schematic diagram showing the configuration of the optical observation device 200 of Modified Example 1. As shown in Figure 3, the optical observation device 200 of Modified Example 1 is equipped with a telecentric lens 14a instead of an objective lens 14 as the imaging optical system. In addition, in the optical observation device 200 of Modified Example 1, the telecentric lens 14a is positioned between the dichroic mirror 13 and the bandpass filter 16. In the optical observation device 200 of Modified Example 1, ultraviolet light (UV) projected from the light source 12 is reflected by the dichroic mirror 13 and incident on the sample S from a direction approximately perpendicular to the surface of the sample S. Then, in the optical observation device 200 of Modified Example 1, the observation light, including fluorescence FL generated from the sample S by ultraviolet light (UV), passes through the dichroic mirror 13 and is imaged as an observation image on the image sensor of the camera 15 by the telecentric lens 14a. The observation light, including the fluorescent FL, that has passed through the telecentric lens 14a passes through the bandpass filter 16, where the wavelength region of the fluorescent FL is transmitted, and the wavelength region other than the wavelength region of the fluorescent FL is attenuated, and an image is formed on the image sensor of the camera 15. Except for these points, the optical observation device 200 and imaging method of Modification 1 have the same configuration as the optical observation device 100 and imaging method of Embodiment 1, and their description can be applied accordingly.

[0049] (Modification 2) In the optical observation apparatus 100 of Embodiment 1, an example was given in which ultraviolet light (UV) projected from the light source 12 is reflected by the dichroic mirror 13 and projected directly onto the surface of the sample S. However, the apparatus is not limited to this, and may be configured so that the ultraviolet light (UV) is focused onto a specific area on the surface of the sample S. In this case, the optical observation apparatus 100 of Embodiment 1 may further include an illumination optical system such as a collimating optical system or a focusing optical system in the optical path between the light source 12 and the dichroic mirror 13 in Figure 1. Figure 4 shows another example of an optical observation apparatus when a collimating lens (collimating optical system) and a focusing lens (focusing optical system) are used as the illumination optical system.

[0050] Figure 4 is a schematic diagram showing the configuration of the optical observation device 300 of Modified Example 2. As shown in Figure 4, the optical observation device 300 of Modified Example 2 includes a collimating lens 19 and a focusing lens 20 as the illumination optical system. In the optical observation device 300 of Modified Example 2, the collimating lens 19 and the focusing lens 20 are arranged in this order between the light source 12 and the dichroic mirror 13, and the dichroic mirror 13 is arranged between the stage 11 and the objective lens 14. As shown in Figure 4, the dichroic mirror 13, objective lens 14, bandpass filter 16, and camera 15 are arranged in this order on the optical axis from the stage 11 side.

[0051] The collimating lens 19 converts ultraviolet (UV) light projected from the light source 12 into parallel light (collimated light). For example, the collimating lens 19 can be a plano-convex lens, an aspherical lens, a cylindrical lens, etc. The position of the collimating lens 19 can be set appropriately, for example, depending on the type of lens that makes up the collimating lens 19.

[0052] The focusing lens 20 converts ultraviolet light (UV), which has been converted into parallel light, into focused light. The focusing lens 20 can be, for example, a convex lens, a spherical lens, an aspherical lens, a cylindrical lens, etc. The position of the focusing lens 20 can be set appropriately, for example, depending on the type of lens that makes up the focusing lens 20. In the optical observation device 300 of Modification 2, the position of the focusing lens 20 is fixed, but the disclosure is not limited thereto, and the position of the focusing lens 20 may be made changeable. In this case, the focusing lens 20 may be made move in the optical axis direction by a drive unit such as an actuator, for example, as detailed in Modification 4 below. By making the position of the focusing lens 20 variable, the optical observation device of the disclosure can change the focal position of ultraviolet light (UV), for example.

[0053] In the optical observation device 300 of Modified Example 2, ultraviolet light (UV) projected from the light source 12 is first transmitted through the collimating lens 19 and converted into parallel light (collimated light). The ultraviolet light converted into parallel light is then transmitted through the focusing lens 20 and converted into focused light. The ultraviolet light converted into focused light is then reflected by the dichroic mirror 13 and incident on the sample S from a direction approximately perpendicular to the surface of the sample S, forming a focal point on the surface of the sample S. In the optical observation device 300 of Modified Example 2, the observation light containing fluorescence FL generated from the sample S by ultraviolet light (UV) is then transmitted through the dichroic mirror 13 and formed as an observation image on the image sensor of the camera 15 by the objective lens 14. The observation light containing fluorescence FL that has passed through the objective lens 14 is then transmitted through the bandpass filter 16, where the wavelength region of fluorescence FL is transmitted and the wavelength region other than the wavelength region of fluorescence FL is attenuated, and an image is formed on the image sensor of the camera 15. Aside from these points, the optical observation device 300 and imaging method of the modified example 2 have the same configuration as the optical observation device 100 and imaging method of the embodiment 1, and their description can be applied accordingly.

[0054] In the optical observation device 300 of Modified Example 2, the illumination optical system and the imaging optical system are configured separately. Therefore, the focusing lens 20 and the objective lens 14 can be operated independently, and as a result, as will be described later, the focal plane FE of ultraviolet light and the focal plane FO of the objective lens can be moved to any position. Thus, in the optical observation device 300 of Modified Example 2, for example, depending on the purpose of optical observation, the focal plane FE of ultraviolet light and the focal plane FO of the objective lens can be set to the same position or to different positions. In this case, for example, as will be described in detail in Embodiment 3 below, by aligning the focal plane FE of ultraviolet light with the surface of the sample S and moving the focal plane FO of the objective lens to the inside of the sample S, an image of the cell background that can be used for more accurate correction can be obtained.

[0055] (Variation 3) In the optical observation apparatus 100 of Embodiment 1, an example was given in which ultraviolet light (UV) projected from the light source 12 is reflected by the dichroic mirror 13 and projected directly onto the surface of the sample S. However, the apparatus is not limited to this, and may be configured so that ultraviolet light (UV) with a uniform light intensity distribution is incident on the surface of the sample S. In this case, the optical observation apparatus 100 of Embodiment 1 may further include, for example, an illumination optical system such as a shaping optical system. Figure 5 shows another example of an optical observation apparatus in which a collimating lens and a homogenizer are used as the illumination optical system.

[0056] Figure 5 is a schematic diagram showing the configuration of the optical observation device 400 of Modified Example 3. As shown in Figure 5, the optical observation device 400 of Modified Example 3 includes a collimating lens 19 and a homogenizer 22 as the illumination optical system. In the optical observation device 400 of Modified Example 3, the collimating lens 19 and the homogenizer 22 are arranged in this order between the light source 12 and the dichroic mirror 13, and the dichroic mirror 13 is arranged between the stage 11 and the objective lens 14. As shown in Figure 5, the dichroic mirror 13, objective lens 14, bandpass filter 16, and camera 15 are arranged in this order on the optical axis from the stage 11 side.

[0057] The homogenizer 22 homogenizes the light intensity distribution (illuminance distribution) of ultraviolet (UV) light that has been transmitted through the collimating lens 19 and converted into parallel light (collimated light). For example, the homogenizer 22 can be a microlens array (fly-eye lens), an aspherical lens, a diffractive optical element (DOE), a rod lens, or an optical guide fiber. The position of the homogenizer 22 can be set appropriately, for example, depending on the type of material that makes up the homogenizer 22.

[0058] In the optical observation device 400 of Modified Example 3, ultraviolet light (UV) projected from the light source 12 is first transmitted through the collimating lens 19 and converted into parallel light. The ultraviolet light converted into parallel light is then transmitted through the homogenizer 22 to equalize the light intensity distribution, and then reflected by the dichroic mirror 13 and incident on the sample S from a direction approximately perpendicular to the surface of the sample S. Then, in the optical observation device 400 of Modified Example 3, the observation light containing fluorescence FL generated from the sample S by ultraviolet light (UV) is transmitted through the dichroic mirror 13 and formed as an observation image on the image sensor of the camera 15 by the objective lens 14. The observation light containing fluorescence FL that has passed through the objective lens 14 is then transmitted through the bandpass filter 16, the wavelength region of fluorescence FL is transmitted, and the wavelength region other than the wavelength region of fluorescence FL is attenuated, and an image is formed on the image sensor of the camera 15. Aside from these points, the optical observation device 400 and imaging method of the modified example 3 have the same configuration as the optical observation device 100 and imaging method of Embodiment 1, and their description can be applied accordingly.

[0059] In the optical observation device 400 of Modified Example 3, the homogenizer 22 is configured to homogenize the light intensity distribution of ultraviolet (UV) light that has been transmitted through the collimating lens 19 and converted into parallel light. Therefore, in the optical observation device 400 of Modified Example 3, ultraviolet (UV) light can be irradiated onto the surface of the sample S as parallel light with a uniform light intensity distribution and no uneven irradiation. As a result, with the optical observation device 400 of Modified Example 3, an observation image with an improved signal-to-noise ratio can be obtained.

[0060] (Embodiment 2) Embodiment 2 relates to a second optical observation apparatus and a second imaging method of the present disclosure.

[0061] This embodiment is an example of a second optical observation device. Figure 6 is a schematic diagram showing the configuration of the optical observation device 500 of Embodiment 2. As shown in Figure 6, the optical observation device 500 of Embodiment 2 has a Z-axis stage 17 as a drive unit, in addition to the configuration of the optical observation device 100 of Embodiment 1. The Z-axis stage 17 is connected to the camera 15 side of the objective lens so that the objective lens 14 can move in the optical axis direction (Z axis direction). The optical observation device 500 of Embodiment 2 also includes a control unit 18.

[0062] The Z-axis stage 17 is capable of moving the position of the objective lens 14 relative to the sample S. The Z-axis stage 17 is controlled by a control unit 18, which will be described later. In the optical observation apparatus 500 of Embodiment 2, the Z-axis stage 17 is used as the drive unit, but the drive unit can be any drive unit that can move the objective lens 14 in the direction of the optical axis. Specific examples include a combination of a motor and a ball screw, a rack and pinion, a timing belt, etc.

[0063] Figure 7 shows the configuration of the control unit 18. Figure 7 is a block diagram showing an example of the configuration of the control unit 18. As shown in Figure 7, the control unit 18 has a configuration similar to that of a personal computer, server computer, workstation, etc.

[0064] The control unit 18 includes a central processing unit (CPU) 18a, main memory 18b, auxiliary storage device 18c, video codec 18e, I / O (input-output) interface 18f, controller (system controller, I / O controller, etc.) 18g, and bus 18h.

[0065] The CPU 18a, in cooperation with other components via the controller 18e, is responsible for the overall control of the optical observation apparatus 500 of Embodiment 2. In the optical observation apparatus 500 of Embodiment 2, the CPU 18a executes, for example, the program 18d of this disclosure and other programs, and also reads and writes various types of information. Specifically, the CPU 18a functions as a drive instruction unit 181, an imaging instruction unit 182, and a correction unit 183. The optical observation apparatus 500 of Embodiment 2 is equipped with a CPU 18a as a computing device, but may also be equipped with other computing devices such as a GPU (Graphics Processing Unit) or an APU (Accelerated Processing Unit), or a combination of the CPU and these.

[0066] The main memory 18b is also called primary memory. When the CPU 18a performs processing, the memory 18b reads various operational programs, such as the program 105 of the present invention, which are stored in the storage device 18c (auxiliary storage device) described later. The CPU 18a then reads and decodes the data from the memory 18b and executes the program. The main memory is, for example, RAM (random access memory). The memory 18b also includes, for example, ROM (read-only memory).

[0067] The auxiliary storage device 18c stores an operating program including the program 18d of this disclosure. The auxiliary storage device 18c includes, for example, a storage medium and a drive for reading from and writing to the storage medium. The storage medium is not particularly limited and may be internal or external, and examples include HD (hard disk), FD (floppy disk), CD-ROM, CD-R, CD-RW, MO, DVD, flash memory, memory card, etc., and the drive is not particularly limited. The auxiliary storage device 18c may be, for example, a hard disk drive (HDD) in which the storage medium and the drive are integrated.

[0068] The video codec 18e includes a GPU (Graphics Processing Unit) that generates a screen to be displayed based on drawing instructions received from the CPU 18a and transmits the screen signal to, for example, a display device 518i outside the optical observation device 500 of Embodiment 2, and a video memory that temporarily stores screen and image data.

[0069] The I / O interface 18e is a device that communicates with the camera 15 and the Z-axis stage 17 to control them or to acquire information such as images. The I / O interface 18e may also include a servo driver (servo controller). Furthermore, the I / O interface 18e may be connected to an input means (input device 18j) outside the optical observation apparatus 500 of Embodiment 2, for example. The input device 18j may be a pointing device such as a touch panel, trackpad, or mouse that can be operated by the user's fingers, a keyboard, or push buttons.

[0070] Bus 18h can also be connected to external devices, for example. Examples of such external devices include external storage devices (external databases, etc.) and printers. The optical observation device 500 of Embodiment 2 can be connected to a communication network, for example, by a communication device connected to bus 18h, and can also be connected to the external devices via the communication network. The communication network is not particularly limited and can use any known network, for example, it may be wired or wireless. Examples of such communication networks include the Internet, WWW (World Wide Web), telephone lines, LAN (Local Area Network), WiFi (Wireless Fidelity), etc.

[0071] The display device 18i may be a monitor that outputs video (for example, various image display devices such as liquid crystal displays (LCDs) and cathode ray tube (CRT) displays).

[0072] Next, the imaging method of Embodiment 2 using the optical observation device 500 of Embodiment 2 will be described with reference to Figures 8 and 9. Figure 8 is a flowchart of the imaging method of Embodiment 2. As shown in Figure 8, the imaging method of Embodiment 2 includes step S4 (correction) in addition to each step (S1 to S3) of the imaging method of Embodiment 1.

[0073] First, steps S1 and S2 are performed in the same manner as the imaging method of Embodiment 1.

[0074] Next, in step S3, an observation image of the sample S is captured at different positions in the optical axis direction. Specifically, the drive instruction unit 181 of the control unit 18 instructs the Z-axis stage 17 to move the objective lens 14 to the first position shown in Figure 9(A). The first position of the objective lens 14 is the position where the image sensor of the camera 15 is at its focal point relative to the surface of the sample S, specifically, the position where the focal point of the image sensor of the camera 15 is set to the surface of the sample S that is in contact with the mounting surface of the stage 11. Then, at the instruction of the imaging instruction unit 182 of the control unit 18, the camera 15 captures the first observation image G1. Next, the drive instruction unit 181 of the control unit 18 instructs the Z-axis stage 17 to move the objective lens 14 to the second position shown in Figure 9(B). The second position of the objective lens 14 is a position in the Z-axis direction that is further toward the sample S than the first position. The second position of the objective lens 14 is the position that sets the focal point of the image sensor of the camera 15 to a position moved from the surface of the sample S toward the inside of the sample. The "outside of the sample" is the direction toward the objective lens 14 in a direction approximately perpendicular to the sample surface (downward in Figure 9(A)), and the "inside of the sample" is the opposite direction from the outside of the sample (upward in Figure 9(A)). Then, according to the instructions of the imaging instruction unit 182 of the control unit 18, the camera 15 captures the second observation image G2. In the optical axis direction, the distance between the first position and the second position can be set to, for example, 10 to 30 μm or about 20 μm. In the imaging method of Embodiment 2, by setting the distance between the first position and the second position to, for example, 40 μm or less, a second observation image G2 suitable for correcting the first observation image G1 can be captured.

[0075] Next, in step S4, the first observation image G1 is corrected using the two first observation images G1 and second observation images G2 acquired in step S3. Specifically, in step S4, the first observation image G1 is corrected by taking the difference (G1-G2) between the first observation image G1 and the second observation image G2. Specifically, the first observation image G1 and the second observation image G2 are decomposed into RGB channels, and a difference process is performed on each channel between the first observation image G1 and the second observation image G2. Then, by merging each channel, the corrected observation image G3 can be obtained. Furthermore, if the pixel values ​​of the image are converted to floating-point format before the difference process is performed, the contours of the object can be enhanced. In this case, the corrected observation image G3 after the difference process may be saved in floating-point format, or the values ​​may be offset and converted to a format such as 8, 16, or 32 bits before saving. As a result, in step S4, the background image of the cell image in the first observation image G1 is canceled, and a corrected observation image G3 is obtained in which the cell image is clarified.

[0076] This concludes the imaging method of Embodiment 2.

[0077] In the optical observation apparatus 500 of Embodiment 2, the objective lens 14 is moved to a first position, and the first observation image G1 is captured with the focal position of the image sensor of the camera 15 set to the surface of the sample S. Furthermore, the objective lens 14 is moved to a second position, and the second observation image G2 is captured with the focal position of the image sensor of the camera 15 set to a position moved inward from the surface of the sample S. The first observation image G1 is captured with the focal position of the image sensor of the camera 15 coinciding with the surface of the sample S. Therefore, in the first observation image G1, the fluorescently stained cells contained in the surface of the sample S appear as a high-brightness image, while the background image of the cells appears as a lower-brightness image. On the other hand, the second observation image G2 is captured with the focal position of the image sensor of the camera 15 coinciding with a position moved inward from the surface of the sample S. In other words, the second observation image G2 is an image captured when the focus does not coincide with the surface of the sample S. Therefore, in the second observation image G2, the fluorescently stained cells contained in the surface of the sample S appear to have a considerably lower brightness than in the first observation image G1. Also, in the image of the second observation image G2, the background of the cells is captured in a direction in which the focal position of the camera 15's image sensor is aligned, compared to when the first observation image G1 was captured, and the image of the background of the cells appears to have a higher brightness than the image of the first observation image G1. Here, when the difference between the first observation image G1 and the second observation image G2 is taken, the difference in brightness of the image showing cells that showed high brightness in the first observation image G1 and low brightness in the second observation image G2 is large. On the other hand, when the same difference is taken, the difference in brightness of the image showing the background of cells that showed low brightness in the first observation image G1 and relatively higher brightness in the second observation image G2 than in the first observation image G1 is small. In other words, according to this principle, the observation image G3 obtained by taking the difference between the first observation image G1 and the second observation image G2 has the background image of the cells canceled out while the contrast of the image showing the cells is increased. By obtaining the observation image G3, which is a corrected observation image G1 in this way, the influence of the background of the fluorescently stained cells of sample S is suppressed, and an observation image in which the image of the cells is clearly defined can be obtained.

[0078] Figure 10 shows photographs of the first observation image G1 before correction and the observation image G3 after correction. A mouse kidney tissue section (2 mm thick) was rinsed with buffer, fixed with ethanol, washed with buffer, and stained with a fluorescent dye. The first observation image G1 (Figure 10(A)) was captured from the stained kidney tissue section at a first position where the kidney tissue surface was the focal point, and the second observation image G2 was captured at a second position 20 μm inward from the position where the first observation image G1 was captured. The difference processing between the first observation image G1 and the second observation image G2 was performed as described above to obtain the corrected observation image G3 (Figure 10(B)). As shown in Figure 10(A), in the first uncorrected observation image G1, the area around the cell containing the granular cell nucleus and cytoplasm (background) is blurred, whereas in the corrected observation image G3, the cell containing the granular cell nucleus and cytoplasm is clearly defined, and the contrast with the background surrounding the cell is increased.

[0079] (Embodiment 3) Embodiment 3 relates to a third optical observation apparatus and a third imaging method of the present disclosure.

[0080] This embodiment is an example of a third optical observation device. Figures 11(A) and (B) are schematic diagrams showing the configuration of the optical observation device 600 of Embodiment 3. As shown in Figures 11(A) and (B), the optical observation device 600 of Embodiment 3 has an illumination optical system such as a focusing optical system in addition to the configuration of the optical observation device 500 of Embodiment 2. The optical observation device 600 of Embodiment 3 includes a collimating lens 19 and a focusing lens 20 as the illumination optical system. The collimating lens 19 and the focusing lens 20 are arranged in this order between the light source 12 and the dichroic mirror 13, and the dichroic mirror 13 is arranged between the stage 11 and the objective lens 14. As shown in Figures 11(A) and (B), the dichroic mirror 13, objective lens 14, Z-axis stage 17, bandpass filter 16, and camera 15 are arranged on the optical axis in this order from the stage 11 side. As shown in Figures 11(A) and (B), in the optical observation apparatus 600 of Embodiment 3, the focusing lens 20 is positioned so that the focal plane FE of ultraviolet light UV is in contact with the surface of the stage 11 and coincides with the surface layer of the sample S.

[0081] Next, the imaging method of Embodiment 3 using the optical observation device 600 of Embodiment 3 will be described. The imaging method of Embodiment 3 includes the same steps as the imaging method of Embodiment 2: S1 (irradiation), S2 (imaging), S3 (imaging), and S4 (correction).

[0082] First, steps S1 and S2 are performed in the same manner as the imaging method of Embodiment 2.

[0083] Next, in step S3, observation images of the sample S are captured at different positions in the optical axis direction. Specifically, the drive instruction unit 181 of the control unit 18 instructs the Z-axis stage 17 to move the objective lens 14 to the first position shown in Figure 11(A). At the first position, the objective lens 14 is positioned so that the focal point of the image sensor of the camera 15 is the surface of the sample S, specifically, the focal point of the image sensor of the camera 15 is set to the surface of the sample S that is in contact with the mounting surface of the stage 11. As shown in Figure 11(A), at the first position, the focal plane FE of ultraviolet light and the focal plane FO of the objective lens coincide. Then, at the instruction of the imaging instruction unit 182 of the control unit 18, the camera 15 captures the first observation image G1. Next, the drive instruction unit 181 of the control unit 18 instructs the Z-axis stage 17 to move the objective lens 14 to the second position shown in Figure 11(B). At the second position, the objective lens 14 is positioned in the Z-axis direction further in the direction of the sample S than at the first position. At the second position, the objective lens 14 is positioned such that the focal point of the image sensor of the camera 15 is located in the direction inward from the surface of the sample S. As shown in Figure 11(B), at the second position, the focal plane FE of ultraviolet light and the focal plane FO of the objective lens are different. Note that "outside the sample" is the direction toward the objective lens 14 (downward in Figure 11) in a direction approximately perpendicular to the sample surface, and "inside the sample" is the opposite direction from the outside of the sample (upward in Figure 11). Then, at the instruction of the imaging instruction unit 182 of the control unit 18, the camera 15 captures the second observation image G2. In the optical axis direction, the distance between the first position and the second position can be set to, for example, 10 to 30 μm or about 20 μm. In the imaging method of Embodiment 3, by setting the distance between the first position and the second position to, for example, 40 μm or less, a second observation image G2 suitable for correcting the first observation image G1 can be acquired.

[0084] Next, step S4 is performed in the same manner as the imaging method of Embodiment 2.

[0085] This concludes the imaging method of Embodiment 3.

[0086] In the optical observation apparatus 600 of Embodiment 3, the objective lens 14 is moved to a first position, and the first observation image G1 is captured with the focal position of the image sensor of the camera 15 set to the surface of the sample S. Furthermore, the objective lens 14 is moved to a second position, and the second observation image G2 is captured with the focal position of the image sensor of the camera 15 set to a position moved inward from the surface of the sample S. As shown in Figure 11(A), the first observation image G1 is captured with the surface of the sample S aligned with the focal plane FE of ultraviolet light and the focal plane FO of the objective lens. Therefore, in the first observation image G1, the fluorescently stained cells contained in the surface of the sample S appear as a high-brightness image, while the background image of the cells appears as a lower-brightness image. On the other hand, as shown in Figure 11(B), the second observation image G2 was captured with the surface of the sample S coinciding with the focal plane FE of ultraviolet light (UV), while the focal plane FO of the objective lens was located inside the sample. Therefore, in the second observation image G2, the fluorescently stained cells contained on the surface of the sample S appear to have a considerably lower brightness than in the first observation image G1. On the other hand, in the image of the second observation image G2, the background image of the cells is caused by the scattering of ultraviolet light (UV) from the surface of the sample S, and since the position of the first observation image and the position of the focal plane FE of ultraviolet light (UV) do not change, the difference in the background image of the cells from the first observation image G1 is small. Here, when taking the difference between the first observation image G1 and the second observation image G2 in the image showing the cells, the difference in brightness of the image showing cells that showed high brightness in the first observation image G1 and low brightness in the second observation image G2 is large. On the other hand, in the image of the cell background, when a similar difference is taken, the difference in brightness of the cell background between the first observation image G1 and the second observation image G2 becomes smaller. That is, by this principle, the observation image G3 obtained by taking the difference between the first observation image G1 and the second observation image G2 has the image of the cell background canceled out, while the contrast of the image showing the cell becomes higher. By obtaining an observation image G3 that corrects observation image G1 in this way, the influence of the background of the fluorescently stained cells of sample S is suppressed, and an observation image in which the cell image is clearly defined can be obtained.Furthermore, in the optical observation device 600 of Embodiment 3, the difference in brightness of the cell background between the first observation image G1 and the second observation image G2 in the image of the cell background is smaller than the difference in brightness of the cell background in the optical observation device 500 of Embodiment 2. Therefore, with the optical observation device 600 of Embodiment 3, the influence of the cell background is further suppressed, and an observation image with a clearer image of the cell can be obtained.

[0087] (Modification 4) The optical observation apparatus of this disclosure is not limited to the form of Embodiment 3, and various modifications are possible. For example, in the optical observation apparatus 600 of Embodiment 3, the case in which the condensing lens 20 is fixed in a predetermined position and the objective lens 14 is moved in the optical axis direction is illustrated, but the apparatus is not limited to this, and the objective lens 14 may be fixed in a predetermined position and the condensing lens 20 may be moved in the optical axis direction. In this case, the optical observation apparatus 600 of Embodiment 3 may further include, for example, a drive unit that can move the condensing lens 20 in the optical axis direction. Figure 12 shows another example of an optical observation apparatus using a drive unit.

[0088] Figure 12 is a schematic diagram showing the configuration of the optical observation device 700 of the modified example 4. As shown in Figure 12, the optical observation device 700 of the modified example 4 includes a drive unit 21.

[0089] The drive unit 21 moves the focusing lens 20 in the optical axis direction, thereby adjusting the position of the focal plane FE of ultraviolet light (UV). The drive unit 21 only needs to be capable of moving the focusing lens 20 in the optical axis direction; for example, an actuator, a cam mechanism, etc., can be used. Examples of the actuator include a servo motor, a stepping motor, etc. The position of the drive unit 21 can be set appropriately according to the position of the focusing lens 20, for example. The drive unit 21 is controlled by the control unit. The control unit can be described by referring to the description of the control unit 18 above.

[0090] In the optical observation apparatus 700 of Modified Example 4, with the focal plane FO of the objective lens aligned with the surface of the sample S, the condensing lens 20 is moved in the optical axis direction using the drive unit 21, and the position of the focal plane FE of ultraviolet light UV is moved from the position on the surface of the sample S ((Figure 12(A))) to the direction outward from the sample (Figure 12(B)) or inward from the sample (Figure 12(C)). Except for these points, the optical observation apparatus 700 and imaging method of Modified Example 4 have the same configuration as the optical observation apparatus 600 and imaging method of Embodiment 3, and their description can be applied accordingly.

[0091] In the optical observation device 700 of Modification 4, the illumination optical system and the imaging optical system are configured separately. Therefore, the focusing lens 20 and the objective lens 14 can be operated independently, and the focal plane FE of ultraviolet light (UV) can be moved to any position while the focal plane FO of the objective lens is aligned with the surface of the sample S. When the position of the focal plane FE of ultraviolet light is moved from the surface of the sample S ((Figure 12(A))) toward the outside of the sample (Figure 12(B)), ultraviolet light that spreads after being focused at the focal plane FE can be irradiated onto the surface of the sample S. Also, when the position of the focal plane FE of ultraviolet light is moved from the surface of the sample S ((Figure 12(A))) toward the inside of the sample (Figure 12(C)), ultraviolet light that spreads before being focused at the focal plane FE can be irradiated onto the surface of the sample S.

[0092] (Variation 5) In the optical observation apparatus 600 of Embodiment 3, the example shows the use of a collimating lens and a focusing lens as the illumination optical system, but it is not limited to this, and the apparatus may be configured so that ultraviolet light (UV) with a uniform light intensity distribution is incident on the surface layer of the sample S. In this case, the optical observation apparatus 600 of Embodiment 3 may, for example, be equipped with an illumination optical system such as the shaping optical system in place of or in addition to the focusing lens. Figure 13 shows another example of an optical observation apparatus in which a collimating lens and a homogenizer are used as the illumination optical system.

[0093] Figure 13 is a schematic diagram showing the configuration of the optical observation device 800 of Modification 5. As shown in Figure 13, the optical observation device 800 of Modification 5 includes a collimating lens 19 and a homogenizer 22 as the illumination optical system. In the optical observation device 800 of Modification 5, the collimating lens 19 and the homogenizer 22 are arranged in this order between the light source 12 and the dichroic mirror 13, and the dichroic mirror 13 is arranged between the stage 11 and the objective lens 14. As shown in Figure 13, the dichroic mirror 13, objective lens 14, bandpass filter 16, and camera 15 are arranged in this order on the optical axis from the stage 11 side.

[0094] In the optical observation device 800 of Modified Example 5, ultraviolet light (UV) projected from the light source 12 is first transmitted through the collimating lens 19 and converted into parallel light (collimated light). The ultraviolet light (UV) converted into parallel light is then transmitted through the homogenizer 22 to equalize the light intensity distribution (illuminance distribution), and then reflected by the dichroic mirror 13 and incident on the sample S from a direction approximately perpendicular to the surface of the sample S. Then, in the optical observation device 800 of Modified Example 5, the observation light containing fluorescence FL generated from the sample S by ultraviolet light (UV) is transmitted through the dichroic mirror 13 and formed as an observation image on the image sensor of the camera 15 by the objective lens 14. The observation light containing fluorescence FL that has passed through the objective lens 14 is then transmitted through the bandpass filter 16, the wavelength region of fluorescence FL is transmitted, and the wavelength region other than the wavelength region of fluorescence FL is attenuated, and an image is formed on the image sensor of the camera 15. Aside from these points, the optical observation device 800 and imaging method of the modified example 5 have the same configuration as the optical observation device 600 and imaging method of the embodiment 3, and their description can be applied accordingly.

[0095] In the optical observation device 800 of Modified Example 5, the homogenizer 22 is configured to homogenize the light intensity distribution of ultraviolet (UV) light that has been transmitted through the collimating lens 19 and converted into parallel light. Therefore, in the optical observation device 800 of Modified Example 5, ultraviolet (UV) light can be irradiated onto the surface layer of the sample S as parallel light with a uniform light intensity distribution and no uneven irradiation. As a result, with the optical observation device 800 of Modified Example 5, an observation image with an improved signal-to-noise ratio can be obtained.

[0096] (Embodiment 4) Embodiment 4 relates to a fourth optical observation apparatus and a fourth imaging method of the present disclosure.

[0097] This embodiment is an example of a fourth optical observation device. Figure 14 is a schematic diagram showing the configuration of the optical observation device 900 of Embodiment 4. As shown in Figure 14, the optical observation device 900 of Embodiment 4 has an illumination optical system such as a focusing optical system in addition to the configuration of the optical observation device 100 of Embodiment 1. The optical observation device 900 of Embodiment 4 includes a collimating lens 19, a focusing lens 20, a first mirror 23a, and a second mirror 23b as the illumination optical system. The collimating lens 19, the focusing lens 20, the first mirror 23a, and the second mirror 23b are arranged in this order on the optical axis between the light source 12 and the stage 11. As shown in Figure 14, the objective lens 14, the bandpass filter 16, and the camera 15 are arranged in this order on the optical axis from the stage 11 side.

[0098] The first mirror 23a reflects ultraviolet (UV) light that has passed through the focusing lens 20 toward the second mirror 23b. The first mirror 23a can be any material capable of reflecting ultraviolet (UV) light, such as a metal mirror, polymer mirror, glass mirror, or polypolymer mirror.

[0099] The second mirror 23b reflects the ultraviolet (UV) light reflected by the first mirror 23a toward the stage 11. The second mirror 23b reflects the ultraviolet (UV) light such that the angle of incidence θ of the ultraviolet (UV) light with respect to the surface of the stage 11 is acute (0° < θ < 90°). The second mirror 23b can be any material capable of reflecting ultraviolet (UV) light, such as a metal mirror, polymer mirror, glass mirror, or polypolymer mirror.

[0100] Next, the imaging method of Embodiment 4 using the optical observation device 900 of Embodiment 4 will be described. The imaging method of Embodiment 4 includes the same steps as the imaging method of Embodiment 1: S1 step (irradiation), S2 step (imaging), and S3 step (imaging).

[0101] In step S1, ultraviolet (UV) light projected from the light source 12 is irradiated onto the sample S placed on the stage 11. Specifically, in step S1, the ultraviolet (UV) light projected from the light source 12 is first transmitted through the collimating lens 19 and converted into parallel light (collimated light). The ultraviolet (UV) light converted into parallel light is then transmitted through the focusing lens 20 and converted into focused light. The ultraviolet (UV) light converted into focused light is reflected by the first mirror 23a toward the second mirror 23b, and then reflected by the second mirror 23b toward the stage 11. The reflected ultraviolet (UV) light is then irradiated onto the surface of the sample S from an oblique direction, forming a focal point on the surface of the sample S. When the sample S is irradiated with ultraviolet (UV) light, the fluorescent dye on the sample S is excited and enters an excited state, and fluorescence FL is emitted when the fluorescent dye returns to its ground state.

[0102] In step S2, the observation light containing fluorescence FL emitted from the sample S by ultraviolet light (UV) in step S1 is imaged onto the image sensor of the camera 15 by the objective lens 14. Specifically, in step S2, the observation light in the direction of the optical axis of the observation light containing fluorescence FL is transmitted through the objective lens 14. At this time, the objective lens 14 images the observation light so as to form an observation image on the image sensor of the camera 15. The observation light transmitted through the objective lens 14 comes into contact with the bandpass filter 16, where the wavelength region of fluorescence FL is transmitted and the wavelength region other than the wavelength region of fluorescence FL is attenuated. The observation light transmitted through the objective lens 14 is imaged onto the image sensor of the camera 15.

[0103] In step S3, the image sensor of camera 15 captures the formed observation image. This allows for the acquisition of an observation image of sample S.

[0104] This concludes the imaging method of Embodiment 4.

[0105] In the optical observation device 900 of Embodiment 4, ultraviolet light (UV) is reflected by a first mirror 23a and a second mirror 23b positioned outside the optical axis between the sample S and the camera 15. Therefore, according to the optical observation device 900 of Embodiment 4, ultraviolet light (UV) can be irradiated onto the surface of the sample S from an oblique direction without passing through the objective lens 14. In this case, since the ultraviolet light (UV) is converted into parallel light in the optical observation device 900 of Embodiment 4, the sample S can be uniformly irradiated with ultraviolet light (UV). Therefore, the optical observation device 900 of Embodiment 4 can suppress the generation of shadows caused by irregularities in the sample S.

[0106] Furthermore, in the optical observation device 900 of Embodiment 4, the illumination optical system and the imaging optical system are configured separately. Therefore, the condensing lens 20 and the objective lens 14 can be operated independently, and as described above, the focal plane FE of ultraviolet light and the focal plane FO of the objective lens can be moved to any position. Accordingly, in the optical observation device 900 of Embodiment 4, for example, depending on the purpose of optical observation, the focal plane FE of ultraviolet light and the focal plane FO of the objective lens can be set to the same position or to different positions.

[0107] (Experimental variation 6) In the optical observation apparatus 900 of Embodiment 4, an example was given in which a collimating lens, a focusing lens, a first mirror, and a second mirror are used as the illumination optical system. However, the apparatus is not limited to this, and the apparatus may be configured so that ultraviolet light (UV) with a uniform light intensity distribution is irradiated onto the surface of the sample S from an oblique direction. In this case, the optical observation apparatus 900 of Embodiment 4 may include, for example, an illumination optical system such as the shaping optical system in addition to the collimating lens, focusing lens, first mirror, and second mirror. Figure 15 shows another example of an optical observation apparatus in which a collimating lens, a homogenizer, a focusing lens, a first mirror, and a second mirror are used as the illumination optical system.

[0108] Figure 15 is a schematic diagram showing the configuration of the optical observation device 1000 of Modification 6. As shown in Figure 15, the optical observation device 1000 of Modification 6 includes a collimating lens 19, a homogenizer 22, a focusing lens 20, a first mirror 23a, and a second mirror 23b as the illumination optical system. In the optical observation device 1000 of Modification 6, the collimating lens 19, homogenizer 22, focusing lens 20, the first mirror 23a, and the second mirror 23b are arranged in this order on the optical axis between the light source 12 and the stage 11. As shown in Figure 15, the objective lens 14, bandpass filter 16, and camera 15 are arranged in this order on the optical axis from the stage 11 side.

[0109] In the optical observation device 1000 of Modified Example 6, ultraviolet light (UV) projected from the light source 12 is first transmitted through the collimating lens 19 and converted into parallel light (collimated light). The ultraviolet light (UV) converted into parallel light is then transmitted through the homogenizer 22 to equalize the light intensity distribution (illuminance distribution), and then transmitted through the focusing lens 20 to be converted into focused light. Except for these points, the optical observation device 1000 and imaging method of Modified Example 6 have the same configuration as the optical observation device 900 and imaging method of Embodiment 4, and their description can be applied accordingly.

[0110] In the optical observation device 1000 of Modified Example 6, ultraviolet light (UV) with a uniform light intensity distribution is reflected by the first mirror 23a and the second mirror 23b, which are positioned outside the optical axis between the sample S and the camera 15. Therefore, according to the optical observation device 1000 of Modified Example 6, ultraviolet light (UV) with a uniform light intensity distribution can be irradiated onto the surface of the sample S from an oblique direction without passing through the objective lens 14. As a result, the optical observation device 900 of Embodiment 4 can suppress the generation of shadows caused by the unevenness of the sample S.

[0111] Although the present invention has been described above with reference to embodiments, the present invention is not limited to the above embodiments. Various modifications to the configuration and details of the present invention can be understood by those skilled in the art within the scope of the present invention.

[0112] This application claims priority based on Japanese Patent Application No. 2024-152772, filed on 4 September 2024, and incorporates all of its disclosures herein.

[0113] The patents, patent applications, and documents cited herein are incorporated herein by reference in the same manner as their contents are specifically described herein.

[0114] <Note> Some or all of the above embodiments and examples may be described as follows, but are not limited to the following. <First Optical Observation Device> (Note 1) A placement unit capable of arranging samples containing cells, A light source capable of projecting ultraviolet light, An optical unit that reflects ultraviolet light incident from the light source onto the sample, irradiating the sample with ultraviolet light, and transmits observation light emitted from the sample by the ultraviolet light, An imaging optical system that forms an image of the aforementioned observation light as an observation image, It includes an imaging unit that captures the formed observation image using an image sensor, An optical observation device in which ultraviolet light reflected by the optical unit is incident on the sample from a direction substantially perpendicular to the surface of the sample. (Note 2) Includes a filter unit that changes the wavelength range in the transmitted observation light, The filter unit is positioned between the optical unit and the imaging unit in the optical observation apparatus as described in Appendix 1. (Note 3) The aforementioned filter unit includes a bandpass filter, as described in Appendix 2 of the optical observation apparatus. (Note 4) The aforementioned filter unit transmits wavelengths in the 400-600 nm range, as described in Appendix 2 or 3 of the optical observation apparatus. (Note 5) The optical unit is an optical observation device as described in any of the appendices 1 to 4, including a dichroic mirror. (Note 6) The optical observation apparatus according to any one of the appendices 1 to 5, wherein the optical unit, the imaging optical system, and the imaging unit are arranged in this order in the optical axis direction of the imaging optical system. (Note 7) The imaging optical system includes an objective lens, The objective lens is positioned between the optical unit and the imaging unit. An optical observation apparatus according to any one of the appendices 1 to 6, wherein the observation light emitted from the sample by the ultraviolet light passes through the optical unit and is imaged as an observation image on the image sensor by the objective lens. (Note 8) The imaging optical system includes a telecentric lens, The telecentric lens is positioned between the optical unit and the imaging unit. An optical observation apparatus according to any one of the appendices 1 to 6, wherein the observation light emitted from the sample by the ultraviolet light is imaged onto the image sensor by the telecentric lens. (Note 9) The optical observation apparatus according to any one of the appendices 1 to 5, wherein the imaging optical system, the optical unit, and the imaging unit are arranged in this order in the optical axis direction of the imaging optical system. (Note 10) The imaging optical system includes an objective lens, The objective lens is positioned between the optical unit and the sample. The reflected ultraviolet light is irradiated onto the sample through the objective lens. An optical observation apparatus according to any one of the appendices 1 to 5, wherein the observation light emitted from the sample by the ultraviolet light is imaged as an observation image on the image sensor by the objective lens. (Note 11) The system includes an illumination optical system that guides ultraviolet light projected from the light source onto the sample, The illumination optical system is an optical observation device according to any one of the appendices 1 to 10, disposed between the light source and the optical unit. (Note 12) The illumination optical system is Collimating optical system that converts ultraviolet light into parallel light, A shaping optical system that changes the light intensity distribution of the ultraviolet light, and / or A focusing optical system for focusing the ultraviolet light onto the sample, An optical observation device, including any of the devices described in Appendix 1 to 11. (Note 13) The optical observation apparatus described in Appendix 12, wherein the illumination optical system includes the collimating optical system and the focusing optical system. (Note 14) The optical observation apparatus according to Appendix 12, wherein the illumination optical system includes the collimating optical system and the shaping optical system. (Note 15) A focusing optical system for focusing the ultraviolet light onto the sample, The light-gathering optical system is provided with a light-gathering optical system drive unit that can move along the ultraviolet light path, Control unit, including, The control unit is The light-gathering optical system drive unit is driven and controlled so that the light-gathering optical system is positioned at a first position on the optical path and a second position on the optical path, where the positional relationship of the ultraviolet light on the optical path is different. Furthermore, the optical observation apparatus according to any one of the appendices 1 to 14, wherein the focusing optical system drive unit is controlled to capture a first observation image formed when the focusing optical system is in the first position, and to capture a second observation image formed when the focusing optical system is in the second position, thereby correcting the first observation image with the second observation image to clarify the image of cells contained in the sample. (Note 16) A drive unit that can move the position of the imaging optical system relative to the sample, Control unit, including, The control unit is The drive unit is controlled to drive the imaging optical system so that it is positioned at a first position relative to the surface of the sample and at a second position relative to the surface of the sample. Furthermore, the imaging unit is controlled so that the imaging optical system is positioned at the first position to capture a first observation image, and the imaging optical system is positioned at the second position to capture a second observation image, thereby correcting the first observation image with the second observation image and clarifying the image of cells contained in the surface layer of the sample. An optical observation device as described in any of the appendices 1 to 15. (Note 17) The first position is a position that sets the focal position of the image sensor of the imaging unit to the surface layer of the sample. The optical observation apparatus as described in Appendix 16, wherein the second position is a position in which the focal position of the image sensor of the imaging unit is moved from the surface layer of the sample toward the inside of the sample. (Note 18) The control unit is An optical observation device according to Appendix 16 or 17, which corrects the background image of the cell in the first observation image relating to the first position by taking the difference between the first observation image relating to the first position and the second observation image relating to the second position. (Note 19) The optical observation apparatus according to any one of the appendices 1 to 18, wherein the wavelength range of the ultraviolet light is 200 to 400 nm. (Note 20) The optical observation apparatus according to any one of the appendices 1 to 19, wherein the wavelength range of the observation light is 400 to 600 nm. (Note 21) The optical observation apparatus according to any one of the appendices 1 to 20, wherein the cells are stained with a fluorescent dye that can be excited by ultraviolet light. <Second optical observation device> (Note 22) A placement unit capable of arranging samples containing cells, A light source capable of projecting ultraviolet light, An optical unit that reflects ultraviolet light incident from the light source onto the sample, irradiating the sample with ultraviolet light, and transmits observation light emitted from the sample by the ultraviolet light, An imaging optical system that forms an image of the aforementioned observation light as an observation image, An imaging unit that captures the formed observation image using an image sensor, A drive unit that can move the position of the imaging optical system relative to the sample, Control unit, including, The control unit is The drive unit is controlled to drive the imaging optical system so that it is positioned at a first position relative to the sample and at a second position relative to the sample. Furthermore, the imaging unit is controlled so that the imaging optical system is positioned at the first position to capture a first observation image, and then positioned at the second position to capture a second observation image, thereby correcting the first observation image with the second observation image to clarify the image of the cells contained in the sample. Optical observation device. (Note 23) Includes a filter unit that changes the wavelength range in the transmitted observation light, The filter unit is positioned between the optical unit and the imaging unit in the optical observation apparatus as described in Appendix 22. (Note 24) The aforementioned filter unit is an optical observation apparatus as described in Appendix 23, including a bandpass filter. (Note 25) The aforementioned filter unit transmits wavelengths in the 400-600 nm range, as described in Appendix 23 or 24 of the optical observation apparatus. (Note 26) The optical unit is an optical observation device according to any one of the appendices 22 to 25, including a dichroic mirror. (Note 27) The optical observation apparatus according to any one of the appendices 22 to 26, wherein the optical unit, the imaging optical system, and the imaging unit are arranged in this order in the optical axis direction of the imaging optical system. (Note 28) The imaging optical system includes an objective lens, The objective lens is positioned between the optical unit and the imaging unit. An optical observation apparatus according to any one of appendices 22 to 27, wherein the observation light emitted from the sample by the ultraviolet light passes through the optical unit and is imaged as an observation image on the image sensor by the objective lens. (Note 29) The imaging optical system includes a telecentric lens, The telecentric lens is positioned between the optical unit and the imaging unit. An optical observation apparatus according to any one of appendices 22 to 27, wherein the observation light emitted from the sample by the ultraviolet light is imaged onto the image sensor by the telecentric lens. (Note 30) The optical observation apparatus according to any one of the appendices 22 to 26, wherein the imaging optical system, the optical unit, and the imaging unit are arranged in this order in the optical axis direction of the imaging optical system. (Note 31) The imaging optical system includes an objective lens, The objective lens is positioned between the optical unit and the sample. The reflected ultraviolet light is irradiated onto the sample through the objective lens. An optical observation apparatus according to any one of appendices 22 to 26, wherein the observation light emitted from the sample by the ultraviolet light is imaged as an observation image on the image sensor by the objective lens. (Note 32) The system includes an illumination optical system that guides ultraviolet light projected from the light source onto the sample, The illumination optical system is an optical observation device according to any one of the appendices 22 to 31, disposed between the light source and the optical unit. (Note 33) The illumination optical system is Collimating optical system that converts ultraviolet light into parallel light, A shaping optical system that changes the light intensity distribution of the ultraviolet light, and / or A focusing optical system for focusing the ultraviolet light onto the sample, An optical observation apparatus as described in Appendix 32, including the one described above. (Note 34) The optical observation apparatus described in Appendix 33, wherein the illumination optical system includes the collimating optical system and the focusing optical system. (Note 35) The optical observation apparatus described in Appendix 33, wherein the illumination optical system includes the collimating optical system and the shaping optical system. (Note 36) A focusing optical system for focusing the ultraviolet light onto the sample, The light-gathering optical system is provided with a drive unit that can move along the optical path of the ultraviolet light, Control unit, including, The control unit is The light-gathering optical system drive unit is driven and controlled so that the light-gathering optical system is positioned at a first position on the optical path and a second position on the optical path, where the positional relationship of the ultraviolet light on the optical path is different. Furthermore, the optical observation apparatus according to any one of the appendices 22 to 35, wherein the focusing optical system drive unit is controlled to capture a first observation image formed when the focusing optical system is in the first position, and to capture a second observation image formed when the focusing optical system is in the second position, thereby correcting the first observation image with the second observation image to clarify the image of cells contained in the sample. (Note 37) The first position is a position that sets the focal position of the image sensor of the imaging unit to the surface of the sample. The optical observation apparatus according to any one of the appendices 22 to 36, wherein the second position is a position in which the focal position of the image sensor of the imaging unit is set to a position moved in the direction toward the inside of the sample from the surface layer of the sample. (Note 38) The control unit is An optical observation device according to any one of the appendices 22 to 37, which corrects the background image of the cell in the first observation image relating to the first position by taking the difference between the first observation image relating to the first position and the second observation image relating to the second position. (Note 39) An optical observation apparatus according to any one of the appendices 22 to 38, wherein ultraviolet light reflected by the optical unit is incident on the sample from a direction substantially perpendicular to the surface of the sample. (Note 40) The optical observation apparatus according to any one of the appendices 22 to 39, wherein the wavelength range of the ultraviolet light is 200 to 400 nm. (Note 41) The optical observation apparatus according to any one of the appendices 22 to 40, wherein the wavelength range of the observation light is 400 to 600 nm. (Note 42) The optical observation apparatus according to any one of the appendices 22 to 41, wherein the cells are stained with a fluorescent dye that can be excited by ultraviolet light. <Third Optical Observation Device> (Note 43) A placement unit capable of arranging samples containing cells, A light source capable of projecting ultraviolet light, A focusing optical system for focusing the ultraviolet light onto the sample, An optical unit that reflects the ultraviolet light onto the sample, irradiates the sample with ultraviolet light, and transmits the observation light emitted from the sample by the ultraviolet light, An imaging optical system that forms an image of the aforementioned observation light as an observation image, An imaging unit that captures the formed observation image using an image sensor, The light-gathering optical system is provided with a light-gathering optical system drive unit that can move along the ultraviolet light path, Control unit, including, The control unit is The light-gathering optical system drive unit is driven and controlled so that the light-gathering optical system is positioned at a first position on the optical path and a second position on the optical path, where the positional relationship of the ultraviolet light on the optical path is different. Furthermore, the focusing optical system drive unit is controlled to capture a first observation image formed when the focusing optical system is in the first position, and to capture a second observation image formed when the focusing optical system is in the second position, thereby correcting the first observation image with the second observation image to clarify the image of the cells contained in the sample. Optical observation device. (Note 44) Includes a filter unit that changes the wavelength range in the transmitted observation light, The filter unit is positioned between the optical unit and the imaging unit in the optical observation apparatus as described in Appendix 42. (Note 45) The aforementioned filter unit includes a bandpass filter, as described in Appendix 43, for the optical observation apparatus. (Note 46) The aforementioned filter unit transmits wavelengths in the 400-600 nm range, as described in Appendix 43 or 44 of the optical observation apparatus. (Note 47) The optical unit is an optical observation device according to any one of the appendices 42 to 45, including a dichroic mirror. (Note 48) The optical observation apparatus according to any one of the appendices 42 to 46, wherein the optical unit, the imaging optical system, and the imaging unit are arranged in this order in the optical axis direction of the imaging optical system. (Note 49) The imaging optical system includes an objective lens, The objective lens is positioned between the optical unit and the imaging unit. An optical observation apparatus according to any one of appendices 42 to 47, wherein the observation light emitted from the sample by the ultraviolet light passes through the optical unit and is imaged as an observation image on the image sensor by the objective lens. (Note 50) The imaging optical system includes a telecentric lens, The telecentric lens is positioned between the optical unit and the imaging unit. An optical observation apparatus according to any one of appendices 42 to 47, wherein the observation light emitted from the sample by the ultraviolet light is imaged onto the image sensor by the telecentric lens. (Note 51) The optical observation apparatus according to any one of the appendices 42 to 46, wherein the imaging optical system, the optical unit, and the imaging unit are arranged in this order in the optical axis direction of the imaging optical system. (Note 52) The imaging optical system includes an objective lens, The objective lens is positioned between the optical unit and the sample. The reflected ultraviolet light is irradiated onto the sample through the objective lens. An optical observation apparatus according to any one of appendices 42 to 46, wherein the observation light emitted from the sample by the ultraviolet light is imaged as an observation image on the image sensor by the objective lens. (Note 53) The system includes an illumination optical system that guides ultraviolet light projected from the light source onto the sample, The illumination optical system includes the focusing optical system, The illumination optical system is an optical observation device according to any one of the appendices 42 to 51, disposed between the light source and the optical unit. (Note 54) The illumination optical system is A collimating optical system that converts the aforementioned ultraviolet light into parallel light, and / or, A reshaping optical system that changes the light intensity distribution of ultraviolet light, An optical observation apparatus as described in Appendix 52, including the one described therein. (Note 55) The optical observation apparatus described in Appendix 53, wherein the illumination optical system includes the collimating optical system and the focusing optical system. (Note 56) The optical observation apparatus described in Appendix 53, wherein the illumination optical system includes the collimating optical system and the shaping optical system. (Note 57) An optical observation apparatus according to any one of appendices 43 to 56, wherein ultraviolet light reflected by the optical unit is incident on the sample from a direction substantially perpendicular to the surface of the sample. <Fourth Optical Observation Device> (Note 58) A placement unit capable of arranging samples containing cells, A light source capable of projecting ultraviolet light, An illumination optical system that guides ultraviolet light projected from the light source onto the sample, An imaging optical system that forms an image of the aforementioned observation light as an observation image, An imaging unit that captures the formed observation image using an image sensor, Includes, The illumination optical system is Includes a collimating optical system that converts the ultraviolet light into parallel light, The parallel light is shone onto the surface of the sample from an oblique direction. Optical observation device. (Note 59) The illumination optical system includes a reflective mirror that reflects the ultraviolet light back onto the sample. The optical observation apparatus described in Appendix 58, wherein the reflective mirror irradiates the sample with the ultraviolet light parallel to the surface of the sample from an oblique direction. (Note 60) The illumination optical system is A shaping optical system that changes the light intensity distribution of the ultraviolet light, and / or A focusing optical system for focusing the ultraviolet light onto the sample, An optical observation apparatus as described in Appendix 58 or 59, including the optical observation apparatus described in Appendix 58 or 59. (Note 61) A focusing optical system for focusing the ultraviolet light onto the sample, The light-gathering optical system is provided with a drive unit that can move along the optical path of the ultraviolet light, Control unit, including, The control unit is The light-gathering optical system drive unit is driven and controlled so that the light-gathering optical system is positioned at a first position on the optical path and a second position on the optical path, where the positional relationship of the ultraviolet light on the optical path is different. Furthermore, the optical observation apparatus according to any one of the appendices 58 to 60, wherein the focusing optical system drive unit is controlled to capture a first observation image formed when the focusing optical system is in the first position, and to capture a second observation image formed when the focusing optical system is in the second position, thereby correcting the first observation image with the second observation image to clarify the image of cells contained in the sample. (Note 62) Includes a filter unit that changes the wavelength range in the transmitted observation light, The filter unit is disposed between the imaging optical system and the imaging unit, and is part of the optical observation apparatus as described in any of appendices 58 to 61. (Note 63) The aforementioned filter unit includes a bandpass filter, as described in Appendix 62, for the optical observation apparatus. (Note 64) The aforementioned filter unit transmits wavelengths in the 400-600 nm range, and is an optical observation apparatus as described in any of appendices 58 to 63. (Note 65) The optical observation apparatus according to any one of the appendices 58 to 64, wherein the imaging optical system and the imaging unit are arranged in this order in the optical axis direction of the imaging optical system. (Note 66) The imaging optical system includes an objective lens, The objective lens is positioned between the arrangement unit and the imaging unit. An optical observation apparatus according to any one of appendices 58 to 65, wherein the observation light emitted from the sample by the ultraviolet light is imaged as an observation image on the image sensor by the objective lens. (Note 67) The imaging optical system includes a telecentric lens, The telecentric lens is positioned between the arrangement unit and the imaging unit. An optical observation apparatus according to any one of appendices 58 to 65, wherein the observation light emitted from the sample by the ultraviolet light is imaged onto the image sensor by the telecentric lens. (Note 68) The optical observation apparatus according to any one of the appendices 58 to 64, wherein the imaging optical system and the imaging unit are arranged in this order in the optical axis direction of the imaging optical system. (Note 69) A drive unit that can move the position of the imaging optical system relative to the sample, Control unit, including, The control unit is The drive unit is controlled to drive the imaging optical system so that it is positioned at a first position relative to the surface of the sample and at a second position relative to the surface of the sample. Furthermore, the imaging unit is controlled so that the imaging optical system is positioned at the first position to capture a first observation image, and the imaging optical system is positioned at the second position to capture a second observation image, thereby correcting the first observation image with the second observation image and clarifying the image of cells contained in the surface layer of the sample. An optical observation device as described in any of the appendices 58 to 68. (Note 70) The first position is a position that sets the focal position of the image sensor of the imaging unit to the surface layer of the sample. The optical observation apparatus as described in Appendix 69, wherein the second position is a position in which the focal position of the image sensor of the imaging unit is set to a position moved in the direction toward the inside of the sample from the surface layer of the sample. (Note 71) The control unit is An optical observation device according to Appendix 69 or 70, which corrects the background image of the cell in the first observation image relating to the first position by taking the difference between the first observation image relating to the first position and the second observation image relating to the second position. (Note 72) The optical observation apparatus according to any one of the appendices 58 to 71, wherein the wavelength range of the ultraviolet light is 200 to 400 nm. (Note 73) The optical observation apparatus according to any one of the appendices 58 to 72, wherein the wavelength range of the observation light is 400 to 600 nm. (Note 74) The optical observation apparatus according to any one of the appendices 58 to 73, wherein the cells are stained with a fluorescent dye that can be excited by ultraviolet light. <First imaging method> (Note 75) An imaging method used in an optical observation apparatus including an arrangement unit, a light source, an optical unit, an imaging optical system, and an imaging unit, For a sample containing cells arranged in the aforementioned arrangement unit, the ultraviolet light projected from the light source is reflected by the optical unit and irradiated onto the sample with the ultraviolet light, and the irradiation step involves the irradiation of the sample from a direction substantially perpendicular to the surface of the sample. An imaging step in which observation light emitted from the sample by the ultraviolet light is transmitted through the optical unit and formed as an observation image by the imaging optical system, The imaging step involves capturing the observation image using the image sensor of the imaging unit, An imaging method, including the imaging method. (Note 76) Includes a filter unit, The imaging method according to Appendix 75, wherein in the imaging step, the observation light transmitted through the optical unit passes through the filter unit, thereby changing the wavelength range of the observation light, and the changed observation light is imaged as the observation image. (Note 77) The imaging method according to Appendix 76, wherein the filter unit includes a bandpass filter. (Note 78) The imaging method according to Appendix 76 or 77, wherein the filter unit transmits wavelengths in the 400-600 nm range. (Note 79) The imaging method according to any one of appendices 75 to 78, wherein the optical unit includes a dichroic mirror. (Note 80) The imaging method according to any one of the appendices 75 to 79, wherein the optical unit, the imaging optical system, and the imaging unit are arranged in this order in the optical axis direction of the imaging optical system. (Note 81) The imaging optical system includes an objective lens, The objective lens is positioned between the optical unit and the imaging unit. The imaging method according to any one of appendices 75 to 80, wherein in the imaging step, observation light emitted from the sample by ultraviolet light passes through the optical unit and is imaged as an observation image on the image sensor by the objective lens. (Note 82) The imaging optical system includes a telecentric lens, The imaging method according to any one of appendices 75 to 81, wherein in the imaging step, the observation light emitted from the sample by the ultraviolet light is imaged onto the image sensor by the telecentric lens. (Note 83) The imaging method according to any one of appendices 75 to 79, wherein the imaging optical system, the optical unit, and the imaging unit are arranged in this order in the optical axis direction of the imaging optical system. (Note 84) The imaging optical system includes an objective lens, In the irradiation step, the ultraviolet light reflected by the optical unit passes through the objective lens and irradiates the sample. The imaging method according to any one of appendices 75 to 80, wherein the imaging step involves forming an image as an observation image using the objective lens. (Note 85) The system includes an illumination optical system that guides ultraviolet light projected from the light source onto the sample, The illumination optical system is positioned between the light source and the optical unit. The imaging method according to any one of appendices 75 to 84, wherein in the irradiation step, ultraviolet light projected from the light source is reflected by the optical unit after passing through the illumination optical system. (Note 86) The illumination optical system is Collimating optical system that converts ultraviolet light into parallel light, A shaping optical system that changes the light intensity distribution of the ultraviolet light, and / or A focusing optical system for focusing the ultraviolet light onto the sample, Includes, The imaging method according to Appendix 85, wherein in the irradiation step, ultraviolet light projected from the light source is reflected by the optical unit after passing through the illumination optical system. (Note 87) The imaging method according to Appendix 86, wherein the illumination optical system includes the collimating optical system and the focusing optical system. (Note 88) The imaging method according to Appendix 86, wherein the illumination optical system includes the collimating optical system and the shaping optical system. (Note 89) A focusing optical system for focusing the ultraviolet light onto the sample, The light-gathering optical system is provided with a light-gathering optical system drive unit that can move along the ultraviolet light path, Includes, In the irradiation step, ultraviolet light projected from the light source is reflected by the optical unit after passing through the focusing optical system. In the imaging step, the focusing optical system drive unit is driven to control the position of the focusing optical system, and the observation images are captured as the first observation image and the second observation image, respectively, when the focusing optical system is positioned at a first position on the optical path and a second position on the optical path, where the positional relationship of the ultraviolet light on the optical path is different. The process includes a correction step to clarify the image of cells contained in the sample by correcting the first observation image at a first position on the optical path based on the second observation image at a second position on the optical path. The imaging method described in any of the appendices 75 to 88. (Note 90) Includes a drive unit that can move the position of the imaging optical system relative to the sample, In the imaging step, the drive unit is driven to control the position of the imaging optical system, and the observation image is captured as the first observation image and the second observation image, respectively, with the imaging optical system positioned at a first position and a second position, which have different positional relationships with respect to the sample. The imaging method according to any one of appendices 75 to 89, comprising a correction step of correcting the first observation image relating to the first position based on the second observation image relating to the second position to clarify the image of cells contained in the sample. (Note 91) In the imaging process, The first position is a position that sets the focal position of the image sensor of the imaging unit to the surface of the sample. The imaging method according to Appendix 90, wherein the second position is a position in which the focal position of the image sensor of the imaging unit is set to a position moved in the direction toward the inside of the sample from the surface layer of the sample. (Note 92) The imaging method according to Appendix 90 or 91, wherein the correction step corrects the background image of the cell in the first observation image at the first position by taking the difference between the first observation image at the first position and the second observation image at the second position. (Note 93) The imaging method according to any one of the appendices 75 to 92, wherein the wavelength range of the ultraviolet light is 200 to 400 nm. (Note 94) The imaging method described in any of appendices 75 to 93, wherein the wavelength range of the observation light is 400 to 600 nm. (Note 95) The imaging method according to any one of the appendices 75 to 94, wherein the cells are cells stained with a fluorescent dye that can be excited by ultraviolet light. <Second imaging method> (Note 96) An imaging method used in an optical observation apparatus including an arrangement unit, a light source, an optical unit, an imaging optical system, an imaging unit, and a drive unit, For a sample containing cells arranged in the aforementioned arrangement unit, the irradiation step involves reflecting ultraviolet light projected from the light source using the optical unit and irradiating the sample with the ultraviolet light; An imaging step in which observation light emitted from the sample by the ultraviolet light is transmitted through the optical unit and formed as an observation image by the imaging optical system, The imaging process involves driving the drive unit to control the position of the imaging optical system, and while the imaging optical system is positioned at a first position and a second position with different positional relationships to the sample, the imaging sensor of the imaging unit captures the observation image as a first observation image and a second observation image, respectively. An imaging method comprising a correction step of correcting the first observation image at the first position with the second observation image at the second position to clarify the image of cells contained in the sample. (Note 97) Includes a filter unit, The imaging method according to Appendix 96, wherein in the imaging step, the observation light transmitted through the optical unit passes through the filter unit, thereby changing the wavelength range of the observation light, and the changed observation light is imaged as the observation image. (Note 98) The imaging method described in Appendix 97, wherein the filter unit includes a bandpass filter. (Note 99) The imaging method according to Appendix 97 or 98, wherein the filter unit transmits wavelengths in the 400-600 nm range. (Note 100) The imaging method according to any one of appendices 96 to 99, wherein the optical unit includes a dichroic mirror. (Note 101) The imaging method according to any one of appendices 96 to 100, wherein the optical unit, the imaging optical system, and the imaging unit are arranged in this order in the optical axis direction of the imaging optical system. (Note 102) The imaging optical system includes an objective lens, The objective lens is positioned between the optical unit and the imaging unit. The imaging method according to any one of the appendices 96 to 101, wherein in the imaging step, observation light emitted from the sample by ultraviolet light passes through the optical unit and is imaged as an observation image on the image sensor by the objective lens. (Note 103) The imaging optical system includes a telecentric lens, The imaging method according to any one of appendices 96 to 101, wherein in the imaging step, the observation light emitted from the sample by the ultraviolet light is imaged onto the image sensor by the telecentric lens. (Note 104) The imaging method according to any one of appendices 96 to 100, wherein the imaging optical system, the optical unit, and the imaging unit are arranged in this order in the optical axis direction of the imaging optical system. (Note 105) The imaging optical system includes an objective lens, In the irradiation step, the ultraviolet light reflected by the optical unit passes through the objective lens and irradiates the sample. The imaging method according to any one of appendices 96 to 100, wherein the imaging step involves forming an image as an observation image using the objective lens. (Note 106) The system includes an illumination optical system that guides ultraviolet light projected from the light source onto the sample, The illumination optical system is positioned between the light source and the optical unit. The imaging method according to any one of appendices 96 to 105, wherein in the irradiation step, ultraviolet light projected from the light source is reflected by the optical unit after passing through the illumination optical system. (Note 107) The illumination optical system is Collimating optical system that converts ultraviolet light into parallel light, A shaping optical system that changes the light intensity distribution of the ultraviolet light, and / or A focusing optical system for focusing the ultraviolet light onto the sample, Includes, The imaging method according to Appendix 106, wherein in the irradiation step, ultraviolet light projected from the light source is reflected by the optical unit after passing through the illumination optical system. (Note 108) The imaging method according to Appendix 107, wherein the illumination optical system includes the collimating optical system and the focusing optical system. (Note 109) The imaging method according to Appendix 107, wherein the illumination optical system includes the collimating optical system and the shaping optical system. (Note 110) A focusing optical system for focusing the ultraviolet light onto the sample, The light-gathering optical system is provided with a light-gathering optical system drive unit that can move along the ultraviolet light path, Includes, In the irradiation step, ultraviolet light projected from the light source is reflected by the optical unit after passing through the focusing optical system. In the imaging step, the focusing optical system drive unit is driven to control the position of the focusing optical system, and the observation images are captured as the first observation image and the second observation image, respectively, when the focusing optical system is positioned at a first position on the optical path and a second position on the optical path, where the positional relationship of the ultraviolet light on the optical path is different. The process includes a correction step to clarify the image of cells contained in the sample by correcting the first observation image at a first position on the optical path based on the second observation image at a second position on the optical path. The imaging method described in any of the appendices 96 to 109. (Note 111) In the imaging process, The first position is a position that sets the focal position of the image sensor of the imaging unit to the surface of the sample. The imaging method according to any one of appendices 96 to 110, wherein the second position is a position in which the focal position of the image sensor of the imaging unit is set to a position moved in the direction toward the inside of the sample from the surface layer of the sample. (Note 112) The imaging method according to any one of appendices 96 to 111, wherein the correction step corrects the background image of the cell in the first observation image at the first position by taking the difference between the first observation image at the first position and the second observation image at the second position. (Note 113) The imaging method according to any one of appendices 96 to 112, wherein the wavelength range of the ultraviolet light is 200 to 400 nm. (Note 114) The imaging method according to any one of the appendices 96 to 113, wherein the wavelength range of the observation light is 400 to 600 nm. (Note 115) The imaging method according to any one of appendices 96 to 114, wherein the cells are cells stained with a fluorescent dye that can be excited by ultraviolet light. <Third imaging method> (Note 116) An imaging method used in an optical observation apparatus including an arrangement unit, a light source, a light-gathering optical system, an optical unit, an imaging optical system, an imaging unit, and a light-gathering optical system drive unit, For a sample containing cells arranged in the aforementioned arrangement unit, the irradiation step involves projecting ultraviolet light from the light source, passing it through the focusing optical system, and then reflecting it off the optical unit to irradiate the sample with the ultraviolet light. An imaging step in which observation light emitted from the sample by the ultraviolet light is transmitted through the optical unit and formed as an observation image by the imaging optical system, The imaging process involves driving the drive unit to control the position of the focusing optical system, and when the focusing optical system is positioned at a first position on the optical path and a second position on the optical path, where the positional relationship of the ultraviolet light on the optical path is different, the imaging sensor of the imaging unit captures the observed image as a first observed image and a second observed image, respectively. An imaging method comprising a correction step of correcting the first observation image at a first position on the optical path with the second observation image at a second position on the optical path to clarify the image of cells contained in the sample. (Note 117) Includes a filter unit, The imaging method according to Appendix 116, wherein in the imaging step, the observation light transmitted through the optical unit passes through the filter unit, thereby changing the wavelength range of the observation light, and the changed observation light is imaged as the observation image. (Note 118) The imaging method described in Appendix 117, wherein the filter unit includes a bandpass filter. (Note 119) The imaging method according to Appendix 117 or 118, wherein the filter unit transmits wavelengths in the 400-600 nm range. (Note 120) The imaging method according to any one of appendices 116 to 119, wherein the optical unit includes a dichroic mirror. (Note 121) The imaging method according to any one of appendices 116 to 120, wherein the optical unit, the imaging optical system, and the imaging unit are arranged in this order in the optical axis direction of the imaging optical system. (Note 122) The imaging optical system includes an objective lens, The objective lens is positioned between the optical unit and the imaging unit. The imaging method according to any one of appendices 116 to 121, wherein in the imaging step, observation light emitted from the sample by ultraviolet light passes through the optical unit and is imaged as an observation image on the image sensor by the objective lens. (Note 123) The imaging optical system includes a telecentric lens, In the imaging step, the observation light emitted from the sample by the ultraviolet light is imaged on the imaging element by the telecentric lens, and the imaging method according to any one of Appendices 116 to 122. (Appendix 124) The imaging optical system, the optical unit, and the imaging unit are arranged in this order in the optical axis direction of the imaging optical system, and the imaging method according to any one of Appendices 116 to 120. (Appendix 125) The imaging optical system includes an objective lens, In the irradiation step, the ultraviolet light reflected by the optical unit passes through the objective lens and irradiates the sample. In the imaging step, an image is formed as an observation image by the objective lens, and the imaging method according to any one of Appendices 116 to 120. (Appendix 126) It includes an illumination optical system for guiding the ultraviolet light projected from the light source to the sample. The illumination optical system includes the condenser optical system. The illumination optical system is arranged between the light source and the optical unit. In the irradiation step, the ultraviolet light projected from the light source is reflected by the optical unit after passing through the illumination optical system, and the imaging method according to any one of Appendices 116 to 125. (Appendix 127) It includes a drive unit capable of moving the position of the imaging optical system with respect to the sample. In the imaging step, the drive unit is driven to control the position of the imaging optical system, and the imaging optical system is in a state where it is located at a first position and a second position with different positional relationships with respect to the sample, and the observation image is respectively imaged as a first observation image and a second observation image. The imaging method according to any one of Appendices 116 to 126 includes a correction step of correcting the first observation image corresponding to the first position based on the second observation image corresponding to the second position to clarify the image of the cells contained in the sample. (Appendix 128) In the imaging step, The first position is a position where the focal position of the imaging element of the imaging unit is set on the surface of the sample. The second position is a position where the focal position of the imaging element of the imaging unit is set at a position moved in the direction inside the sample from the surface of the sample, according to the imaging method described in Supplementary Note 127. (Supplementary Note 129) In the correction step, by taking the difference between the first observation image related to the first position and the second observation image related to the second position, the background image of the cells in the first observation image related to the first position is corrected, according to the imaging method described in Supplementary Note 127 or 128. (Supplementary Note 130) The wavelength range of the ultraviolet light is 200 to 400 nm, according to the imaging method described in any one of Supplementary Notes 116 to 129. (Supplementary Note 131) The wavelength range of the observation light is 400 to 600 nm, according to the imaging method described in any one of Supplementary Notes 116 to 130. (Supplementary Note 132) The cells are cells stained with a fluorescent dye that can be excited by the ultraviolet light, according to the imaging method described in any one of Supplementary Notes 116 to 131. <Fourth imaging method> (Supplementary Note 133) An imaging method used in an optical observation apparatus including an arrangement unit, a light source, an illumination optical system, an imaging optical system, and an imaging unit, The illumination optical system includes a collimating optical system that converts the ultraviolet light into parallel light, For a sample including cells arranged in the arrangement unit, an irradiation step of irradiating the sample with the ultraviolet light projected from the light source, after converting the ultraviolet light into parallel light by the collimating optical system, obliquely from the sample surface; An imaging step of imaging the observation light emitted from the sample by the ultraviolet light as an observation image by the imaging optical system; An imaging step of imaging the observation image by the imaging element of the imaging unit; including: (Supplementary Note 134) The illumination optical system includes a reflective mirror that reflects the ultraviolet light back onto the sample. The imaging method according to Appendix 133, wherein in the irradiation step, the reflective mirror irradiates the sample with parallel ultraviolet light from an oblique direction relative to the surface of the sample. (Note 135) The illumination optical system is A shaping optical system that changes the light intensity distribution of the ultraviolet light, and / or A focusing optical system for focusing the ultraviolet light onto the sample, Includes, The imaging method according to Appendix 134, wherein in the irradiation step, ultraviolet light projected from the light source is irradiated onto the sample after passing through the illumination optical system. (Note 136) A focusing optical system for focusing the ultraviolet light onto the sample, The light-gathering optical system is provided with a light-gathering optical system drive unit that can move along the ultraviolet light path, Includes, In the irradiation step, ultraviolet light projected from the light source passes through the focusing optical system and is then irradiated onto the surface of the sample from an oblique direction. In the imaging step, the focusing optical system drive unit is driven to control the position of the focusing optical system, and the observation images are captured as the first observation image and the second observation image, respectively, when the focusing optical system is positioned at a first position on the optical path and a second position on the optical path, where the positional relationship of the ultraviolet light on the optical path is different. The process includes a correction step to clarify the image of cells contained in the sample by correcting the first observation image at a first position on the optical path based on the second observation image at a second position on the optical path. The imaging method described in any of the appendices 133 to 135. (Note 137) Includes a filter unit, The imaging method according to any one of appendices 133 to 136, wherein in the imaging step, the observation light passes through the filter unit to change the wavelength range of the observation light, and the changed observation light is imaged as the observation image. (Note 138) The imaging method described in Appendix 137, wherein the filter unit includes a bandpass filter. (Note 139) The imaging method according to Appendix 137 or 138, wherein the filter unit transmits wavelengths in the 400-600 nm range. (Note 140) The imaging method according to any one of appendices 133 to 139, wherein the imaging optical system and the imaging unit are arranged in this order in the optical axis direction of the imaging optical system. (Note 141) The imaging optical system includes an objective lens, The objective lens is positioned between the arrangement unit and the imaging unit. The imaging method according to any one of appendices 133 to 140, wherein in the imaging step, observation light emitted from the sample by ultraviolet light is imaged as an observation image on the image sensor by the objective lens. (Note 142) The imaging optical system includes a telecentric lens, The imaging method according to any one of appendices 133 to 140, wherein in the imaging step, the observation light emitted from the sample by the ultraviolet light is imaged onto the image sensor by the telecentric lens. (Note 143) The imaging method according to any one of appendices 133 to 139, wherein the imaging optical system and the imaging unit are arranged in this order in the optical axis direction of the imaging optical system. (Note 144) Includes a drive unit that can move the position of the imaging optical system relative to the sample, In the imaging step, the drive unit is driven to control the position of the imaging optical system, and the observation image is captured as the first observation image and the second observation image, respectively, with the imaging optical system positioned at a first position and a second position, which have different positional relationships with respect to the sample. An imaging method according to any one of Appendices 133 to 143, including a correction step of correcting the first observation image related to the first position based on the second observation image related to the second position to clarify an image of cells contained in the sample. (Appendix 145) In the imaging step, The first position is a position where the focal position of the imaging element of the imaging unit is set on the surface layer surface of the sample, The second position is a position where the focal position of the imaging element of the imaging unit is set at a position moved in the direction inside the sample from the surface layer surface of the sample. The imaging method according to Appendix 144. (Appendix 146) In the correction step, by taking the difference between the first observation image related to the first position and the second observation image related to the second position, the background image of the cells in the first observation image related to the first position is corrected. The imaging method according to Appendix 144 or 145. (Appendix 147) The wavelength range of the ultraviolet light is 200 to 400 nm. The imaging method according to any one of Appendices 133 to 146. (Appendix 148) The wavelength range of the observation light is 400 to 600 nm. The imaging method according to any one of Appendices 133 to 147. (Appendix 149) The cells are cells stained with a fluorescent dye that can be excited by the ultraviolet light. The imaging method according to any one of Appendices 133 to 148.

Explanation of Reference Signs

[0115] 11 Stage 12 Light source 13 Dichroic mirror 14 Objective lens 15 Camera 16 Band-pass filter 17 Z-axis stage 18 Control unit 181 Drive instruction unit 182 Imaging instruction unit 183 Correction Unit 18a CPU 18b Main Memory 18c Auxiliary storage device 18d Program 18e video codec 18f I / O Interface 18g controller 18h bus 18i display device 18j Input Device 19. Collimating lenses 20 Focusing lenses 21 Drive unit 22 Homogenizer 23a First Mirror 23b The second mirror Optical observation devices for 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 meter readings.

Claims

1. A placement unit capable of arranging samples containing cells, A light source capable of projecting ultraviolet light, An optical unit that reflects ultraviolet light incident from the light source onto the sample, irradiating the sample with ultraviolet light, and transmits observation light emitted from the sample by the ultraviolet light, An imaging optical system that forms an image of the aforementioned observation light as an observation image, It includes an imaging unit that captures the formed observation image using an image sensor, An optical observation device in which ultraviolet light reflected by the optical unit is incident on the sample from a direction substantially perpendicular to the surface of the sample.

2. Includes a filter unit that changes the wavelength range in the transmitted observation light, The optical observation apparatus according to claim 1, wherein the filter unit is disposed between the optical unit and the imaging unit.

3. The imaging optical system includes an objective lens, The objective lens is positioned between the optical unit and the sample. The reflected ultraviolet light is irradiated onto the sample through the objective lens. The optical observation apparatus according to claim 1 or 2, wherein the observation light emitted from the sample by the ultraviolet light is imaged as an observation image on the image sensor by the objective lens.

4. The imaging optical system includes a telecentric lens, The telecentric lens is positioned between the optical unit and the imaging unit. The optical observation apparatus according to claim 1 or 2, wherein the observation light emitted from the sample by the ultraviolet light is imaged onto the image sensor by the telecentric lens.

5. The system includes an illumination optical system that guides ultraviolet light projected from the light source onto the sample, The optical observation apparatus according to claim 1 or 2, wherein the illumination optical system is disposed between the light source and the optical unit.

6. The illumination optical system is Collimating optical system that converts ultraviolet light into parallel light, A shaping optical system that changes the light intensity distribution of the ultraviolet light, and / or, A focusing optical system for focusing the ultraviolet light onto the sample, The optical observation apparatus according to claim 5, including the following:

7. The optical observation apparatus according to claim 6, wherein the illumination optical system includes the collimating optical system and the focusing optical system.

8. The optical observation apparatus according to claim 6, wherein the illumination optical system includes the collimating optical system and the shaping optical system.

9. A drive unit that can move the position of the imaging optical system relative to the sample, Control unit, including, The control unit is The drive unit is controlled to drive the imaging optical system so that it is positioned at a first position relative to the surface of the sample and at a second position relative to the surface of the sample. Furthermore, the imaging unit is controlled so that the imaging optical system is positioned at the first position to capture a first observation image, and the imaging optical system is positioned at the second position to capture a second observation image, thereby correcting the first observation image with the second observation image and clarifying the image of cells contained in the surface layer of the sample. The optical observation apparatus according to claim 1 or 2.

10. The first position is a position that sets the focal position of the image sensor of the imaging unit to the surface layer of the sample. The optical observation apparatus according to claim 9, wherein the second position is a position in which the focal position of the image sensor of the imaging unit is set to a position moved in the direction toward the inside of the sample from the surface layer of the sample.

11. The control unit is The optical observation apparatus according to claim 9, wherein the background image of the cell in the first observation image at the first position is corrected by taking the difference between the first observation image at the first position and the second observation image at the second position.

12. The optical observation apparatus according to claim 1 or 2, wherein the wavelength range of the ultraviolet light is 200 to 400 nm.

13. A placement unit capable of arranging samples containing cells, A light source capable of projecting ultraviolet light, An optical unit that reflects ultraviolet light incident from the light source onto the sample, irradiating the sample with ultraviolet light, and transmits observation light emitted from the sample by the ultraviolet light, An imaging optical system that forms an image of the aforementioned observation light as an observation image, An imaging unit that captures the formed observation image using an image sensor, A drive unit that can move the position of the imaging optical system relative to the sample, Control unit, including, The control unit is The drive unit is controlled to drive the imaging optical system so that it is positioned at a first position relative to the sample and at a second position relative to the sample. Furthermore, the imaging unit is controlled so that the imaging optical system is positioned at the first position to capture a first observation image, and the imaging optical system is positioned at the second position to capture a second observation image, thereby correcting the first observation image with the second observation image to clarify the image of the cells contained in the sample. Optical observation device.

14. An imaging method used in an optical observation apparatus including an arrangement unit, a light source, an optical unit, an imaging optical system, and an imaging unit, For a sample containing cells arranged in the aforementioned arrangement unit, the ultraviolet light projected from the light source is reflected by the optical unit and irradiated onto the sample with the ultraviolet light, and the irradiation step involves the irradiation of the sample from a direction substantially perpendicular to the surface of the sample. An imaging step in which observation light emitted from the sample by the ultraviolet light is transmitted through the optical unit and formed as an observation image by the imaging optical system, The imaging step involves capturing the observation image using the image sensor of the imaging unit, An imaging method, including the imaging method.

15. An imaging method used in an optical observation apparatus including an arrangement unit, a light source, an optical unit, an imaging optical system, an imaging unit, and a drive unit, For a sample containing cells arranged in the aforementioned arrangement unit, the irradiation step involves reflecting ultraviolet light projected from the light source using the optical unit and irradiating the sample with the ultraviolet light; An imaging step in which observation light emitted from the sample by the ultraviolet light is transmitted through the optical unit and formed as an observation image by the imaging optical system, The imaging process involves driving the drive unit to control the position of the imaging optical system, and while the imaging optical system is positioned at a first position and a second position with different positional relationships to the sample, the imaging sensor of the imaging unit captures the observation image as a first observation image and a second observation image, respectively. An imaging method comprising a correction step of correcting the first observation image at the first position with the second observation image at the second position to clarify the image of cells contained in the sample.

Images