Sample observation device and sample observation method

Abstract

The sample observation device (1) comprises: an irradiation optical system (3) for irradiating a sample (S) with planar light (L2); a scanning unit (4) for scanning the sample (S) relative to an irradiation surface (R) of the planar light (L2); an imaging optical system (5) having an observation axis (P2) inclined relative to the irradiation surface (R) for imaging observation light (L3) generated on the sample (S) by irradiation with the planar light (L2); an image acquisition unit (6) for acquiring multiple partial image data corresponding to a portion of the light image of the observation light (L3) imaged by the imaging optical system (5); and an image generation unit (8) for generating observation image data of the sample (S) based on the multiple partial image data generated by the image acquisition unit (6).

Description

Technical Field

[0001] The present invention relates to a sample observation device and a sample observation method. Background Art

[0002] SPIM (Selective Plane Illumination Microscopy) is known as one method for observing the interior of samples with three-dimensional structures, such as cells. For example, the tomographic imaging observation device described in Patent Document 1 discloses the basic principle of SPIM: planar light is irradiated onto a sample, and fluorescence or scattered light generated within the sample is imaged on an imaging plane, thereby acquiring observation image data of the sample's interior.

[0003] Another example of a sample observation apparatus using planar light is the SPIM microscope described in Patent Document 2. In this conventional SPIM microscope, the surface on which the sample is placed is irradiated with planar light at a predetermined inclination angle, and the observation light from the sample is imaged by an observation optical system having an observation axis perpendicular to the irradiation surface of the planar light.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Laid-Open No. 62-180241

[0007] Patent Document 2: Japanese Patent Application Laid-Open No. 2014-202967 Summary of the Invention

[0008] Problems to be solved by the invention

[0009] In the sample observation device described in the above-mentioned Patent Document 2, by irradiating the entire surface of the focal plane of the observation optical system with planar light, an image of a cross-sectional plane in the direction of the observation axis can be obtained by a single imaging. Therefore, in order to obtain three-dimensional information of the sample, it is necessary to scan the sample along the direction of the observation axis and obtain images of multiple cross-sectional planes in the direction of the observation axis. In this conventional sample observation device, until images of all cross-sectional planes are obtained, it is necessary to repeatedly select the cross-sectional planes to be imaged (scanning and stopping of the sample) and obtain images. In addition, in the case where the area where the observation object exists is wider than the imaging area, in addition to the action of obtaining a cross-sectional image in the direction of the observation axis, it is also necessary to move the platform in a direction different from the observation axis direction to select the imaging field of view. Therefore, the time required until the observation image data is obtained becomes a technical problem.

[0010] An object of the embodiment is to provide a sample observation device and a sample observation method.

[0011] Technical means to solve problems

[0012] One aspect of the embodiment involves a sample observation device comprising: an irradiation optical system that irradiates planar light onto the sample; a scanning unit that scans the sample relative to the irradiation surface of the planar light; an imaging optical system that has an observation axis inclined relative to the irradiation surface and images the observation light generated on the sample by the irradiation of the planar light; an image acquisition unit that multiple times acquires partial image data corresponding to a portion of the light image of the observation light imaged by the imaging optical system; and an image generation unit that generates observation image data of the sample based on the multiple partial image data generated by the image acquisition unit.

[0013] In this specimen observation device, the specimen is scanned relative to the surface illuminated by the planar light. Furthermore, the observation axis of the imaging optical system is tilted relative to the surface illuminated by the planar light. Consequently, the image acquisition unit can sequentially acquire partial image data of a tomographic plane along the optical axis of the planar light, and the image generation unit can generate observed image data of the specimen based on these multiple partial image data. This specimen observation device eliminates the need for field of view selection, allowing simultaneous specimen scanning and image acquisition. This improves throughput until observation image data is acquired.

[0014] Alternatively, the sample can be held in a sample container having an input surface for planar light, and the optical axis of the planar light from the irradiation optical system can be arranged so as to be orthogonal to the input surface of the sample container. In this case, multiple samples can be scanned simultaneously using the sample container. Furthermore, by arranging the optical axis of the planar light orthogonal to the input surface of the sample container, positional correction of partial image data acquired by the image acquisition unit is eliminated, thereby simplifying the generation and processing of observation image data.

[0015] Alternatively, the scanning unit may scan the sample in a direction perpendicular to the optical axis of the planar light of the irradiation optical system. In this case, image processing such as position correction of the partial image data acquired by the image acquisition unit is unnecessary, and the generation process of the observation image data can be simplified.

[0016] Furthermore, the inclination angle of the observation axis of the imaging optical system relative to the irradiation surface of the planar light may be 10° to 80°. Within this range, the resolution of the observed image can be sufficiently ensured.

[0017] Furthermore, the inclination angle of the observation axis of the imaging optical system relative to the surface illuminated by the planar light can be set to 20° to 70°. Within this range, the resolution of the observed image can be further sufficiently ensured. Furthermore, the angular variation of the field of view relative to the observation axis can be suppressed, ensuring field of view stability.

[0018] Furthermore, the inclination angle of the observation axis of the imaging optical system relative to the irradiation surface of the planar light may be 30° to 65°. Within this range, the resolution of the observed image and the stability of the visual field can be more appropriately ensured.

[0019] Alternatively, the image acquisition unit may include a two-dimensional imaging device, and extract image data corresponding to a portion of the optical image of the observation light from the data output from the two-dimensional imaging device as partial image data.

[0020] Furthermore, the image acquisition unit may include a line sensor that captures a portion of the optical image of the observation light and outputs partial image data. With this configuration, partial image data can be acquired with high accuracy.

[0021] Alternatively, the image acquisition unit may include a slit for passing a portion of the observation light image and a photodetector for detecting the light image passing through the slit, and generate partial image data based on data output from the photodetector.

[0022] Furthermore, the image generating unit may generate observed image data of the sample on a plane perpendicular to the optical axis of the planar light based on the plurality of partial image data. This allows obtaining a cross-sectional image of the sample with reduced background influence as the observed image.

[0023] Furthermore, the sample observation device may further include an analysis unit that analyzes the observation image data and generates an analysis result. Since the analysis unit analyzes the observation image data generated by the image generation unit, the throughput of the analysis can also be improved.

[0024] In addition, one aspect of the embodiment involves a sample observation method comprising: an irradiation step of irradiating the sample with planar light; a scanning step of scanning the sample relative to the irradiation surface of the planar light; an imaging step of imaging the observation light generated on the sample by irradiation with the planar light using an imaging optical system having an observation axis inclined relative to the irradiation surface; an image acquisition step of acquiring partial image data corresponding to a portion of the light image of the observation light imaged by the imaging optical system multiple times; and an image generation step of generating observation image data of the sample based on multiple partial image data.

[0025] In this specimen observation method, the specimen is scanned relative to the surface illuminated by planar light. Furthermore, an imaging optical system is used whose observation axis is tilted relative to the surface illuminated by the planar light. Consequently, in the image acquisition step, partial image data of a tomographic plane along the optical axis of the planar light can be sequentially acquired, and in the image generation step, observed image data of the specimen can be generated based on the plurality of partial image data. This specimen observation method eliminates the need for field of view selection, enabling simultaneous scanning of the specimen and image acquisition. Consequently, throughput can be improved until observed image data is obtained.

[0026] Effects of the Invention

[0027] According to the sample observation device and the sample observation method, an improvement in throughput until observation image data is obtained is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic configuration diagram showing one embodiment of a sample observation device.

[0029] Figure 2 It is an enlarged view of the main part showing the vicinity of the sample.

[0030] Figure 3 This is a diagram showing an example of an image acquisition unit.

[0031] Figure 4 This is a flowchart showing an example of a sample observation method using a sample observation device.

[0032] Figure 5 FIG. 1 is a diagram showing an example of generation of observation image data by the image generating unit.

[0033] Figure 6 It is a diagram showing the state of image acquisition in a comparative example.

[0034] Figure 7 It is a diagram showing the state of image acquisition in the embodiment.

[0035] Figure 8 It is a diagram showing an example of calculation of the field of view of the sample observation device.

[0036] Figure 9 This is a diagram showing the relationship between the tilt angle of the observation axis and the resolution.

[0037] Figure 10 This is a diagram showing the relationship between the tilt angle of the observation axis and the stability of the visual field.

[0038] Figure 11 This is a graph showing the relationship between the tilt angle of the observation axis and the transmittance of the observation light from the sample.

[0039] Figure 12 It is a diagram showing a modified example of the imaging optical system. DETAILED DESCRIPTION

[0040] Hereinafter, preferred embodiments of a sample observation device and a sample observation method will be described in detail with reference to the accompanying drawings.

[0041] [Structure of sample observation device]

[0042] Figure 1 This is a schematic structural diagram showing an embodiment of a sample observation device. The sample observation device 1 is a device that irradiates a planar light L2 onto a sample S, images at least one of the fluorescence, scattered light, and diffuse reflected light generated inside the sample S on an imaging surface, and obtains observation image data of the inside of the sample S. As such a sample observation device 1, there is a slide scanner that obtains and displays an image of the sample S held on a glass slide, or a plate reader that obtains and analyzes image data of the sample S held on a microplate. Figure 1 As shown, the sample observation device 1 includes a light source 2 , an irradiation optical system 3 , a scanning unit 4 , an imaging optical system 5 , an image acquisition unit 6 , and a computer 7 .

[0043] Examples of the sample S to be observed include human or animal cells, tissues, organs, animals or plants themselves, and plant cells and tissues. The sample S may also be contained in a solution, gel, or a substance having a different refractive index from that of the sample S.

[0044] Light source 2 is a light source that outputs light L1 that irradiates sample S. Examples of light source 2 include laser light sources such as laser diodes and solid-state laser light sources. Alternatively, light source 2 may be a light-emitting diode, a superluminescent diode, or a lamp-type light source. Light L1 output from light source 2 is guided to irradiation optical system 3.

[0045] The irradiation optical system 3 is an optical system that shapes the light L1 output from the light source 2 into planar light L2, and irradiates the sample S with the shaped planar light L2 along the optical axis P1. In the following description, the optical axis P1 of the irradiation optical system 3 is sometimes referred to as the optical axis of the planar light L2. The irradiation optical system 3 is composed of, for example, a light shaping element such as a cylindrical lens, a conical lens, or a spatial light modulator, and is optically coupled to the light source 2. The irradiation optical system 3 may also be composed of an objective lens. The planar light L2 formed by the irradiation optical system 3 is irradiated onto the sample S. In the sample S irradiated with the planar light L2, observation light L3 is generated on the irradiation surface R of the planar light L2. The observation light L3 is, for example, at least one of fluorescence excited by the planar light L2, scattered light of the planar light L2, and diffused reflected light of the planar light L2.

[0046] When observing along the thickness direction of the sample S, the planar light L2 is preferably a thin planar light with a thickness of 2 mm or less, considering resolution. Furthermore, when the sample S is very thin, that is, when observing a sample S with a thickness below the Z-direction resolution described below, the thickness of the planar light L2 does not affect the resolution. Therefore, a planar light L2 with a thickness exceeding 2 mm may also be used.

[0047] The scanning unit 4 is a mechanism for scanning the sample S relative to the irradiation surface R of the planar light L2. In this embodiment, the scanning unit 4 is composed of a moving stage 12 for moving a sample container 11 holding the sample S. The sample container 11 is, for example, a microplate, a slide, a dish, etc. In this embodiment, a microplate is exemplified. Figure 2 As shown, the sample container 11 includes a plate-shaped main body 14 having a plurality of wells 13 containing the sample S arranged in a straight line (or array), and a plate-shaped transparent member 15 provided on one side of the main body 14 to block one end of the wells 13 .

[0048] When the sample S is placed in the hole 13, the hole 13 may be filled with a medium such as water. The transparent member 15 has an input surface 15a for the planar light L2 of the sample S placed in the hole 13. The material of the transparent member 15 is not particularly limited as long as it is transparent to the planar light L2, but may be glass, quartz, or a synthetic resin, for example. The sample container 11 is positioned relative to the movable stage 12 so that the input surface 15a is orthogonal to the optical axis P1 of the planar light L2. The other end of the hole 13 is open to the outside. The sample container 11 may also be fixed relative to the movable stage 12.

[0049] like Figure 1 As shown, the movable stage 12 scans the sample container 11 along a predetermined direction based on a control signal from the computer 7. In this embodiment, the movable stage 12 scans the sample container 11 along a direction within a plane perpendicular to the optical axis P1 of the planar light L2. In the following description, the direction of the optical axis P1 of the planar light L2 is referred to as the Z-axis, the scanning direction of the sample container 11 by the movable stage 12 is referred to as the Y-axis, and the direction perpendicular to the Y-axis within the plane perpendicular to the optical axis P1 of the planar light L2 is referred to as the X-axis. The irradiation surface R of the planar light L2 with respect to the sample S is a surface within the XZ plane.

[0050] The imaging optical system 5 is an optical system that forms an image of the observation light L3 generated on the sample S by the irradiation of the planar light L2. Figure 2As shown, the imaging optical system 5 is composed of, for example, an objective lens 16 and an imaging lens. The optical axis of the imaging optical system 5 becomes the observation axis P2 of the observation light L3. The observation axis P2 of the imaging optical system 5 is inclined at an inclination angle θ relative to the irradiation surface R of the planar light L2 in the sample S. The inclination angle θ is also consistent with the angle formed by the optical axis P1 of the planar light L2 toward the sample S and the observation axis P2. The inclination angle θ is 10° to 80°. From the viewpoint of improving the resolution of the observed image, the inclination angle θ is preferably 20° to 70°. In addition, from the viewpoint of improving the resolution of the observed image and the stability of the field of view, the inclination angle θ is further preferably 30° to 65°.

[0051] like Figure 1 As shown, the image acquisition unit 6 is a device that repeatedly acquires partial image data corresponding to a portion of the optical image of the observation light L3 formed by the imaging optical system 5. The image acquisition unit 6 is configured, for example, to include an imaging device that captures the optical image of the observation light L3. Examples of the imaging device include area image sensors such as CMOS image sensors and CCD image sensors. These area image sensors are placed on the imaging surface of the imaging optical system 5 and capture the optical image using, for example, a global shutter or a rolling shutter, and output two-dimensional image data to the computer 7.

[0052] Regarding the method of obtaining the partial image data of the light image of the observation light L3, various methods can be used. Figure 3 As shown in (A), a subarray can also be set on the imaging surface of the area image sensor 21. When reading the subarray in the area image sensor, only the set pixel columns in the full pixel column can be read, which can increase the frame rate. Therefore, in this case, since only the pixel columns 21a included in the subarray are read, it is possible to capture a portion of the light image of the observation light L3 and obtain partial image data. In addition, as shown in FIG. Figure 3 As shown in (B), all pixel rows of the area image sensor 21 may be used as a reading area, and a portion of the two-dimensional image may be extracted by subsequent image processing to obtain partial image data.

[0053] Furthermore, if Figure 3 As shown in (C), a line sensor 22 may be used instead of the area image sensor 21 to limit the imaging surface itself to a pixel column and obtain partial image data. Figure 3 As shown in FIG. 2D , a slit 23 that transmits only a portion of the observation light L3 may be disposed in front of the area image sensor (photodetector) 21, and image data of the pixel row 21a corresponding to the slit 23 may be acquired as partial image data. Furthermore, when the slit 23 is used, a point sensor such as a photomultiplier tube may be used in place of the area image sensor 21.

[0054] The computer 7 is physically composed of a memory such as RAM and ROM, a processor (calculation circuit) such as a CPU, a communication interface, a storage unit such as a hard disk, and a display unit such as a display. Examples of the computer 7 include a personal computer, a cloud server, a smart device (smartphone, tablet terminal, etc.), and a microcomputer. The computer 7 functions as a controller for controlling the operation of the light source 2 and the moving stage 12, an image generating unit 8 for generating observation image data of the sample S, and an analyzing unit 10 for analyzing the observation image data by executing a program stored in the memory by the CPU of the computer system (see Figure 1 ).

[0055] The computer 7, acting as a controller, receives input from the user regarding a measurement start operation and synchronously drives the light source 2, the moving stage 12, and the image acquisition unit 6. In this case, the computer 7 can control the light source 2 so that the light source 2 continuously outputs the light L1 while the sample S is moving on the moving stage 12, or can control the output of the light L1 from the light source 2 to be turned on / off (ON / OFF) according to the image captured by the image acquisition unit 6. In addition, if the irradiation optical system 3 includes a light shutter (not shown), the computer 7 can also control the light shutter to turn on / off the irradiation of the planar light L2 onto the sample S.

[0056] Furthermore, the computer 7, serving as the image generating unit 8, generates observation image data of the specimen S based on the plurality of partial image data generated by the image acquiring unit 6. The image generating unit 8 generates observation image data of the specimen S, for example, on a plane (XY plane) perpendicular to the optical axis P1 of the planar light L2, based on the plurality of partial image data output from the image acquiring unit 6. The image generating unit 8 performs operations such as storing the generated observation image data and displaying it on a monitor, etc., in accordance with predetermined user operations.

[0057] The computer 7, serving as the analysis unit 10, performs analysis based on the observed image data generated by the image generation unit 8 and generates analysis results. The analysis unit 10 performs operations such as storing the generated analysis results and displaying them on a monitor, etc., in response to user-defined operations. Alternatively, the observation image data generated by the image generation unit may not be displayed on a monitor, and only the analysis results generated by the analysis unit 10 may be displayed on the monitor, etc.

[0058] [Sample observation method]

[0059] Figure 4 This is a flowchart illustrating an example of a sample observation method using a sample observation device. As shown in the figure, the sample observation method includes an irradiation step (step S01), a scanning step (step S02), an imaging step (step S03), an image acquisition step (step S04), an image generation step (step S05), and an analysis step (step S06).

[0060] In the irradiation step S01, planar light L2 is irradiated onto the sample S. When the user inputs an operation to start measurement, the light source 2 is driven based on a control signal from the computer 7, and light L1 is output from the light source 2. The light L1 output from the light source 2 is shaped into planar light L2 by the irradiation optical system 3 and irradiated onto the sample S.

[0061] In the scanning step S02, the sample S is scanned relative to the irradiation surface R of the planar light L2. When the user inputs an operation to start measurement, the moving stage 12 is driven synchronously with the driving of the light source 2 based on a control signal from the computer 7. As a result, the sample container 11 is driven linearly along the Y-axis direction at a constant speed, and the sample S in the aperture 13 is scanned relative to the irradiation surface R of the planar light L2.

[0062] In the imaging step S03, the observation light L3 generated by the irradiation of the planar light L2 on the specimen S is imaged on the imaging surface of the image acquisition unit 6 using the imaging optical system 5 having an observation axis P2 inclined relative to the irradiation surface R. In the image acquisition step S04, partial image data corresponding to a portion of the optical image of the observation light L3 formed by the imaging optical system 5 is acquired multiple times. The partial image data is sequentially output from the image acquisition unit 6 to the image generation unit 8.

[0063] In the image generation step S05, observation image data of the sample S is generated based on the plurality of partial image data. Figure 1 and Figure 2 As shown in FIG. 1 , the irradiation surface R of the planar light L2 with respect to the sample S is a surface within the XZ plane, and the irradiation surface R is scanned along the Y-axis direction with respect to the sample S. Therefore, in the image generation unit 8, as shown in FIG. Figure 5 As shown in (A), by acquiring XZ cross-sectional image data 31 as partial image data multiple times along the Y-axis direction, three-dimensional information of the sample S is accumulated. In the image generating unit 8, the data is reconstructed using the multiple XZ cross-sectional images, for example, Figure 5 As shown in (B), an XY cross-sectional image having an arbitrary thickness at an arbitrary position in the Z-axis direction of the sample S is generated as observation image data 32 with the background suppressed.

[0064] In analysis step S06, the analysis unit 10 analyzes the observed image data and generates analysis results. For example, in drug development screening, a sample S and a reagent are placed in a sample container 11, and observed image data is acquired. The analysis unit 10 then evaluates the reagent based on the observed image data, generating evaluation data as analysis results.

[0065] [Effects]

[0066] like Figure 6As shown in (A), the sample observation device 100 involved in the comparative example has an observation axis P2 that is orthogonal to the irradiation surface R of the planar light L2. In this sample observation device 100, by irradiating the entire focal plane of the observation optical system with the planar light L2, an image of a cross-section plane in the sample S that is orthogonal to the direction of the observation axis P2 can be obtained by a single camera shot. Therefore, in order to obtain three-dimensional information of the sample S, it is necessary to scan the sample S along the direction of the observation axis P2 and obtain images of multiple cross-section planes that are orthogonal to the direction of the observation axis P2. In the sample observation device 100 involved in this comparative example, as Figure 6 As shown in (B), until images of all cross-sectional planes are acquired, the selection of cross-sectional planes (scanning and stopping of the specimen S) and image acquisition must be repeated. Furthermore, if the area of ​​observation is wider than the image capture area, in addition to acquiring cross-sectional images in the direction of the observation axis P2, it is also necessary to select the imaging field of view by moving the stage in a direction different from the observation axis.

[0067] In contrast, in the sample observation device 1 according to the embodiment, Figure 7 As shown in FIG. 1A , the image acquisition unit 6 acquires an image of the specimen S while scanning the specimen S relative to the irradiation surface R of the planar light L2. Furthermore, the observation axis P2 of the imaging optical system 5 is tilted relative to the irradiation surface R of the planar light L2. Therefore, the image acquisition unit 6 can sequentially acquire partial image data of the tomographic plane in the direction of the optical axis P1 (Z-axis direction) of the planar light L2, and the image generation unit 8 can generate observed image data 32 of the specimen S based on the plurality of partial image data.

[0068] In the sample observation device 1, as Figure 7 As shown in (B), it is possible to sequentially acquire images while scanning the sample S. In the operation of the sample observation device 100 of the comparative example, each time the movable stage 12 is driven and stopped, time is lost due to inertia and other factors. On the other hand, in the sample observation device 1, by reducing the number of times the movable stage 12 is driven and stopped and performing the scanning operation of the sample S and image acquisition simultaneously, throughput is improved until the observation image data 32 is obtained.

[0069] In addition, in the sample observation device 1, as Figure 2As shown, the sample S is held by a sample container 11 having an input surface 15a for the planar light L2, and is arranged so that the optical axis P1 of the planar light L2 from the irradiation optical system 3 is perpendicular to the input surface 15a of the sample container 11. Furthermore, in the sample observation device 1, the scanning unit 4 scans the sample S in a direction (Y-axis direction) perpendicular to the optical axis P1 (Z-axis direction) of the planar light L2 from the irradiation optical system 3. This eliminates the need for image processing such as position correction of partial image data obtained by the image acquisition unit 6, and facilitates the generation of observation image data.

[0070] In the sample observation apparatus 1 , the inclination angle θ of the observation axis P2 of the imaging optical system 5 relative to the irradiation surface R of the planar light L2 in the sample S is 10° to 80°, preferably 20° to 70°, and more preferably 30° to 65°. This point will be discussed below.

[0071] Figure 8 This figure shows an example calculation of the field of view in a sample observation device. In the example shown in this figure, the imaging optical system is located in medium A with a refractive index of n1, and the irradiation surface of planar light is located in medium B with a refractive index of n2. If the field of view in the imaging optical system is V, the irradiation surface is V', the inclination angle of the observation axis relative to the irradiation surface is θ, the refraction angle at the boundary between media A and B is θ', and the distance between media A and B at the interface when the field of view V is tilted at an angle of θ is L, the following equations (1) to (3) hold true.

[0072] (number 1)

[0073] L=V / cosθ…(1)

[0074] (Number 2)

[0075] sinθ'=(n1 / n2)sinθ…(2)

[0076] (Number 3)

[0077] V'=L / tanθ'…(3)

[0078] Figure 9 This graph shows the relationship between the tilt angle of the observation axis and resolution. In this graph, the horizontal axis is the tilt angle θ of the observation axis, and the vertical axis is the relative value V' / V of the field of view. For example, when the refractive index n1 of medium A is set to 1 (air), and the refractive index n2 of medium B is varied from 1.0 to 2.0 in 0.1 increments, the V' / V values ​​are plotted against the tilt angle θ. Smaller V' / V values ​​indicate higher resolution in the depth direction of the specimen (hereinafter referred to as "Z-direction resolution"), while larger V' / V values ​​indicate lower Z-direction resolution.

[0079] from Figure 9The results shown show that when the refractive index n1 of medium A and the refractive index n2 of medium B are equal, the value of V' / V is inversely proportional to the tilt angle θ. Furthermore, when the refractive index n1 of medium A and the refractive index n2 of medium B are different, the value of V' / V describes a parabola with respect to the tilt angle θ. These results demonstrate that Z-direction resolution can be controlled by the refractive index of the sample arrangement space, the refractive index of the imaging optical system arrangement space, and the tilt angle θ of the observation axis. Consequently, it can be seen that better Z-direction resolution is achieved when the tilt angle θ ranges from 10° to 80°, compared to when the tilt angle θ is less than 10° and exceeds 80°.

[0080] In addition, from Figure 9 The results show that the tilt angle θ, at which the Z-direction resolution is maximized, tends to decrease as the difference between the refractive indices n1 and n2 increases. Within the refractive index n2 range of 1.1 to 2.0, the tilt angle θ, at which the Z-direction resolution is maximized, falls within a range of approximately 47° to 57°. For example, when the refractive index n2 is 1.33 (water), the tilt angle θ at which the Z-direction resolution is maximized is estimated to be approximately 52°. Furthermore, when the refractive index n2 is 1.53 (glass), the tilt angle θ at which the Z-direction resolution is maximized is estimated to be approximately 48°.

[0081] Figure 10 This is a graph showing the relationship between the tilt angle of the observation axis and the stability of the field of view. In this graph, the horizontal axis is the tilt angle θ of the observation axis, and the vertical axis is the stability of the field of view. The stability is represented by the ratio of the difference between V' / V in the tilt angle θ+1 and V' / V in the tilt angle θ-1 relative to V' / V in the tilt angle θ, and is calculated based on the following formula (4). The closer the stability is to 0%, the smaller the change in the field of view relative to the change in the tilt angle is, and the field of view can be evaluated as stable. In this Figure 10 In, with Figure 9 Similarly, the stability is plotted when the refractive index n1 of medium A is set to 1 (air) and the refractive index n2 of medium B is changed from 1.0 to 2.0 in increments of 0.1.

[0082] (Number 4)

[0083] Stability (%) = ((V' / V) θ+1 -(V' / V) θ-1 ) / (V' / V) θ …(4)

[0084] from Figure 10The results show that when the tilt angle θ is less than 10° and greater than 80°, the stability exceeds ±20%, making it difficult to control the field of view. On the other hand, when the tilt angle θ is between 10° and 80°, the stability is less than ±20%, and the field of view can be controlled. Furthermore, when the tilt angle θ is between 20° and 70°, the stability is less than ±10%, and the field of view can be easily controlled.

[0085] Figure 11 This is a graph showing the relationship between the tilt angle of the observation axis and the transmittance of the observation light from the sample. In this graph, the horizontal axis is the tilt angle θ of the observation axis, the left vertical axis is the relative value of the field of view, and the right vertical axis is the transmittance. Figure 11 In the example, the sample container is kept in a sample state, and the refractive index n1 of medium A is set to 1 (air), the refractive index n2 of medium B is set to 1.53 (glass), and the refractive index n3 of medium C is set to 1.33 (water). The transmittance is the product of the transmittances of the interface between mediums B and C and the interface between mediums A and B. Figure 11 In the figure, the transmittance of P wave, the transmittance of S wave, and the angular dependence of their average values ​​are plotted. Figure 11 In FIG, the relative value of the field of view of medium C is also plotted.

[0086] from Figure 11 The results shown here demonstrate that by varying the tilt angle θ of the observation axis, the transmittance of the observation light from the sample to the imaging optical system can be varied. It can be seen that within the tilt angle θ range of 80° or less, a transmittance of at least 50% can be achieved. Furthermore, within the tilt angle θ range of 70° or less, a transmittance of at least 60% can be achieved, and within the tilt angle θ range of 65° or less, a transmittance of at least 75% can be achieved.

[0087] The above results indicate that when high resolution in the Z direction of the sample is required, the tilt angle θ is preferably selected from the range of 30° to 65° so that, for example, the value of V' / V, which is the relative value of the field of view, is less than 3, the stability is less than 5%, and the transmittance of the observation light (the average of the P and S waves) is 75% or greater. Furthermore, when high resolution in the Z direction of the sample is not required, the tilt angle θ can be appropriately selected from the range of 10° to 80°. From the perspective of ensuring the field of view per pixel, it is preferably selected from the range of 10° to 30° or 65° to 80°.

[0088] For example, the optical axis P1 of the planar light L2 and the input surface 15a of the sample container 11 do not necessarily need to be perpendicular to each other, nor do the optical axis P1 of the planar light L2 and the scanning direction of the sample S by the scanner 4 need to be perpendicular to each other.

[0089] In addition, for example, in the above-described embodiment, a transparent member 15 is provided in the sample container 11 so as to block one end of the hole 13, and the planar light L2 is input from the input surface 15a of the transparent member 15. However, a configuration may also be adopted in which the planar light L2 is input from the other end of the hole 13. In this case, the number of interfaces between media with different refractive indices is reduced, and the number of refractions of the observation light L3 can be reduced. Furthermore, the sample S may be held in a solid such as a gel instead of the sample container 11, or the sample S may be moved by flowing a fluid such as a sheath fluid in a transparent container as in a flow cytometer. In the case of a flow cytometer, a sheath fluid containing a liquid containing a specimen serving as the sample S is flowed using a flow cell. Thus, since the specimen is arranged while being moved, the flow cell can be aligned with the scanning unit.

[0090] In addition, multiple pairs of imaging optical systems 5 and image acquisition units 6 may be configured. In this case, in addition to being able to enlarge the observation range, it is also possible to observe observation light L3 of multiple different wavelengths. In addition, multiple image acquisition units 6 may be configured relative to one imaging optical system 5, or one image acquisition unit 6 may be configured relative to multiple imaging optical systems 5. Multiple image acquisition units 6 may also be combined with different types of light detectors or imaging devices. The light source 2 may also be composed of multiple light sources that output light of different wavelengths. In this case, the sample S can be irradiated with excitation light of different wavelengths.

[0091] In order to alleviate astigmatism, a prism may be arranged in the imaging optical system 5. In this case, for example, Figure 12 As shown, a prism 41 may be disposed on the rear side of the objective lens 16 (between the objective lens 16 and the image acquisition unit 6). To counteract defocus, the imaging surface of the imaging device in the image acquisition unit 6 may be tilted relative to the observation axis P2. Alternatively, for example, a dichroic mirror or prism may be disposed between the imaging optical system 5 and the image acquisition unit 6 to perform wavelength separation of the observation light L3.

[0092] Furthermore, as described above, the observation light L3 may include fluorescence excited by the planar light L2, scattered light from the planar light L2, or diffusely reflected light from the planar light L2. Therefore, the image acquisition unit 6 may also acquire image data for different types of observation light L3. In this case, at least two types of observation light, namely, fluorescence excited by the planar light L2, scattered light from the planar light L2, and diffusely reflected light from the planar light L2, can be used as the target.

[0093] Explanation of symbols

[0094] 1…sample observation device, 3…irradiation optical system, 4…scanning unit, 5…imaging optical system, 6…image acquisition unit, 8…image generation unit, 10…analysis unit, 11…sample container, 15a…input surface, 21…area image sensor (camera), 22…line sensor, 23…slit, 31…partial image data, 32…observation image data, L2…planar light, L3…observation light, P2…observation axis, R…irradiation surface, S…sample, θ…tilt angle.

Claims

1. A SPIM selective plane illumination microscope, wherein: have: an irradiation optical system for irradiating a sample with planar light; a scanning unit configured to scan the sample relative to an irradiation surface of the planar light; an imaging optical system having an observation axis inclined with respect to the irradiation surface and forming an image of observation light generated on the sample by irradiation with the planar light; an image acquisition unit that acquires, a plurality of times, partial image data corresponding to a portion of the optical image of the observation light formed by the imaging optical system; as well as an image generating unit that generates observation image data of the sample based on the plurality of partial image data generated by the image acquiring unit, In the sample, the observation axis is tilted relative to the planar light, The scanning unit scans the sample in a direction orthogonal to the optical axis of the planar light of the irradiation optical system.

2. The SPIM selective plane illumination microscope according to claim 1, wherein: The sample is held by a sample container having an input surface for the planar light. The optical axis of the planar light of the irradiation optical system is arranged to be perpendicular to the input surface of the sample container.

3. The SPIM selective plane illumination microscope according to claim 1, wherein: The observation axis of the imaging optical system is inclined at an angle of 10° to 80° with respect to the irradiation surface of the planar light.

4. The SPIM selective plane illumination microscope according to claim 2, wherein: The observation axis of the imaging optical system is inclined at an angle of 10° to 80° with respect to the irradiation surface of the planar light.

5. The SPIM selective plane illumination microscope according to any one of claims 1 to 4, wherein: The observation axis of the imaging optical system is inclined at an angle of 20° to 70° with respect to the irradiation surface of the planar light.

6. The SPIM selective plane illumination microscope according to any one of claims 1 to 4, wherein: The observation axis of the imaging optical system has an inclination angle of 30° to 65° with respect to the irradiation surface of the planar light.

7. The SPIM selective plane illumination microscope according to claim 5, wherein: The observation axis of the imaging optical system has an inclination angle of 30° to 65° with respect to the irradiation surface of the planar light.

8. The SPIM selective plane illumination microscope according to any one of claims 1 to 4, wherein: The image acquisition unit includes a two-dimensional imaging device, and extracts image data corresponding to a portion of the optical image of the observation light from data output from the two-dimensional imaging device as the partial image data.

9. The SPIM selective plane illumination microscope according to claim 5, wherein: The image acquisition unit includes a two-dimensional imaging device, and extracts image data corresponding to a portion of the optical image of the observation light from data output from the two-dimensional imaging device as the partial image data.

10. The SPIM selective plane illumination microscope according to claim 6, wherein: The image acquisition unit includes a two-dimensional imaging device, and extracts image data corresponding to a portion of the optical image of the observation light from data output from the two-dimensional imaging device as the partial image data.

11. The SPIM selective plane illumination microscope according to claim 7, wherein: The image acquisition unit includes a two-dimensional imaging device, and extracts image data corresponding to a portion of the optical image of the observation light from data output from the two-dimensional imaging device as the partial image data.

12. The SPIM selective plane illumination microscope according to any one of claims 1 to 4, wherein: The image acquisition unit includes a line sensor that captures a portion of the optical image of the observation light and outputs the partial image data.

13. The SPIM selective plane illumination microscope according to claim 5, wherein: The image acquisition unit includes a line sensor that captures a portion of the optical image of the observation light and outputs the partial image data.

14. The SPIM selective plane illumination microscope according to claim 6, wherein: The image acquisition unit includes a line sensor that captures a portion of the optical image of the observation light and outputs the partial image data.

15. The SPIM selective plane illumination microscope according to claim 7, wherein: The image acquisition unit includes a line sensor that captures a portion of the optical image of the observation light and outputs the partial image data.

16. The SPIM selective plane illumination microscope according to any one of claims 1 to 4, wherein: The image acquisition unit includes a slit that allows a portion of the optical image of the observation light to pass therethrough, and a photodetector that detects the optical image that has passed through the slit, and generates the partial image data based on data output from the photodetector.

17. The SPIM selective plane illumination microscope according to claim 5, wherein: The image acquisition unit includes a slit that allows a portion of the optical image of the observation light to pass therethrough, and a photodetector that detects the optical image that has passed through the slit, and generates the partial image data based on data output from the photodetector.

18. The SPIM selective plane illumination microscope according to claim 6, wherein: The image acquisition unit includes a slit that allows a portion of the optical image of the observation light to pass therethrough, and a photodetector that detects the optical image that has passed through the slit, and generates the partial image data based on data output from the photodetector.

19. The SPIM selective plane illumination microscope according to claim 7, wherein: The image acquisition unit includes a slit that allows a portion of the optical image of the observation light to pass therethrough, and a photodetector that detects the optical image that has passed through the slit, and generates the partial image data based on data output from the photodetector.

20. The SPIM selective plane illumination microscope according to any one of claims 1 to 4, wherein: The image generating unit generates observation image data of the sample on a plane perpendicular to the optical axis of the planar light based on the plurality of partial image data.

21. The SPIM selective plane illumination microscope according to claim 5, wherein: The image generating unit generates observation image data of the sample on a plane perpendicular to the optical axis of the planar light based on the plurality of partial image data.

22. The SPIM selective plane illumination microscope according to claim 6, wherein: The image generating unit generates observation image data of the sample on a plane perpendicular to the optical axis of the planar light based on the plurality of partial image data.

23. The SPIM selective plane illumination microscope according to claim 7, wherein: The image generating unit generates observation image data of the sample on a plane perpendicular to the optical axis of the planar light based on the plurality of partial image data.

24. The SPIM selective plane illumination microscope according to claim 8, wherein: The image generating unit generates observation image data of the sample on a plane perpendicular to the optical axis of the planar light based on the plurality of partial image data.

25. The SPIM selective plane illumination microscope according to claim 9, wherein: The image generating unit generates observation image data of the sample on a plane perpendicular to the optical axis of the planar light based on the plurality of partial image data.

26. The SPIM selective plane illumination microscope according to claim 10, wherein: The image generating unit generates observation image data of the sample on a plane perpendicular to the optical axis of the planar light based on the plurality of partial image data.

27. The SPIM selective plane illumination microscope according to claim 11, wherein: The image generating unit generates observation image data of the sample on a plane perpendicular to the optical axis of the planar light based on the plurality of partial image data.

28. The SPIM selective plane illumination microscope according to claim 12, wherein: The image generating unit generates observation image data of the sample on a plane perpendicular to the optical axis of the planar light based on the plurality of partial image data.

29. The SPIM selective plane illumination microscope according to claim 13, wherein: The image generating unit generates observation image data of the sample on a plane perpendicular to the optical axis of the planar light based on the plurality of partial image data.

30. The SPIM selective plane illumination microscope according to claim 14, wherein: The image generating unit generates observation image data of the sample on a plane perpendicular to the optical axis of the planar light based on the plurality of partial image data.

31. The SPIM selective plane illumination microscope according to claim 15, wherein: The image generating unit generates observation image data of the sample on a plane perpendicular to the optical axis of the planar light based on the plurality of partial image data.

32. The SPIM selective plane illumination microscope according to claim 16, wherein: The image generating unit generates observation image data of the sample on a plane perpendicular to the optical axis of the planar light based on the plurality of partial image data.

33. The SPIM selective plane illumination microscope according to claim 17, wherein: The image generating unit generates observation image data of the sample on a plane perpendicular to the optical axis of the planar light based on the plurality of partial image data.

34. The SPIM selective plane illumination microscope according to claim 18, wherein: The image generating unit generates observation image data of the sample on a plane perpendicular to the optical axis of the planar light based on the plurality of partial image data.

35. The SPIM selective plane illumination microscope according to claim 19, wherein: The image generating unit generates observation image data of the sample on a plane perpendicular to the optical axis of the planar light based on the plurality of partial image data.

36. The SPIM selective plane illumination microscope according to any one of claims 1 to 4, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

37. The SPIM selective plane illumination microscope according to claim 5, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

38. The SPIM selective plane illumination microscope according to claim 6, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

39. The SPIM selective plane illumination microscope according to claim 7, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

40. The SPIM selective plane illumination microscope according to claim 8, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

41. The SPIM selective plane illumination microscope according to claim 9, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

42. The SPIM selective plane illumination microscope according to claim 10, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

43. The SPIM selective plane illumination microscope according to claim 11, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

44. The SPIM selective plane illumination microscope according to claim 12, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

45. The SPIM selective plane illumination microscope according to claim 13, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

46. ​​The SPIM selective plane illumination microscope according to claim 14, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

47. The SPIM selective plane illumination microscope according to claim 15, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

48. The SPIM selective plane illumination microscope of claim 16, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

49. The SPIM selective plane illumination microscope according to claim 17, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

50. The SPIM selective plane illumination microscope according to claim 18, wherein The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

51. The SPIM selective plane illumination microscope of claim 19, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

52. The SPIM selective plane illumination microscope according to claim 20, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

53. The SPIM selective plane illumination microscope according to claim 21, wherein The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

54. The SPIM selective plane illumination microscope of claim 22, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

55. The SPIM selective plane illumination microscope of claim 23, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

56. The SPIM selective plane illumination microscope of claim 24, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

57. The SPIM selective plane illumination microscope of claim 25, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

58. The SPIM selective plane illumination microscope of claim 26, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

59. The SPIM selective plane illumination microscope of claim 27, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

60. The SPIM selective plane illumination microscope of claim 28, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

61. The SPIM selective plane illumination microscope of claim 29, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

62. The SPIM selective plane illumination microscope of claim 30, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

63. The SPIM selective plane illumination microscope of claim 31 , wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

64. The SPIM selective plane illumination microscope of claim 32, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

65. The SPIM selective plane illumination microscope of claim 33, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

66. The SPIM selective plane illumination microscope of claim 34, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

67. The SPIM selective plane illumination microscope of claim 35, wherein: The device further includes an analyzing unit that analyzes the observation image data and generates an analysis result.

68. A method for observing a sample using a SPIM selective plane illumination microscope, wherein: have: an irradiation step of irradiating the sample with planar light; a scanning step of scanning the sample relative to an irradiation surface of the planar light; an imaging step of imaging observation light generated on the sample by irradiation with the planar light using an imaging optical system having an observation axis inclined with respect to the irradiation surface; an image acquisition step of acquiring, a plurality of times, partial image data corresponding to a portion of the optical image of the observation light formed by the imaging optical system; and an image generating step of generating observation image data of the sample based on the plurality of partial image data, In the sample, the observation axis is tilted relative to the planar light, In the scanning step, the sample is scanned in a direction orthogonal to the optical axis of the planar light.

Images