JP2020194190A - Sample observation device and sample observation method - Google Patents

Abstract

To provide a sample observation device and a sample observation method that improve throughput until observation image data is obtained.SOLUTION: A sample observation device 1 comprises: an irradiation optical system 3 that irradiates a sample S with planar light L2; a scanning part 4 that scans the sample S with respect to a surface R irradiated with the planar light L2; an image formation optical system 5 that has an observation axis P2 inclined with respect to the irradiated surface R and forms observation light L3 into an image, the observation light generated from the sample S through the irradiation with the planar light L2; an image acquisition part 6 that acquires a plurality of pieces of partial image data corresponding to part of an optical image formed from the observation light L3 by the image formation optical system 5; and an image creation part 8 that creates observation image data of the sample S on the basis of the plurality of pieces of partial image data created by the image acquisition part 6.SELECTED DRAWING: Figure 1

Description

The present disclosure relates to a sample observation device and a sample observation method.

SPIM (Selective Plane Illumination Microscopy) is known as one of the methods for observing the inside of a sample having a three-dimensional structure such as a cell. For example, the tomographic image observing apparatus described in Patent Document 1 discloses the basic principle of SPIM, irradiates a sample with planar light, and emits fluorescence or scattered light generated inside the sample to an imaging surface. An image is formed and the observation image data inside the sample is acquired.

Examples of other sample observation devices using planar light include the SPIM microscope described in Patent Document 2. In this conventional SPIM microscope, the surface of the sample is irradiated with planar light at a constant inclination angle, and the observation light from the sample is observed by an observation optical system having an observation axis orthogonal to the irradiation surface of the planar light. To image.

Japanese Unexamined Patent Publication No. 62-180241 Japanese Unexamined Patent Publication No. 2014-202967

In the sample observation apparatus described in Patent Document 2 described above, by irradiating the entire surface of the focus surface of the observation optical system with planar light, an image of the tomographic surface in the observation axis direction can be acquired by one imaging. Therefore, in order to acquire the three-dimensional information of the sample, it is necessary to scan the sample in the observation axis direction and acquire images of a plurality of tomographic planes in the observation axis direction. In such a conventional sample observation device, it is necessary to repeat selection (scanning and stopping of the sample) and image acquisition of the tomographic planes to be imaged before acquiring images of all the tomographic planes. When the area where the observation target exists is wider than the imaging area, in addition to the operation of acquiring the cross-sectional image in the observation axis direction, the operation of moving the stage in a direction different from the observation axis direction to select the imaging field. Etc. were needed. Therefore, it has been a problem that it takes time to obtain the observation image data.

The present disclosure has been made to solve the above problems, and an object of the present disclosure is to provide a sample observation device and a sample observation method capable of improving the throughput until the observation image data is obtained.

The sample observation device according to one aspect of the present disclosure includes an irradiation optical system for irradiating a sample with planar light, a scanning unit for scanning the sample with respect to the surface light irradiation surface, and an observation tilted with respect to the irradiation surface. Partial image data that has an axis and forms an image of the observation light generated in the sample by irradiation with planar light, and a part of the optical image of the observation light formed by the imaging optical system. It is provided with an image acquisition unit for acquiring a plurality of images, and an image generation unit for generating observation image data of a sample based on a plurality of partial image data generated by the image acquisition unit.

In this sample observation device, the sample is scanned against the surface illuminated by the planar light, and the observation axis of the imaging optical system is tilted with respect to the irradiated surface of the planar light. Therefore, the image acquisition unit can sequentially acquire the partial image data of the tomographic plane in the optical axis direction of the planar light, and the image generation unit can acquire the observation image data of the sample based on the plurality of partial image data. Can be generated. In this sample observation device, the visual field selection operation becomes unnecessary, and the scanning of the sample and the acquisition of the image can proceed at the same time, so that the throughput until the observation image data is obtained can be improved.

Further, the sample may be held by a sample container having a planar light input surface, and the optical axis of the planar light by the irradiation optical system may be arranged so as to be orthogonal to the input surface of the sample container. In this case, the sample container makes it possible to scan a plurality of samples at once. Further, by making the optical axis of the planar light orthogonal to the input surface of the sample container, it is not necessary to correct the position of the partial image data acquired by the image acquisition unit, and the process of generating the observation image data can be facilitated.

Further, the scanning unit may scan the sample in a direction orthogonal to the optical axis of the planar light produced by the irradiation optical system. In this case, image processing such as position correction of the partial image data acquired by the image acquisition unit becomes unnecessary, and the generation process of the observation image data can be facilitated.

Further, the inclination angle of the observation axis of the imaging optical system with respect to the irradiation surface of the planar light may be 10 ° to 80 °. In this range, the resolution of the observed image can be sufficiently secured.

Further, the inclination angle of the observation axis of the imaging optical system with respect to the irradiation surface of the planar light may be 20 ° to 70 °. In this range, the resolution of the observed image can be further sufficiently secured. In addition, the change in the visual field with respect to the amount of change in the angle of the observation axis can be suppressed, and the stability of the visual field can be ensured.

Further, the inclination angle of the observation axis of the imaging optical system with respect to the irradiation surface of the planar light may be 30 ° to 65 °. In this range, the resolution of the observed image and the stability of the visual field can be more preferably secured.

Further, the image acquisition unit is configured to include a two-dimensional image pickup device, and image data corresponding to a part of the optical image of the observation light may be extracted as partial image data from the data output from the two-dimensional image pickup device. .. With such a configuration, partial image data can be acquired with high accuracy.

Further, the image acquisition unit may include a line sensor that captures a part of the light image by the observation light and outputs the partial image data. With such a configuration, partial image data can be acquired with high accuracy.

Further, the image acquisition unit includes a slit for passing a part of the light image by the observation light and a photodetector for detecting the light image passing through the slit, and the partial image is based on the data output from the photodetector. Data may be generated. With such a configuration, partial image data can be acquired with high accuracy.

Further, the image generation unit may generate observation image data of the sample on a plane orthogonal to the optical axis of the planar light based on the plurality of partial image data. As a result, a cross-sectional image of the sample in which the influence of the background is suppressed can be obtained as an observation image.

Further, the sample observation device may further include an analysis unit that analyzes the observation image data and generates an analysis result. Since the observation image data generated by the image generation unit is analyzed by the analysis unit, the analysis throughput can also be improved.

Further, the sample observation method according to one aspect of the present invention includes an irradiation step of irradiating the sample with planar light, a scanning step of scanning the sample against the irradiated surface of the planar light, and an inclination with respect to the irradiated surface. Using an imaging optical system with an observation axis, the imaging step of imaging the observation light generated in the sample by irradiation with planar light and a part of the optical image by the observation light imaged by the imaging optical system It includes an image acquisition step of acquiring a plurality of corresponding partial image data, and an image generation step of generating observation image data of a sample based on the plurality of partial image data.

In this sample observation method, an imaging optical system is used in which the sample is scanned against the surface illuminated by planar light and the observation axis is inclined with respect to the irradiated surface of planar light. Therefore, in the image acquisition step, it is possible to sequentially acquire the partial image data of the tomographic plane in the optical axis direction of the planar light, and in the image generation step, the observation image data of the sample is based on the plurality of partial image data. Can be generated. In this sample observation method, the visual field selection operation becomes unnecessary, and the scanning of the sample and the acquisition of the image can proceed at the same time, so that the throughput until the observation image data is obtained can be improved.

According to this sample observation device and sample observation method, the throughput until the observation image data is obtained can be improved.

It is a schematic block diagram which shows one Embodiment of a sample observation apparatus. It is an enlarged view of the main part which shows the vicinity of a sample. It is a figure which shows an example of an image acquisition part. It is a flowchart which shows an example of the sample observation method using a sample observation apparatus. It is a figure which shows an example of the generation of observation image data by an image generation part. It is a figure which shows the state of the image acquisition in the comparative example. It is a figure which shows the state of image acquisition in an Example. It is a figure which shows the calculation example of the field of view in a sample observation apparatus. It is a figure which shows the relationship between the tilt angle of an observation axis, and a resolution. It is a figure which shows the relationship between the inclination angle of an observation axis, and the stability of a visual field. It is a figure which shows the relationship between the inclination angle of the observation axis, and the transmittance of the observation light from a sample. It is a figure which shows the modification of the imaging optical system.

Hereinafter, preferred embodiments of the sample observation apparatus and the sample observation method according to one aspect of the present disclosure will be described in detail with reference to the drawings.
[Configuration of sample observation device]

FIG. 1 is a schematic configuration diagram showing an embodiment of a sample observation device. This sample observation device 1 is a device that irradiates the sample S with planar light L2 and forms an image of fluorescence or scattered light generated inside the sample S on the imaging surface to acquire observation image data inside the sample S. is there. As this type of sample observation device 1, a slide scanner that acquires and displays an image of sample S held on a slide glass, or a plate that acquires image data of sample S held on a microplate and analyzes the image data. There is a reader and so on. As shown in FIG. 1, 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. There is.

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

The light source 2 is a light source that outputs the light L1 that is applied to the sample S. Examples of the light source 2 include a laser light source such as a laser diode and a solid-state laser light source. Further, the light source 2 may be a light emitting diode, a superluminescent diode, or a lamp-based light source. The light L1 output from the light source 2 is guided to the irradiation optical system 3.

The irradiation optical system 3 is an optical system in which the light L1 output from the light source 2 is shaped into a planar light L2, and the shaped planar light L2 is irradiated to the sample S along the optical axis P1. In the following description, the optical axis P1 of the irradiation optical system 3 may be referred to as the optical axis of the planar light L2. The irradiation optical system 3 includes an optical shaping element such as a cylindrical lens, an axicon lens, or a spatial light modulator, and is optically coupled to the light source 2. The irradiation optical system 3 may be configured to include an objective lens. The planar light L2 formed by the irradiation optical system 3 irradiates the sample S. In the sample S irradiated with the planar light L2, the observation light L3 is generated on the irradiation surface R of the planar light L2. The observation light L3 is, for example, fluorescence excited by the planar light L2, scattered light of the planar light L2, or diffusely reflected light of the planar light L2.

When observing the sample S in the thickness direction, the planar light L2 is preferably a thin planar light having a thickness of 2 mm or less in consideration of resolution. Further, when the thickness of the sample S is very small, that is, when observing the sample S having a thickness equal to or lower than the resolution in the Z direction described later, the thickness of the planar light L2 does not affect the resolution. Therefore, planar light L2 having a thickness of more than 2 mm may be used.

The scanning unit 4 is a mechanism for scanning the sample S with respect to the irradiation surface R of the planar light L2. In the present embodiment, the scanning unit 4 is composed of a moving stage 12 for moving the sample container 11 holding the sample S. The sample container 11 is, for example, a microplate, a slide glass, a petri dish, or the like. In this embodiment, a microplate is illustrated. As shown in FIG. 2, the sample container 11 has a plate-shaped main body 14 in which a plurality of wells 13 in which the sample S is arranged are arranged in a straight line (or matrix), and wells on one surface side of the main body 14. It has a plate-shaped transparent member 15 provided so as to close one end side of the 13.

When arranging the sample S in the well 13, the well 13 may be filled with a medium such as water. The transparent member 15 has an input surface 15a of planar light L2 with respect to the sample S arranged in the well 13. The material of the transparent member 15 is not particularly limited as long as it is a member having transparency to the planar light L2, and is, for example, glass, quartz, or synthetic resin. The sample container 11 is arranged with respect to the moving 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 well 13 is open to the outside. The sample container 11 may be fixed to the moving stage 12.

As shown in FIG. 1, the moving stage 12 scans the sample container 11 in a preset direction according to a control signal from the computer 7. In the present embodiment, the moving stage 12 scans the sample container 11 in one direction in a plane orthogonal to the optical axis P1 of the planar light L2. In the following description, the optical axis P1 direction of the planar light L2 is the Z axis, the scanning direction of the sample container 11 by the moving stage 12 is the Y axis, and the plane light L2 is orthogonal to the Y axis in a plane orthogonal to the optical axis P1. The direction of light 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 in the XZ plane.

The imaging optical system 5 is an optical system that forms an image of the observation light L3 generated in the sample S by irradiation with the planar light L2. As shown in FIG. 2, the imaging optical system 5 includes, for example, an objective lens 16 and an imaging lens. The optical axis of the imaging optical system 5 is the observation axis P2 of the observation light L3. The observation axis P2 of the imaging optical system 5 is inclined with an inclination angle θ with respect to the irradiation surface R of the planar light L2 in the sample S. The inclination angle θ also coincides with the angle formed by the optical axis P1 and the observation axis P2 of the planar light L2 toward the sample S. 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 °. Further, from the viewpoint of improving the resolution of the observed image and the stability of the visual field, the inclination angle θ is more preferably 30 ° to 65 °.

As shown in FIG. 1, the image acquisition unit 6 is a device that acquires a plurality of partial image data corresponding to a part of an optical image formed by the observation light L3 imaged by the imaging optical system 5. The image acquisition unit 6 includes, for example, an imaging device that captures an optical image by the observation light L3. Examples of the image pickup device include area image sensors such as a CMOS image sensor and a CCD image sensor. These area image sensors are arranged on the imaging surface of the imaging optical system 5, capture an optical image with, for example, a global shutter or a rolling shutter, and output two-dimensional image data to the computer 7.

Various modes can be adopted for the method of acquiring the partial image data of the optical image by the observation light L3. For example, as shown in FIG. 3A, a sub-array may be set on the imaging surface of the area image sensor 21. In this case, since only the pixel sequence 21a included in the sub-array can be read out, a part of the optical image by the observation light L3 can be imaged to acquire partial image data. Further, as shown in FIG. 3B, all the pixel strings of the area image sensor 21 may be used as a readout area, and a part of the two-dimensional image may be extracted by subsequent image processing to acquire partial image data. ..

Further, as shown in FIG. 3C, a line sensor 22 may be used instead of the area image sensor 21, and the imaging surface itself may be limited to one pixel sequence to acquire partial image data. As shown in FIG. 3D, a slit 23 that transmits only a part of the observation light L3 is arranged in front of the area image sensor (photodetector) 21, and image data of the pixel sequence 21a corresponding to the slit 23 is displayed. It may be acquired as partial image data. When the slit 23 is used, a point sensor (photodetector) such as a photomultiplier tube may be used instead of the area image sensor 21.

The computer 7 is physically configured to include a memory such as a RAM and a ROM, a processor (arithmetic 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 such a computer 7 include a personal computer, a cloud server, a smart device (smartphone, tablet terminal, etc.) and the like. The computer 7 has a controller that controls the operations of the light source 2 and the moving stage 12 by executing a program stored in the memory on the CPU of the computer system, an image generation unit 8 that generates observation image data of the sample S, and an observation. It functions as an analysis unit 10 that analyzes image data (see FIG. 1).

The computer 7 as a controller receives an input of a measurement start operation by the user, and drives the light source 2, the moving stage 12, and the image acquisition unit 6 in synchronization with each other. In this case, the computer 7 may control the light source 2 so that the light source 2 continuously outputs the light L1 while the sample S is being moved by the moving stage 12, and the light source 2 may be matched with the image captured by the image acquisition unit 6. The ON / OFF of the output of the optical L1 may be controlled. When the irradiation optical system 3 is provided with an optical shutter (not shown), the computer 7 may turn on / off the irradiation of the planar light L2 to the sample S by controlling the optical shutter.

Further, the computer 7 as the image generation unit 8 generates the observation image data of the sample S based on the plurality of partial image data generated by the image acquisition unit 6. The image generation unit 8 generates observation image data of the sample S on a plane (XY plane) orthogonal to the optical axis P1 of the planar light L2, for example, based on the plurality of partial image data output from the image acquisition unit 6. .. The image generation unit 8 executes the storage of the generated observation image data, the display on the monitor, and the like according to a predetermined operation by the user.

The computer 7 as the analysis unit 10 executes the analysis based on the observation image data generated by the image generation unit 8 and generates the analysis result. The analysis unit 10 stores the generated analysis result, displays it on a monitor, or the like according to a predetermined operation by the user. The observation image data generated by the image generation unit may not be displayed on the monitor or the like, and only the analysis result generated by the analysis unit 10 may be displayed on the monitor or the like.
[Sample observation method]

FIG. 4 is a flowchart showing an example of a sample observation method using a sample observation device. As shown in the figure, this 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), and an image generation step (step S05). , And an analysis step (step S06).

In the irradiation step S01, the sample S is irradiated with the planar light L2. When the operation to start measurement is input by the user, the light source 2 is driven based on the control signal from the computer 7, and the light L1 is output from the light source 2. The light L1 output from the light source 2 is shaped by the irradiation optical system 3 to become planar light L2, which is irradiated to the sample S.

In the scanning step S02, the sample S is scanned against the irradiation surface R of the planar light L2. When the operation to start measurement is input by the user, the moving stage 12 is driven in synchronization with the drive of the light source 2 based on the control signal from the computer 7. As a result, the sample container 11 is linearly driven in the Y-axis direction at a constant speed, and the sample S in the well 13 is scanned with respect to the irradiation surface R of the planar light L2.

In the imaging step S03, the imaging optical system 5 having the observation axis P2 inclined with respect to the irradiation surface R is used, and the observation light L3 generated in the sample S by the irradiation of the planar light L2 is imaged by the image acquisition unit 6. An image is formed on a surface. In the image acquisition step S04, a plurality of partial image data corresponding to a part of the optical image formed by the observation light L3 imaged by the imaging optical system 5 is acquired. The partial image data is sequentially output from the image acquisition unit 6 to the image generation unit 8.

In the image generation step S05, the observation image data of the sample S is generated based on the plurality of partial image data. In the present embodiment, as shown in FIGS. 1 and 2, the irradiation surface R of the planar light L2 with respect to the sample S is a surface in the XZ plane, and the irradiation surface R with respect to the sample S is in the Y-axis direction. It is scanned. Therefore, as shown in FIG. 5A, the image generation unit 8 accumulates the three-dimensional information of the sample S by acquiring a plurality of XZ cross-sectional image data 31 which are partial image data in the Y-axis direction. .. In the image generation unit 8, data is reconstructed using a plurality of XZ cross-sectional images, and as shown in FIG. 5B, for example, XY having an arbitrary thickness at an arbitrary position in the Z-axis direction in the sample S. The cross-sectional image is generated as an observation image 32 with a suppressed background.

In the analysis step S06, the analysis unit 10 analyzes the observed image data and generates an analysis result. For example, in drug discovery screening, sample S and reagents are placed in a sample container 11 and observation image data is acquired. Then, the analysis unit 10 evaluates the reagent based on the observed image data, and generates the evaluation data as the analysis result.
[Action effect]

As shown in FIG. 6A, the sample observation device 100 according to the comparative example has an observation axis P2 orthogonal to the irradiation surface R of the planar light L2. In the sample observation device 100, by irradiating the entire surface of the focus surface of the observation optical system with the planar light L2, it is possible to acquire an image of the tomographic plane orthogonal to the observation axis P2 direction in the sample S by one imaging. Therefore, in order to acquire the three-dimensional information of the sample S, it is necessary to scan the sample S in the observation axis P2 direction and acquire images of a plurality of tomographic planes orthogonal to the observation axis P2 direction. In the sample observation device 100 according to such a comparative example, as shown in FIG. 6B, selection of the tomographic plane to be acquired (scanning and stopping of the sample S) is performed before the images of all the tomographic planes are acquired. ) And image acquisition had to be repeated. When the area where the observation target exists is wider than the imaging, the imaging field of view is selected by moving the stage in a direction different from the observation axis direction in addition to the operation of acquiring the cross-sectional image in the observation axis P2 direction. Operation was required.

On the other hand, in the sample observation device 1 according to the embodiment, as shown in FIG. 7A, the image acquisition unit 6 acquires an image while scanning the sample S with respect to the irradiation surface R of the planar light L2. Further, the observation axis P2 of the imaging optical system 5 is inclined with respect to the irradiation surface R of the planar light L2. Therefore, the image acquisition unit 6 can sequentially acquire partial image data of the fault plane in the optical axis P1 direction (Z-axis direction) of the planar light L2, and the image generation unit 8 can acquire a plurality of partial image data. The observation image data 32 of the sample S can be generated based on the above.

In this sample observation device 1, as shown in FIG. 7B, it is possible to sequentially acquire images while scanning the sample S. In the operation of the sample observation device 100 according to the comparative example, a time loss occurs due to the influence of inertia and the like each time the moving stage 12 is driven and stopped. On the other hand, in the sample observation device 1, the number of times of driving and stopping the moving stage 12 is reduced, and the scanning operation of the sample S and the image acquisition are simultaneously performed, so that the throughput until the observation image data 32 is obtained is improved. Be done.

Further, in the sample observation device 1, as shown in FIG. 2, the sample S is held by the sample container 11 having the input surface 15a of the planar light L2, and the optical axis P1 of the planar light L2 by the irradiation optical system 3 is held. It is arranged so as to be orthogonal to the input surface 15a of the sample container 11. Further, in the sample observation device 1, the scanning unit 4 scans the sample S in a direction (Y-axis direction) orthogonal to the optical axis P1 (Z-axis direction) of the planar light L2 by the irradiation optical system 3. This eliminates the need for image processing such as position correction of the partial image data acquired by the image acquisition unit 6, and facilitates the generation processing of the observation image data.

Further, in the sample observation device 1, the inclination angle θ of the observation axis P2 of the imaging optical system 5 with respect to the irradiation surface R of the planar light L2 in the sample S is 10 ° to 80 °, preferably 20 ° to 70 °. It is preferably 30 ° to 65 °. This point will be considered below.

FIG. 8 is a diagram showing an example of calculating the field of view in the sample observation device. In the example shown in the figure, the imaging optical system is located in the medium A having a refractive index n1, and the irradiation surface of the planar light is located in the medium B having a refractive index n2. The field of view in the imaging optical system is V, the irradiation surface is V', the inclination angle of the observation axis with respect to the irradiation surface is θ, the refraction angle at the interface between the media A and B is θ', and the medium A at the inclination angle θ of the field V. When the distance at the interface between the medium B and the medium B is L, the following equations (1) to (3) hold.
[Number 1]

L = V / cosθ ... (1)
[Number 2]

sinθ'= (n1 / n2) sinθ ... (2)
[Number 3]

V'= L / tanθ'... (3)

FIG. 9 is a diagram showing the relationship between the tilt angle of the observation axis and the resolution. In the figure, the horizontal axis is the tilt angle θ of the observation axis, and the vertical axis is the relative value V'/ V of the visual field. Then, the refractive index n1 of the medium A is set to 1 (air), and the value of V'/ V when the refractive index n2 of the medium B is changed from 1.0 to 2.0 in increments of 0.1 is set to the inclination angle θ. It is plotted against. The smaller the value of V'/ V, the higher the resolution in the depth direction of the sample (hereinafter referred to as "Z direction resolution"), and the larger the value, the lower the resolution in the Z direction.

From the results shown in FIG. 9, it can be seen that when the refractive index n1 of the medium A and the refractive index n2 of the medium B are equal, the value of V'/ V is inversely proportional to the inclination angle θ. Further, when the refractive index n1 of the medium A and the refractive index n2 of the medium B are different, it can be seen that the value of V'/ V draws a parabola with respect to the inclination angle θ. From this result, it can be seen that the resolution in the Z direction can be controlled by the refractive index of the arrangement space of the sample, the refractive index of the arrangement space of the imaging optical system, and the inclination angle θ of the observation axis. Then, it can be seen that in the range of the inclination angle θ of 10 ° to 80 °, better Z-direction resolution can be obtained as compared with the range of the inclination angle θ of less than 10 ° and more than 80 °.

Further, from the results shown in FIG. 9, it can be seen that the inclination angle θ at which the resolution in the Z direction is maximized tends to decrease as the difference between the refractive index n1 and the refractive index n2 increases. When the refractive index n2 is in the range of 1.1 to 2.0, the inclination angle θ at which the resolution in the Z direction is maximized is in the range of about 47 ° to about 57 °. For example, when the refractive index n2 is 1.33 (water), the inclination angle θ at which the resolution in the Z direction is maximized is estimated to be about 52 °. Further, for example, when the refractive index n2 is 1.53 (glass), the inclination angle θ at which the resolution in the Z direction is maximized is estimated to be about 48 °.

FIG. 10 is a diagram showing the relationship between the tilt angle of the observation axis and the stability of the visual field. In the figure, the horizontal axis is the tilt angle θ of the observation axis, and the vertical axis is the stability of the visual field. The stability is expressed by the ratio of the difference value between V'/ V at the tilt angle θ + 1 and V'/ V at the tilt angle θ-1 with respect to V'/ V at the tilt angle θ, and is expressed by the following equation (4). It is calculated based on. It can be evaluated that the closer the stability is to 0%, the smaller the change in the visual field with respect to the change in the tilt angle, and the more stable the visual field is. In FIG. 10, similarly to FIG. 9, the refractive index n1 of the medium A is 1 (air), and the refractive index n2 of the medium B is stable when changed from 1.0 to 2.0 in 0.1 steps. Degrees are plotted.
[Number 4]

Stability (%) = ((V'/ V) _{θ + 1-} (V'/ V) _θ-1 ) / (V'/ V) _θ ... (4)

From the results shown in FIG. 10, it can be seen that the stability exceeds ± 20% in the range where the inclination angle θ is less than 10 ° and exceeds 80 °, and it is difficult to control the visual field. On the other hand, when the inclination angle θ is in the range of 10 ° to 80 °, the stability is ± 20% or less, and the visual field can be controlled. Further, when the inclination angle θ is in the range of 20 ° to 70 °, the stability is ± 10% or less, and the visual field can be easily controlled.

FIG. 11 is a diagram showing the relationship between the tilt angle of the observation axis and the transmittance of the observation light from the sample. In the figure, the horizontal axis is the tilt angle θ of the observation axis, the vertical axis on the left side is the relative value of the field of view, and the vertical axis on the right side is the transmittance. In FIG. 11, in consideration of the holding state of the sample in the sample container, the refractive index n1 of the medium A is 1 (air), the refractive index n2 of the medium B is 1.53 (glass), and the refractive index n3 of the medium C is 1. It is set to .33 (water), and the value of the transmittance is the product of the transmittances of the interfaces of the media B and C and the interfaces of the media A and B. In FIG. 11, the transmittance of the P wave, the transmittance of the S wave, and the angle dependence of the average value thereof are plotted. Further, in FIG. 11, the relative values of the visual fields in the medium C are also plotted.

From the results shown in FIG. 11, it can be seen that the transmittance of the observation light from the sample to the imaging optical system becomes variable by changing the inclination angle θ of the observation axis. It can be seen that at least 50% or more transmittance can be obtained in the range where the inclination angle θ is 80 ° or less. Further, it can be seen that at least 60% or more transmittance is obtained in the range where the inclination angle θ is 70 ° or less, and at least 75% or more is obtained in the range where the inclination angle θ is 65 ° or less.

From the above results, when the Z-direction resolution of the sample is required, for example, the value of V'/ V, which is the relative value of the visual field, is 3 or less, the stability is less than 5%, and the transmittance of the observed light. It is preferable to select the inclination angle θ from the range of 30 ° to 65 ° so that (the average value of the P wave and the S wave) is 75% or more. When the Z-direction resolution of the sample is not required, the inclination angle θ may be appropriately selected from the range of 10 ° to 80 °, and from the viewpoint of securing the range of the visual field per pixel, 10 ° to 30 °. Alternatively, it is preferable to select from the range of 65 ° to 80 °.

The present invention is not limited to the above embodiment. For example, the optical axis P1 of the planar light L2 and the input surface 15a of the sample container 11 do not necessarily have to be orthogonal to each other, and the optical axis P1 of the planar light L2 and the scanning direction of the sample S by the scanning unit 4 are different. It does not necessarily have to be orthogonal.

Further, for example, in the above embodiment, the transparent member 15 is provided in the sample container 11 so as to close one end side of the well 13, and the planar light L2 is input from the input surface 15a of the transparent member 15. The planar light L2 may be input from the other end side of 13. In this case, the number of interfaces between media having different refractive indexes is reduced, and the number of refractions of the observation light L3 can be reduced. Further, instead of the sample container 11, the sample S may be held in a solid substance such as a gel, and the sample S may be moved by flowing a fluid such as water into the transparent container like a flow cytometer. May be good.

Further, a plurality of pairs of the imaging optical system 5 and the image acquisition unit 6 may be arranged. In this case, the observation range can be expanded, and a plurality of observation lights L3 having different wavelengths can be observed. Further, a plurality of image acquisition units 6 may be arranged with respect to the imaging optical system 5, or an image acquisition unit 6 may be arranged with respect to the plurality of imaging optical systems 5. The plurality of image acquisition units 6 may combine different types of photodetectors or image pickup devices. The light source 2 may be composed of a plurality of light sources that output light having different wavelengths. In this case, the sample S can be irradiated with excitation light having different wavelengths.

Further, in order to alleviate astigmatism, a prism may be arranged in the imaging optical system 5. In this case, for example, as shown in FIG. 12, the prism 41 may be arranged on the rear side of the objective lens 16 (between the objective lens 16 and the image acquisition unit 6). As a measure against defocusing, the imaging surface of the imaging device in the image acquisition unit 6 may be tilted with respect to the observation axis P2. In addition, for example, a dichroic mirror or a prism may be arranged between the imaging optical system 5 and the image acquisition unit 6 to separate the wavelengths of the observation light L3.

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 (imaging device), 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 (12)

An irradiation optical system that irradiates the sample with planar light,
A scanning unit that scans the sample against the surface illuminated by the planar light,
An imaging optical system having an observation axis inclined with respect to the irradiation surface and forming an image of the observation light generated in the sample by irradiation with the planar light.
An image acquisition unit that acquires a plurality of partial image data corresponding to a part of an optical image formed by the observation light imaged by the imaging optical system, and an image acquisition unit.
A sample observation device including an image generation unit that generates observation image data of the sample based on a plurality of partial image data generated by the image acquisition unit. The sample is held by a sample container having the planar light input surface.
The sample observation device according to claim 1, wherein the optical axis of the planar light by the irradiation optical system is arranged so as to be orthogonal to the input surface of the sample container. The sample observation device according to claim 1 or 2, wherein the scanning unit scans the sample in a direction orthogonal to the optical axis of the planar light by the irradiation optical system. The sample observation device according to any one of claims 1 to 3, wherein the angle of inclination of the observation axis of the imaging optical system with respect to the irradiation surface of the planar light is 10 ° to 80 °. The sample observation device according to any one of claims 1 to 4, wherein the angle of inclination of the observation axis of the imaging optical system with respect to the irradiation surface of the planar light is 20 ° to 70 °. The sample observation device according to any one of claims 1 to 5, wherein the angle of inclination of the observation axis of the imaging optical system with respect to the irradiation surface of the planar light is 30 ° to 65 °. The image acquisition unit includes a two-dimensional image pickup device, and extracts image data corresponding to a part of an optical image of the observation light from the data output from the two-dimensional image pickup device as the partial image data. The sample observation apparatus according to any one of 6. The sample observation device according to any one of claims 1 to 6, wherein the image acquisition unit includes a line sensor that captures a part of an optical image by the observation light and outputs the partial image data. The image acquisition unit includes a slit through which a part of the light image by the observation light is passed and a photodetector for detecting the light image passing through the slit, and is based on the data output from the photodetector. The sample observation device according to any one of claims 1 to 6, which generates the partial image data. The sample according to any one of claims 1 to 9, wherein the image generation unit generates observation image data of the sample on a plane orthogonal to the optical axis of the planar light based on the plurality of partial image data. Observation device. The sample observation apparatus according to any one of claims 1 to 10, further comprising an analysis unit that analyzes the observation image data and generates an analysis result. An irradiation step of irradiating the sample with planar light,
A scanning step of scanning the sample against the surface illuminated by the planar light,
An imaging step of imaging the observation light generated in the sample by irradiation of 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 partial image data corresponding to a part of an optical image formed by the observation light imaged by the imaging optical system, and an image acquisition step.
A sample observation method comprising an image generation step of generating observation image data of the sample based on the plurality of partial image data.

Images