WO2023007827A1

WO2023007827A1 - Sample observation device and sample observation method

Info

Publication number: WO2023007827A1
Application number: PCT/JP2022/012214
Authority: WO
Inventors: 諭山本; なつみ加藤; 春樹鈴木; 俊毅山田; 慎通茅根; 拓海岩村
Original assignee: 浜松ホトニクス株式会社
Priority date: 2021-07-26
Filing date: 2022-03-17
Publication date: 2023-02-02
Also published as: JPWO2023007827A1

Abstract

A sample observation device 1 comprises: an irradiation optical system 3 that irradiates a sample S with planar light L2; a scanning unit 4 that has a rotating shaft 12 for rotating the sample S at a predetermined angular speed with respect to an irradiation plane V of the planar light L2; an image formation optical system 5 that has an observation axis P2 inclined with respect to the irradiation plane V, and forms an image of observation light L3 generated by the sample S by irradiation with the planar light L2; an image acquisition unit 6 that acquires image data of a light image of the observation light L3 an image of which has been formed by the image formation optical system 5 while the sample S is being scanned; and an image generation unit 8 that, on the basis of the image data, generates observation image data 24 of the sample S.

Description

Specimen observation device and specimen observation method

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 samples with a three-dimensional structure such as cells. For example, a tomographic image observation apparatus described in Patent Document 1 discloses the basic principle of SPIM. In this conventional apparatus, a sample is irradiated with planar light, and fluorescence or scattered light generated inside the sample is imaged on an imaging plane to obtain observation image data of the inside of the sample.

As another sample observation device using planar light, there is a sample observation device described in Patent Document 2, for example. This sample observation apparatus includes an irradiation optical system that irradiates a sample with planar light, and a scanning unit that scans the sample with respect to the irradiation surface of the planar light. An imaging optical system that has an observation axis that is inclined with respect to the irradiation surface and that forms an image of observation light generated in the sample by the irradiation of the planar light; An image acquisition unit that acquires a plurality of partial image data corresponding to a part of an image, and an image generation unit that generates observation image data of the sample based on the plurality of partial image data generated by the image acquisition unit. .

JP-A-62-180241 JP 2018-063292 A

In the sample observation device of Patent Document 2 described above, in acquiring a three-dimensional image of the sample as observation image data, the sample is scanned on two axes with respect to the irradiation surface of the planar light. On the other hand, when actually observing a sample with a sample observing apparatus, a sample container such as a petri dish on which the sample is placed is often circular. Therefore, there has been a demand for the development of a technique that can efficiently scan a sample when acquiring observation image data.

The present disclosure has been made to solve the above problems, and aims to provide a sample observation device and a sample observation method that can efficiently scan a sample when acquiring observation image data.

A sample observation apparatus according to one aspect of the present disclosure includes an irradiation optical system that irradiates a sample with planar light, a scanning unit that has a rotating shaft that rotates the sample at a predetermined angular velocity with respect to the irradiation surface of the planar light, An imaging optical system that has an observation axis that is inclined with respect to the irradiation surface and forms an image of the observation light generated by the sample due to the irradiation of the planar light, and an observation that is imaged by the imaging optical system during scanning of the sample. An image acquisition unit that acquires image data of an optical image using light, and an image generation unit that generates observation image data of a sample based on the image data.

In this sample observation device, the sample is scanned with the planar light by rotating the sample around the rotation axis at a predetermined angular velocity with respect to the irradiation surface of the planar light. As a result, when the sample container in which the sample is placed has a circular shape, the sample can be scanned without waste, and the sample can be scanned more efficiently in acquiring observation image data.

When the width direction of the planar light is the X axis, the thickness direction of the planar light is the Y axis, the optical axis of the planar light is the Z axis, and the tangential direction of rotation of the rotation axis is the R axis, the image generation unit A plurality of XZ image data acquired by the image acquisition unit are integrated in the Z-axis direction to generate a plurality of X image data, and XY image data obtained by reconstructing the plurality of X image data in the R-axis direction are generated. It may be generated as observation image data. Thereby, XY image data having an arbitrary thickness at an arbitrary position in the Z-axis direction can be generated as observation image data. Also, the background of the generated observation image data can be suppressed.

The image generation unit corrects the amount of data assigned to each pixel based on at least the angular velocity of rotation of the rotation axis of the scanning unit and the field size of the image acquisition unit, thereby generating XY image data from a plurality of X image data. may be generated. When the sample S is scanned with respect to the planar light by the rotating shaft, even if the angular velocity of the rotating shaft is constant, the tangential velocity of each part of the sample S changes according to the distance from the rotating shaft. Therefore, by correcting the amount of data assigned to each pixel based on the angular velocity of the rotating shaft and the size of the field of view of the image acquisition unit, it is possible to appropriately convert a plurality of X image data into XY image data.

The rotation axis may be located within the cross section of the planar light. In this case, observation image data can be generated without complicating the correction of the amount of data assigned to each pixel.

When the width direction of the planar light is the X axis, the thickness direction of the planar light is the Y axis, and the optical axis of the planar light is the Z axis, the rotation axis is the XZ plane including the X axis and the Z axis, and It may be parallel to at least one of the YZ planes including the Y axis and the Z axis. In this case, observation image data can be generated without complicating the correction of the amount of data assigned to each pixel.

A sample observation method according to one aspect of the present disclosure includes an irradiation step of irradiating a sample with planar light, a scanning step of rotating the sample around a rotation axis at a predetermined angular velocity with respect to the irradiation surface of the planar light, and an irradiation step. An image forming step of forming an image of observation light generated in a sample by irradiation with planar light using an image forming optical system having an observation axis inclined with respect to the plane; an image acquisition step of acquiring image data of an optical image by the observed light; and an image generating step of generating observation image data of the sample based on the image data.

In this specimen observation method, the specimen is scanned with the planar light by rotating the specimen at a predetermined angular velocity around the rotation axis with respect to the irradiation surface of the planar light. As a result, when the sample container in which the sample is placed has a circular shape, the sample can be scanned without waste, and the sample can be scanned more efficiently in acquiring observation image data.

Assuming that the width direction of the planar light is the X axis, the thickness direction of the planar light is the Y axis, the optical axis of the planar light is the Z axis, and the tangential direction of the rotation of the rotation axis is the R axis, in the image generation step, A plurality of XZ image data acquired in the image acquisition step are integrated in the Z-axis direction to generate a plurality of X image data, and an XY image data obtained by reconstructing the plurality of X image data in the R-axis direction is observed. It may be generated as image data. Thereby, XY image data having an arbitrary thickness at an arbitrary position in the Z-axis direction can be generated as observation image data. Also, the background of the generated observation image data can be suppressed.

In the image generation step, XY image data is generated from a plurality of X image data by correcting the amount of data assigned to each pixel based on at least the angular velocity of the rotation axis in the scanning step and the field size in the image acquisition step. You may When the sample S is scanned with respect to the planar light by the rotating shaft, even if the angular velocity of the rotating shaft is constant, the tangential velocity of each part of the sample S changes according to the distance from the rotating shaft. Therefore, by correcting the amount of data assigned to each pixel based on the angular velocity of the rotating shaft and the size of the field of view of the image acquisition unit, it is possible to appropriately convert a plurality of X image data into XY image data.

In the scanning step, the rotation axis may be positioned within the cross section of the planar light. In this case, observation image data can be generated without complicating the correction of the amount of data assigned to each pixel.

The width direction of the planar light is the X axis, the thickness direction of the planar light is the Y axis, and the optical axis of the planar light is the Z axis. It may be parallel to at least one of the YZ planes including the Z axis. In this case, observation image data can be generated without complicating the correction of the amount of data assigned to each pixel.

According to the present disclosure, it is possible to make the scanning of the sample more efficient when acquiring observation image data.

[Correction under Rule 91 30.03.2022]
1 is a schematic configuration diagram showing an embodiment of a sample observation device according to the present disclosure; FIG. 2 is a flow chart showing an example of a sample observation method using the sample observation apparatus shown in FIG. 1; (A) is a schematic diagram showing an example of the positional relationship between the rotation axis and planar light when viewed from the Z-axis direction, and (B) and (C) when viewed from the Z-axis direction. FIG. 10 is a schematic diagram showing another example of the arrangement relationship between the rotational axis of and planar light. (A) is a schematic diagram showing an example of the positional relationship between the rotation axis and planar light when viewed from the Y-axis direction; It is a typical figure which shows an example of the arrangement|positioning relationship with light. (A) is a schematic diagram showing another example of the arrangement relationship between the rotation axis and planar light when viewed from the Y-axis direction; FIG. 11 is a schematic diagram showing another example of the arrangement relationship with the shape light. FIG. 4 is a schematic diagram showing a plurality of XZ image data acquired by an image acquisition unit; FIG. 4 is a schematic diagram showing how XY image data (observation image data) is generated from a plurality of XZ image data; FIG. 4 is a schematic diagram showing the amount of data assigned to each pixel of the image acquisition unit; (A) is a schematic diagram showing the relationship between the irradiation surface of planar light with respect to the sample and the observation axis, and (B) is a schematic diagram showing the relationship between the scanning operation and image acquisition in this mode. It is a diagram. (A) and (B) are schematic diagrams showing modifications of the positional relationship between the rotation axis and the planar light when viewed from the Z-axis direction. (A) and (B) are schematic diagrams showing another modification of the positional relationship between the rotation axis and the planar light when viewed from the Z-axis direction. FIG. 11 is a schematic diagram showing still another modified example of the positional relationship between the rotation axis and the planar light when viewed from the Z-axis direction;

Preferred embodiments of a sample observation device and a sample observation method according to one aspect of the present disclosure will be described below in detail with reference to the drawings.

FIG. 1 is a schematic configuration diagram showing one embodiment of the sample observation device according to the present disclosure. This sample observation apparatus 1 is an apparatus that acquires observation image data of the inside of the sample S by irradiating the sample S with planar light L2 and forming an image of fluorescence or scattered light generated inside the sample S on an imaging plane. It is configured. A sample observation apparatus 1 of this type may be a slide scanner that acquires and displays an image of a sample S held on a slide glass, or acquires image data of a sample S held on a microplate and analyzes the image data. A plate reader etc. are mentioned. Examples of the sample S to be observed include human or animal cells, tissues, organs, animal or plant itself, and plant cells and tissues. Moreover, the sample S may be contained in a solution, a gel, or a substance having a different refractive index from that of the sample S.

As shown in FIG. 1, the sample observation apparatus 1 includes a light source 2, an irradiation optical system 3, a scanning section 4, an imaging optical system 5, an image acquisition section 6, and a computer 7. there is The light source 2 is a light source that outputs light L1 with which the sample S is irradiated. Examples of the light source 2 include laser light sources such as laser diodes and solid-state laser light sources. Also, the light source 2 may be a light-emitting diode, a super-luminescent diode, or a lamp-based light source. Light L<b>1 output from the light source 2 is guided to the irradiation optical system 3 .

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 may be referred to as the optical axis of the planar light L2. The irradiation optical system 3 includes a light 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 including an objective lens. The sample S is irradiated with the planar light L2 formed by the irradiation optical system 3 . In the sample S irradiated with the planar light L2, the observation light L3 is generated on the irradiation surface V 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 diffuse reflected light of the planar light L2.

When observing the sample S in the thickness direction, the planar light L2 is preferably thin planar light with 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 with a thickness equal to or less than the resolution in the Z-axis direction, the thickness of the planar light L2 does not affect the resolution. Therefore, planar light L2 having a thickness exceeding 2 mm may be used.

The scanning unit 4 is a mechanism that scans the sample S with respect to the irradiation surface V of the planar light L2. In this embodiment, the scanning unit 4 has a rotating shaft 12 that rotates a sample container 11 holding the sample S at a predetermined angular velocity. The sample container 11 is configured by, for example, a petri dish having a circular shape in plan view. The sample container 11 is made of a member having transparency to the planar light L2. Examples of such members include glass, quartz, and synthetic resin. The sample container 11 is attached to the rotating shaft 12 so that the input surface of the planar light L2 (here, the bottom surface of the petri dish) is orthogonal to the optical axis P1 of the planar light L2.

The scanning unit 4 scans the sample container 11 with respect to the planar light L2 by rotating the rotation shaft 12 in a preset rotation direction according to the control signal from the computer 7 . In the following description, the width direction of the planar light L2 is the X axis, the thickness direction of the planar light L2 is the Y axis, the optical axis P1 direction of the planar light L2 is the Z axis, and the scanning direction of the sample S by the scanning unit 4. (the tangential direction of the rotation of the rotating shaft 12) is called the R-axis. The irradiation surface V of the planar light L2 with respect to the sample S is a surface within the XZ plane.

The imaging optical system 5 is an optical system that forms an image of the observation light L3 generated at the sample S by the irradiation of the planar light L2. The imaging optical system 5 includes, for example, an objective lens 16, a bandpass filter 17, a relay lens 18, and the like. The optical axis of the imaging optical system 5 is the optical axis of the observation light L3 (hereinafter "observation axis P2"). The observation axis P2 is inclined with respect to the irradiation plane V of the sample S with the planar light L2 at an inclination angle θ. The tilt angle θ also coincides with the angle formed by the optical axis P1 of the planar light L2 directed toward the sample S and the observation axis P2. The inclination angle θ is, for example, 10° to 80°. From the viewpoint of improving the resolution of the observed image, the tilt angle θ is preferably 20° to 70°. Further, from the viewpoint of improving the resolution of the observed image and stabilizing the field of view, the tilt angle θ is more preferably 30° to 65°.

The image acquisition unit 6 is a device that acquires image data of an optical image formed by the observation light L3 formed by the imaging optical system 5 while the sample S is being scanned. The image acquisition unit 6 includes, for example, an imaging device that captures an optical image using the observation light L3. Examples of imaging devices include area image sensors such as CMOS image sensors and CCD image sensors. These area image sensors are arranged on the imaging plane of the imaging optical system 5 and output two-dimensional image data to the computer 7 . The readout method of the imaging device may be a global shutter method in which the exposure period of each pixel row is the same, or may be a rolling shutter method in which the exposure period of each pixel row is shifted by a predetermined time.

The computer 7 is physically configured with memories such as RAM and 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. Such computers 7 include, for example, personal computers, cloud servers, smart devices (smartphones, tablet terminals, etc.). The computer 7 includes a controller that controls the operation of the light source 2 and the rotating shaft 12, an image generator 8 that generates observation image data of the sample S, and an observation It functions as an analysis unit 10 that analyzes image data.

The computer 7 as a controller receives the user's operation to start measurement, and drives the light source 2, the scanning unit 4, and the image acquisition unit 6 in synchronization. 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 rotating shaft 12. ON/OFF of the output of the light L1 may be controlled. If the irradiation optical system 3 includes an optical shutter (not shown), the computer 7 may turn on/off the irradiation of the sample S with the planar light L2 by controlling the optical shutter.

The computer 7 as the image generation unit 8 generates observed image data of the sample S based on the 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) perpendicular to the optical axis P1 of the planar light L2, for example, based on the image data output from the image acquisition unit 6 . The image generation unit 8 stores the generated observation image data and displays it on a monitor or the like, for example, according to a predetermined operation by the user.

FIG. 2 is a flow chart 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 planar light L2. 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 the light source 2 outputs light L1. The light L1 output from the light source 2 is shaped by the irradiation optical system 3 to become planar light L2, and the sample S is irradiated with the planar light L2.

In the scanning step S02, the sample S is scanned with respect to the irradiation surface V of the planar light L2. When the user inputs an operation to start measurement, the rotating shaft 12 rotates in synchronization with the driving of the light source 2 based on the control signal from the computer 7 . As a result, the sample container 11 rotates in the R-axis direction at a predetermined angular velocity, and the sample S in the sample container 11 is scanned with respect to the irradiation surface V of the planar light L2.

In the irradiation step S01 and the scanning step S02, it is preferable to arrange the irradiation optical system 3 and the scanning unit 4 so that the rotating shaft 12 is positioned within the cross section of the planar light L2. In the example of FIG. 3A, the center C of the sample container 11 and the rotation axis 12 are aligned in plan view from the Z-axis direction. The width W of the planar light L2 is, for example, the same as the radius r of the sample container 11 or slightly larger than the radius r of the sample container 11 . The planar light L2 extends radially from the center C of the sample container 11 so as to straddle the rotating shaft 12 and the edge of the sample container 11 in plan view in the Z-axis direction. Therefore, in the example of FIG. 3A, the scanning of the sample S with the planar light L2 is completed when the sample container 11 makes one rotation about the rotating shaft 12 .

The rotating shaft 12 does not necessarily have to be positioned within the cross section of the planar light L2. For example, as shown in FIG. 3B, the rotating shaft 12 may be displaced from the center C of the sample container 11 in plan view in the Z-axis direction. In the example of FIG. 3B, the planar light L2 extends radially from the center C of the sample container 11 so as to straddle the center C and the edge of the sample container 11, while the rotation axis 12 is It is slightly shifted in the Y-axis direction with respect to the center C of the sample container 11 . Further, as shown in FIG. 3C, the planar light L2 may be displaced from the center C of the sample container 11 in a plan view in the Z-axis direction. In the example of FIG. 3C, while the rotation axis 12 is aligned with the center C of the sample container 11, the planar light L2 is slightly shifted in the Y-axis direction with respect to the center C of the sample container 11. there is

In the configurations shown in FIGS. 3B and 3C, when the shift amount of the rotating shaft 12 with respect to the center C or the shift amount of the planar light L2 with respect to the center C is d, the shift amount d is Accordingly, there may be a region in the vicinity of the edge of the sample container 11 that is not irradiated with the planar light L2. In this case, by making the width W of the planar light L2 longer than the radius r of the sample container 11 by at least d (W≧r+d), the area of the sample container 11 not irradiated with the planar light L2 can be reduced. In addition, in the configuration shown in FIG. 3B or 3C, there may be a region around the rotating shaft 12 that is not irradiated with the planar light L2. In this case, by grasping in advance the amount of deviation of the rotating shaft 12 with respect to the center C of the sample container 11, in the image generation step S05 described later, an image is generated of the area around the rotating shaft 12 which is not irradiated with the planar light L2. A correction can be made to exclude from

In the irradiation step S01 and the scanning step S02, the rotation axis 12 is preferably parallel to at least one of the XZ plane including the X and Z axes and the YZ plane including the Y and Z axes. . In this embodiment, as shown in FIG. 4A, when the planar light L2 is viewed from the Y-axis direction, the rotation axis 12 and the optical axis P1 of the planar light L2 are parallel. Further, as shown in FIG. 4B, when the planar light L2 is viewed from the X-axis direction, the rotation axis 12 and the optical axis P1 of the planar light L2 are parallel. That is, in this embodiment, the rotating shaft 12 is parallel to both the XZ plane and the YZ plane.

The rotating shaft 12 may be non-parallel to one of the XZ plane and the YZ plane. For example, as shown in FIG. 5A, when the planar light L2 is viewed from the Y-axis direction, the optical axis P1 of the planar light L2 obliquely intersects the rotation axis 12, while ), when the planar light L2 is viewed from the X-axis direction, the rotation axis 12 and the optical axis P1 of the planar light L2 may be parallel. Also, although not shown, in contrast to FIGS. 5A and 5B, when the planar light L2 is viewed from the Y-axis direction, the rotation axis 12 and the optical axis P1 of the planar light L2 are aligned. While being parallel, the optical axis P1 of the planar light L2 may obliquely cross the rotation axis 12 when the planar light L2 is viewed from the X-axis direction.

Even with such a configuration, by grasping in advance the oblique angle of the optical axis of the planar light L2 with respect to the rotation axis 12, the amount of data assigned to each pixel is corrected in the image generation step S05 described later. can do. As described above, when the optical axis P1 of the planar light L2 is oblique to the rotation axis 12 when viewed from the X-axis direction, the observation light L3 from the sample S is inclined in the Y-axis direction. A light image of the observation light L3 can be acquired at a position deviated from the coordinates of the sample S of . Therefore, in the image generation step S05, which will be described later, when generating the X image data 22 from the XZ image data 21, each pixel is in the tilt axis direction tilted from the Z axis direction by the oblique angle of the optical axis of the planar light L2. By accumulating the luminance values, the amount of data assigned to each pixel can be corrected. Also, by generating two-dimensional image data in consideration of the projection width of the observation light L3 in the Y-axis direction instead of the X image data 22, the amount of data assigned to each pixel can be corrected.

In the imaging step S03, the imaging optical system 5 having the observation axis P2 inclined with respect to the irradiation surface V 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 plane. In the image acquisition step S04, as shown in FIG. 6, a plurality of XZ image data 21 corresponding to the optical image formed by the observation light L3 formed by the imaging optical system 5 during scanning of the sample S are acquired in the R-axis direction. . A plurality of XZ image data 21 are sequentially output from the image acquisition section 6 to the image generation section 8 .

In the image generation step S05, observation image data of the sample S is generated based on a plurality of XZ image data 21. Here, as shown in FIG. 7, the brightness value of each pixel included in the plurality of XZ image data 21 obtained in the image acquisition step S04 is integrated in the Z-axis direction to generate a plurality of X image data 22. FIG. XY image data 23 is generated by reconstructing the plurality of X image data 22 in the R-axis direction, and the XY image data 23 is generated as observed image data 24 of the sample S. FIG.

In generating the X image data 22, the luminance values of each pixel in an arbitrary range in the Z-axis direction in the plurality of XZ image data 21 may be integrated in the Z-axis direction. In generating the XY image data 23 from the plurality of X image data 22, the XY image data 23 may be directly generated by reconstructing the plurality of X image data 22. Combined XR image data may be generated, and the XY image data 23 may be generated by reconstructing the XR image data.

In the image generation step S05, when generating the XY image data 23 from the plurality of X image data 22, each pixel is assigned based on at least the angular velocity of the rotating shaft 12 in the scanning unit 4 and the field size of the image acquisition unit 6. Correct the amount of data. In the present embodiment, since the sample S is scanned with respect to the planar light L2 by the rotation shaft 12, even if the angular velocity of the rotation shaft 12 is constant, the tangential velocity of each part of the sample S changes from the center of the rotation shaft 12 to It will change according to the distance of In correcting the amount of data assigned to each pixel, the shift amount d of the rotation axis 12 with respect to the planar light L2, the exposure time of the image acquisition section 6, the resolution, etc. may be further taken into consideration. When the rotation axis 12 is non-parallel to one of the XZ plane and the YZ plane, the oblique angle of the optical axis P1 of the planar light L2 with respect to the rotation axis 12 may be further taken into consideration.

In FIG. 8 , the n-th to n+2-th X image data 22 are shown in association with the pixels 25 of the image acquisition unit 6 . As shown in FIG. 8, the data amount [data/pix] per pixel is larger for pixels closer to the position corresponding to the rotation axis 12 and smaller for pixels farther from the position corresponding to the rotation axis 12 . In the example of FIG. 8, in the _fourth pixel 254 as seen from the position corresponding to the rotation axis 12, the data amount per pixel is 3.2 [data/pix]. The data amount per pixel is 1 [data/pix] at the _11th pixel 25-11 when viewed from the position corresponding to . Therefore, by correcting (for example, dividing) the amount of data assigned to each pixel 25 based on the angular velocity of the rotating shaft 12 and the size of the field of view of the image acquisition unit 6, the plurality of X image data 22 are converted to the XY image data 23. Appropriate conversions can be performed.

In the analysis step S06, the analysis unit 10 analyzes the observation image data 24 and generates an analysis result. For example, in drug discovery screening, a sample S and a reagent are placed in the sample container 11 and observation image data 24 are acquired. Then, the analysis unit 10 evaluates the reagent based on the observed image data 24 and generates evaluation data as an analysis result.

In the image formation step S03 and the image acquisition step S04 described above, as shown in FIG. In this state, the image acquisition unit 6 acquires an image while scanning the sample S against the irradiation surface V of the planar light L2. Therefore, the image acquisition unit 6 can sequentially acquire the XZ image data 21 of the tomographic plane in the optical axis P1 direction (Z-axis direction) of the planar light L2, and the image generation unit 8 can generate a plurality of XZ images. Observation image data 24 of the sample S can be generated based on the data 21 .

For example, in the apparatus configuration (comparative example) having an observation axis orthogonal to the irradiation plane V of the planar light L2, selection of a tomographic plane for acquiring an image ( scanning and pausing) and image acquisition must be repeated. Further, when the area where the observation target exists is larger than the imaging area, in addition to the operation of acquiring the cross-sectional image in the observation axis direction, the operation of selecting the imaging field of view by moving the stage in a direction different from the observation axis direction. need to do On the other hand, when adopting a structure in which the observation axis P2 of the imaging optical system 5 is inclined with respect to the irradiation surface V of the planar light L2 as in the sample observation apparatus 1, as shown in FIG. , image acquisition can be performed sequentially while the sample S is continuously scanned. By reducing the number of times the rotating shaft 12 is driven and stopped, and by performing the scanning operation of the sample S and the acquisition of the image at the same time, the throughput until the observation image data 24 is obtained can be improved.

As described above, in the sample observation apparatus 1, the sample S rotates around the rotation axis 12 at a predetermined angular velocity with respect to the irradiation surface V of the planar light L2, thereby scanning the sample S with the planar light L2. done. As a result, when the sample container 11 in which the sample S is placed has a circular shape, the sample S can be scanned without waste, and the scanning of the sample S can be made efficient in obtaining the observation image data 24. .

In the sample observation device 1, when the width direction of the planar light L2 is the X axis, the optical axis P1 of the planar light L2 is the Z axis, and the tangential direction of the rotation of the rotating shaft 12 by the scanning unit 4 is the R axis, an image is generated. A unit 8 integrates a plurality of XZ image data 21 acquired by the image acquisition unit 6 in the Z-axis direction to generate a plurality of X image data 22, and combines the plurality of X image data 22 in the R-axis direction. The obtained XY image data 23 is generated as observation image data 24 . Thereby, XY image data 23 having an arbitrary thickness at an arbitrary position in the Z-axis direction can be generated as observation image data 24 . Also, the background of the generated observation image data 24 can be suppressed.

In the sample observing apparatus 1, a plurality of X image data 22 are obtained by correcting the amount of data assigned to each pixel based on at least the angular velocity of rotation of the rotating shaft 12 in the scanning section 4 and the field size of the image acquisition section 6. XY image data 23 is generated from the When the sample S is scanned with respect to the planar light L2 by the rotating shaft 12, even if the angular velocity of the rotating shaft 12 is constant, the tangential velocity of each part of the sample S changes according to the distance from the rotating shaft 12. It will happen. Therefore, by correcting the amount of data assigned to each pixel based on the angular velocity of the rotating shaft 12 and the size of the field of view of the image acquisition unit 6, the plurality of X image data 22 are appropriately converted into the XY image data 23. can.

In the sample observation device 1, the rotating shaft 12 is positioned within the cross section of the planar light L2. Further, the rotation axis is parallel to both the width direction (X-axis) of the planar light L2 and the thickness direction (Y-axis) of the planar light L2. This makes it possible to generate the observed image data 24 without complicating the correction of the amount of data assigned to each pixel.

The present disclosure is not limited to the above embodiments. For example, in the above embodiment, the width W of the planar light L2 is approximately the same as the radius r of the sample container 11. However, as shown in FIG. , or may be slightly larger than the diameter 2r of the sample container 11 . If the width W of the planar light L2 is approximately the same as the radius r of the sample container 11, the resolution of the observation image data 24 can be improved. , the scanning of the sample S with the planar light L2 is completed by rotating the sample container 11 halfway around the rotating shaft 12, so that the time required for scanning the sample S can be shortened. .

When the width W of the planar light L2 is approximately the same as the diameter 2r of the sample container 11, two

image acquisition units

6A and 6B may be used as shown in FIG. 10(B). In the example of FIG. 10B, the

image acquisition units

6A and 6B are arranged at rotationally symmetrical (two-fold symmetrical) positions with respect to the planar light L2 passing through the center C of the sample container 11 . In the configuration of FIG. 10A, one image acquisition unit 6 captures the optical image of the observation light L3 corresponding to the entire width W of the planar light L2, so the image size is large. On the other hand, in the configuration of FIG. 10B, since the two

image acquisition units

6A and 6B capture the optical image of the observation light L3 corresponding to the entire width W of the planar light L2, the image size can be made relatively small. , high-speed reading becomes possible. Therefore, the time required for scanning the sample S can be shortened.

Also, in the above embodiment, the single planar light L2 is used to scan the sample S, but a plurality of planar lights L2 may be used to scan the sample S. In the example of FIG. 11A, two planar lights L2A and L2B with different wavelengths and two

image acquisition units

6A and 6B with different detection wavelength bands are used. Both of the planar lights L2A and L2B have a width W of the planar light L2 that is approximately the same as the radius r of the sample container 11, and are 90° The sample container 11 is irradiated with the phase angle. The image acquisition unit 6A acquires the XZ image data 21 of the optical image based on the observation light L3 generated on the sample S by the irradiation of the planar light L2A, and the image acquisition unit 6B acquires the XZ image data 21 on the sample S by irradiation of the planar light L2B. XZ image data 21 of an optical image based on the generated observation light L3 is acquired.

In the example of FIG. 11(B), the planar lights L2A and L2B are arranged around the rotation axis 12 with a phase angle of 180° in plan view from the Z-axis direction. That is, in a plan view in the Z-axis direction, the planar lights L2A and L2B are arranged to irradiate the sample container 11 as linear light passing through the rotating shaft 12 . The two

image acquisition units

6A and 6B are arranged at rotationally symmetrical (two-fold symmetrical) positions with the planar lights L2A and L2B passing through the center C of the sample container 11 interposed therebetween.

Alternatively, as shown in FIG. 12, a plurality of sample containers 11 may be scanned on a circular orbit F around the rotation axis 12. In the example of FIG. 12, four sample containers 11 are arranged on a circular orbit F around the rotating shaft 12 . The planar light L2 has a width slightly larger than the diameter 2r of the sample container 11 . The radius rr of the circular orbit F is not particularly limited, but is appropriately set according to the diameter of the sample container 11 and the number of sample containers 11 arranged on the circular orbit F, for example. The planar light L2 is irradiated toward the sample container 11 at a predetermined point on the circular orbit F so as to be perpendicular to the tangent line of the circular orbit F at the predetermined point. Rotation of the rotating shaft 12 causes the sample container 11 scanned on the circular orbit F to pass the position of the planar light L2, so that the sample S in the sample container 11 is aligned with the irradiation surface V of the planar light L2. Scanned. According to this aspect, the plurality of sample containers 11 can be continuously scanned with the planar light L2, thereby improving the throughput until obtaining the observation image data 24 of the plurality of samples S. .

DESCRIPTION OF SYMBOLS 1... Sample observation apparatus 3... Irradiation optical system 4... Scanning part 5... Imaging optical system 6... Image acquisition part 8... Image generation part 12... Rotating shaft 21... XZ image data (image data) , 22... X image data, 23... XY image data, 24... Observation image data, L2... Planar light, L3... Observation light, P2... Observation axis, S... Sample, V... Irradiation surface.

Claims

an irradiation optical system for irradiating a sample with planar light;
a scanning unit having a rotating shaft for rotating the sample at a predetermined angular velocity with respect to the irradiation surface of the planar light;
an imaging optical system that has an observation axis that is inclined with respect to the irradiation surface and that forms an image of observation light generated in the sample by irradiation with the planar light;
an image acquisition unit that acquires image data of an optical image formed by the imaging optical system during scanning of the sample by the observation light;
and an image generator that generates observation image data of the sample based on the image data.
When the width direction of the planar light is the X axis, the thickness direction of the planar light is the Y axis, the optical axis of the planar light is the Z axis, and the tangential direction of the rotation of the rotation axis is the R axis,
The image generation unit integrates the plurality of XZ image data acquired by the image acquisition unit in the Z-axis direction to generate a plurality of X image data, and reconstructs the plurality of X image data in the R-axis direction. 2. A sample observation apparatus according to claim 1, wherein the XY image data obtained by said observation image data is generated as said observation image data.
The image generation unit corrects the amount of data assigned to each pixel based on at least the angular velocity of the rotation axis in the scanning unit and the field size of the image acquisition unit, thereby converting the plurality of X image data into the 3. A specimen observing apparatus according to claim 2, which generates XY image data.
The sample observation apparatus according to any one of claims 1 to 3, wherein the rotating shaft is positioned within the cross section of the planar light.
When the width direction of the planar light is the X axis, the thickness direction of the planar light is the Y axis, and the optical axis of the planar light is the Z axis,
5. The rotation axis is parallel to at least one of an XZ plane including the X axis and the Z axis and a YZ plane including the Y axis and the Z axis. The sample observation device according to item 1.
an irradiation step of irradiating the sample with planar light;
a scanning step of rotating the sample around a rotation axis at a predetermined angular velocity with respect to the irradiation surface of the planar light;
an image forming step of forming an image of the observation light generated in the sample by the irradiation of the planar light using an image forming optical system having an observation axis inclined with respect to the irradiation surface;
an image acquisition step of acquiring image data of an optical image of the observation light formed by the imaging optical system during scanning of the sample;
and an image generating step of generating observation image data of the sample based on the image data.
When the width direction of the planar light is the X axis, the thickness direction of the planar light is the Y axis, the optical axis of the planar light is the Z axis, and the tangential direction of the rotation of the rotation axis is the R axis,
In the image generation step, the plurality of XZ image data acquired in the image acquisition step are integrated in the Z-axis direction to generate a plurality of X image data, and the plurality of X image data are reconstructed in the R-axis direction. 7. A sample observation method according to claim 6, wherein the obtained XY image data is generated as said observation image data.
In the image generating step, the plurality of X images are generated by correcting the amount of data assigned to each pixel based on at least the angular velocity of rotation of the rotation axis in the scanning step and the field size in the image acquiring step. 8. A sample observation method according to claim 7, wherein the XY image data is generated from the data.
The sample observation method according to any one of claims 6 to 8, wherein in the scanning step, the rotation axis is positioned within the cross section of the planar light.
When the width direction of the planar light is the X axis, the thickness direction of the planar light is the Y axis, and the optical axis of the planar light is the Z axis,
10. The scanning step is parallel to at least one of an XZ plane including the X axis and the Z axis and a YZ plane including the Y axis and the Z axis. Specimen observation method described.