WO2021166069A1 - Light-sheet microscope, control method, and program - Google Patents

Abstract

This light-sheet microscope (1) is provided with: a lighting optical system (20); an observation optical system (30); a parallel-planar plate (11); and a control device (80). The lighting optical system (20) comprises an objective lens (21) having a first optical axis, and forms a light sheet on a sample (S). The observation optical system (30) comprises an objective lens (31) having a second optical axis intersecting with the first optical axis, and forms an optical image of the sample (S) on the basis of light coming from the sample (S). The parallel-planar plate (11) regulates the incident position of light entering the objective lens (21) in a direction intersecting with the first optical axis. The control device (80) controls the parallel-planar plate (11) on the basis of the refractive indices of two or more media so as to make an observation plane and the light sheet parallel to each other. The two or more media have mutually different refractive indices, are arranged on a light path extending from the objective lens (21) up to the objective lens (31) by way of the sample (S), and each have, on the optical path, an interface so as not to be orthogonal to the first axis or the second axis.

Description

Lightsheet microscope, control method, and program

The disclosure of this specification relates to a light sheet microscope, a control method, and a program.

In recent years, research using cultured cells such as spheroids and organoids has attracted attention. As a method for observing the internal structure of such a three-dimensional sample, there is known a method in which a sample is subjected to a transparent treatment and the transparent sample is observed using a light sheet microscope. This method is expected to be applied not only to basic biological research but also to drug discovery screening and clinical diagnosis.

In a light sheet microscope, a sample contained in a container is illuminated via the side surface of the container and observed via the bottom surface of the container by using an illumination objective lens and an observation objective lens having optical axes orthogonal to each other. The configuration is generally adopted. However, in the configuration in which the sample is illuminated from the side surface of the container, the containers that can be used are limited. For example, the use of a container having a side surface having a low transmittance or a container having a side surface having a curved surface is not desirable because it causes deterioration of lighting performance. Also, a container consisting of a plurality of horizontally aligned elements, such as a multi-well plate, is not suitable for use in a light sheet microscope having a general configuration for the same reason.

Such restrictions on the container can be avoided by using, for example, a light sheet microscope as described in Patent Document 1. Patent Document 1 describes a light sheet microscope that illuminates and observes light through the bottom surface of a container by obliquely incident light on the bottom surface of the container.

Japanese Unexamined Patent Publication No. 2014-202967

In a light sheet microscope, a good observation image can be obtained by matching the observation surface to be observed via the observation objective lens with the light sheet formed by the illumination objective lens.

However, in the light sheet microscope described in Patent Document 1, since light is obliquely incident on the bottom surface of the container, when the refractive index of the medium in contact with the container (for example, the culture solution and the immersion liquid) is different, the light sheet is used. The parallel relationship between the observation surfaces is broken, and the observation image is significantly deteriorated.

From the above circumstances, an object of the present invention is to provide a technique for obtaining a good observation image by using a light sheet microscope regardless of the shape of the container and the difference in the refractive index between the media in contact with the container. be.

The light sheet microscope according to one aspect of the present invention includes a first objective lens having a first optical axis, and has an illumination optical system that forms a light sheet on a sample and a second optical axis that intersects the first optical axis. An observation optical system that includes a second objective lens and forms an optical image of the sample based on the light from the sample, and an incident position of light incident on the first objective lens intersects the first optical axis. A first adjusting unit that adjusts in a direction and a control unit that makes the observation surface parallel to the light sheet by controlling the first adjusting unit based on the refractive index of two or more media. The above-mentioned medium is a medium having different refractive indexes and arranged on an optical path from the first objective lens to the second objective lens via the sample, and the first light is on the optical path. It includes a control unit that has an interface that is not orthogonal to either the axis and the second optical axis.

The control method according to one aspect of the present invention includes an illumination optical system that forms a light sheet on a sample, including a first objective lens having a first optical axis, and a second optical axis that intersects the first optical axis. A control method for a light sheet microscope including an observation optical system that forms an optical image of the sample based on light from the sample, including a second objective lens. This control method is a step of acquiring the refractive index of two or more media, and the two or more media are on the optical path from the first objective lens to the second objective lens via the sample. A step of having media having different refractive indexes from each other and having an interface on the optical path that is not orthogonal to either the first optical axis and the second optical axis, and the two or more media. This includes a step of making the observation surface parallel to the light sheet by adjusting the incident position of the light incident on the first objective lens in a direction intersecting the first optical axis based on the refractive index of the first objective lens.

A program according to one aspect of the present invention includes an illumination optical system that forms a light sheet on a sample, including a first objective lens having a first optical axis, and a second optical axis that intersects the first optical axis. A process of acquiring the refractive indexes of two or more media in a light sheet microscope including an observation optical system that forms an optical image of the sample based on light from the sample, including two objective lenses. The two or more media are media having different refractive indexes arranged on the optical path from the first objective lens to the second objective lens via the sample, and are said to be on the optical path. Based on the process of having an interface that is not orthogonal to either the first optical axis and the second optical axis and the refractive index of the two or more media, the position of light incident on the first objective lens is determined by the first. The process of making the observation surface parallel to the light sheet by adjusting in the direction intersecting the optical axis is executed.

According to the above aspect, a good observation image can be obtained by using a light sheet microscope regardless of the shape of the container and the difference in the refractive index between the media in contact with the container.

It is a figure for demonstrating the arrangement of the objective lens in a light sheet microscope. It is a figure for demonstrating the influence which light passing obliquely through an interface has on a light sheet microscope. It is another figure for demonstrating the influence which light passing obliquely through an interface has on a light sheet microscope. It is a figure which illustrated the structure of the light sheet microscope which concerns on 1st Embodiment. It is a figure which illustrated the structure of the control device shown in FIG. It is a figure which illustrated the structure of the light sheet microscope which concerns on 2nd Embodiment. It is a figure which illustrated the structure of the light sheet microscope which concerns on 3rd Embodiment. It is a figure which illustrated the structure of a varifocal lens. It is a figure which showed the modification of the multi-well plate.

FIG. 1 is a diagram for explaining the arrangement of the objective lens in the light sheet microscope. 2 and 3 are diagrams for explaining the effect of light passing obliquely through the interface on the light sheet microscope. Hereinafter, with reference to FIGS. 1 to 3, the effects caused by the oblique light incident on the interface in the light sheet microscope and the countermeasures thereof will be described.

In the light sheet microscope, the objective lens OB1 for illumination and the objective lens OB2 for observation are arranged so that the optical axis X1 of the objective lens OB1 and the optical axis X2 of the objective lens OB2 intersect with each other. Specifically, as shown in FIG. 1, assuming that the angle formed by the horizontal plane HP and the optical axis X1 is an angle α and the angle formed by the horizontal plane HP and the optical axis X2 is an angle β, the objective lens OB1 and the objective lens OB2 are In other words, the optical axis X1 and the optical axis X2 are generally arranged so as to satisfy the relationship of α + β = 90 °. In the most typical light sheet microscope, the angle α is 0 °, and a configuration is adopted in which illumination is performed from the horizontal direction and observation is performed from the vertical direction.

As a result, if the optical path from the objective lens OB1 to the objective lens OB2 is filled with a single medium, the optical axis X1 formed by the illumination optical system including the objective lens OB1 is as shown in FIG. Since the light sheet LS0 parallel to the light sheet LS0 is parallel to the observation surface OS of the observation optical system including the objective lens OB2, it is possible to easily match them. Therefore, it is possible to obtain a good observation image with a light sheet microscope.

Further, even when the optical path from the objective lens OB1 to the objective lens OB2 is not filled with a single medium, the interface F of these media is orthogonal to either the optical axis X1 or the optical axis X2 on the optical path. If this is the case, the light sheet LS0 formed by the illumination optical system and the observation surface OS of the observation optical system are parallel to each other. This is because the refraction of light rays does not occur at the interface F. Therefore, it is possible to easily match the light sheet LS0 and the observation surface OS as in the case of being filled with a single medium, and it is possible to obtain a good observation image. For example, the most typical configuration of a light sheet microscope that satisfies α = 0 ° meets this condition.

On the other hand, when the optical path from the objective lens OB1 to the objective lens OB2 is not filled with a single medium, and the interface F of these media is orthogonal to both the optical axis X1 and the optical axis X2 on the optical path. If not, the light sheet formed by the illumination optical system and the observation surface of the observation optical system are not parallel to each other. This point will be described in more detail with reference to FIG.

The interface F shown in FIG. 2 is the interface between the medium M1 and the medium M2 having different refractive indexes. Here, the case where the refractive index of the medium M1 <the refractive index of the medium M2 is shown. As shown in FIG. 2, when the interface F is not orthogonal to either the optical axis X1 or the optical axis X2 on the optical path, the light parallel to the optical axis is obliquely incident on the interface F. Therefore, focusing on the illumination light, the light L0 emitted from the objective lens OB1 along the optical axis X1 is refracted at the interface F, and as a result, the illumination optical system forms a light sheet LS1 that is not parallel to the optical axis X1. .. Focusing on the observation light, the light L2 incident on the imaging device of the light sheet microscope is also refracted at the interface F, so that the observation surface OS formed by tracking the imaging surface with the back light is not orthogonal to the optical axis X2. That is, an observation surface OS that is not parallel to the optical axis X1 is formed. Further, the light sheet LS1 and the observation surface OS have directions in which the optical axis X1 is rotated in opposite directions. Therefore, the light sheet LS1 and the observation surface OS are not parallel.

If the parallel relationship between the light sheet and the observation surface is broken, the amount of deviation between the observation surface and the light sheet will be different at each position in the field of view. Therefore, it is difficult to match the observation surface and the light sheet over the entire field of view, and it is difficult to obtain a good observation image. As described above, with a conventional light sheet microscope, it is difficult to obtain a good observation image while avoiding the restrictions of the container.

Therefore, in order to deal with such a problem, the light sheet microscope according to the embodiment of the present invention includes a first adjusting unit for adjusting the orientation of the light sheet. The first adjusting unit adjusts the incident position of the light incident on the objective lens OB1 which is an example of the first objective lens in the direction intersecting the optical axis X1 which is an example of the first optical axis. More specifically, the incident position in the direction parallel to the optical axis X2, which is an example of the second optical axis, is adjusted. This makes it possible to rotate the light sheet around the rotation axis in the width direction of the light sheet to adjust the orientation of the light sheet.

The specific configuration of the first adjustment unit is not particularly limited. The first adjusting unit may include, for example, a transparent parallel flat plate arranged on the optical path leading to the objective lens OB1. The first adjusting unit may change the shift amount generated in the parallel plane plate with respect to the light directed to the objective lens OB1 by rotating the parallel plane plate, and adjust the shift amount to change the shift amount of the objective lens OB1. The incident position on the light beam, particularly the incident position in the direction parallel to the optical axis X2, may be adjusted. Further, the first adjusting unit may include a reflecting surface that reflects light toward the objective lens OB1. The reflecting surface may be provided on the mirror or the prism. The first adjusting unit may shift the light from the reflecting surface toward the objective lens OB1 by moving the reflecting surface, and by changing the position of the reflecting surface and changing the reflecting position, the objective lens OB1 The incident position on the light beam, particularly the incident position in the direction parallel to the optical axis X2, may be adjusted.

The light sheet microscope according to the embodiment of the present invention includes a control unit that controls the first adjustment unit in addition to the first adjustment unit. The control unit controls the first adjustment unit based on the refractive index of two or more media to make the observation surface OS and the light sheet parallel. These two or more media are media having different refractive indexes arranged on the optical path from the objective lens OB1 to the objective lens OB2 via the sample, and the optical axis X1 and the optical axis are on the optical path. It has an interface F that is not orthogonal to either of X2. That is, in the example shown in FIG. 2, the two or more media are the medium M1 and the medium M2.

More specifically, the control unit first acquires the refractive indexes of the two or more media described above in order to control the first adjustment unit based on the operation of the user. In the example shown in FIG. 2, the control unit acquires the refractive index of the medium M1 and the refractive index of the medium M2. Here, for example, the user may input the refractive index values of two or more media into the light sheet microscope, and the control unit may acquire the input values as the refractive indexes of the two or more media. Further, the user selects identification information (for example, a name) of two or more media, the light sheet microscope specifies the refractive index from the identification information, and the control unit acquires the refractive index of two or more media. You may.

From these refractive indexes, the amount of refraction of the illumination light and the observation light at the interface F can be calculated. Further, the angle γ required to form the light sheet LS2 parallel to the observation surface OS by the geometric calculation using these refractive indexes, that is, the angle formed by the light L1 emitted from the objective lens OB1 with respect to the interface F. Can also be calculated. Further, the above-mentioned incident position that realizes the angle γ from the angle γ can also be calculated. That is, it is possible to calculate the incident position from the refractive indexes of the two or more media described above. Utilizing such a relationship, the control unit calculates the incident position where the observation surface OS and the light sheet are parallel based on the acquired refractive indexes of the two or more media, and the light is emitted to the calculated incident position. The first adjusting unit is controlled so as to be incident. As a result, as shown in FIG. 2, the light sheet LS2 is formed by the illumination optical system, and the observation surface OS and the light sheet LS2 are parallel to each other.

The specific configuration of the control unit is not particularly limited, but the control unit is, for example, an electronic circuit (circuitry) including one or more processors. The control unit may be included in the light sheet microscope, and may be, for example, a microcomputer provided in the microscope main body or a general-purpose computer connected to the microscope main body.

As described above, according to the light sheet microscope according to the embodiment of the present invention, by providing the first adjustment unit and the control unit, at least one of the illumination light and the observation light is oblique to the interface of the medium having a different refractive index. The light sheet and the observation surface can be made parallel even when the light is incident on the light sheet. Therefore, the observation surface and the light sheet can be made parallel over the entire field of view, and a good observation image can be obtained. In other words, a good observation image can be obtained by using a light sheet microscope regardless of the shape of the container and the difference in the refractive index between the media in contact with the container.

The light sheet microscope according to the embodiment of the present invention may further include a second adjusting unit that adjusts the relative distance between the observation surface and the light sheet. Even if the observation surface and the light sheet are parallel to each other, if the observation surface and the light sheet are separated from each other, they will not be in focus and the observation image will be deteriorated. By using the refractive indexes of the two or more media described above, it is possible to calculate the position of the light sheet and the position of the observation surface, and it is possible to calculate the relative distance between the light sheet and the observation surface. Therefore, the control unit may bring the observation surface closer to the light sheet surface by controlling the second adjustment unit based on the refractive index of the two or more media described above. As a result, blurring of the observed image is suppressed and an observed image having high contrast can be obtained.

The specific configuration of the second adjustment unit is not particularly limited. The second adjusting unit may be moved closer to the observation surface by, for example, moving the light sheet in the thickness direction of the light sheet. More specifically, the second adjusting unit may include an optical deflector that changes the angle of the light incident on the pupil of the objective lens OB1 and changes the deflection direction of the light in the optical deflector to the pupil. The light sheet may be moved in the thickness direction by changing the angle of the incident light. The optical deflector is not particularly limited, but may be a galvanometer mirror that mechanically deflects the light, an AO deflector that deflects the light using an acoustic optical effect, or an electro-optical effect. It may be an EO deflector that deflects light. Further, the second adjusting unit may be brought closer to the light sheet by, for example, moving the observation surface in the normal direction of the observation surface. More specifically, the second adjusting unit may include a driving unit that moves the objective lens OB2 in the direction of the optical axis X2, and by moving the objective lens OB2 in the optical axis direction, the observation surface is moved in the normal direction. You may move it.

The light sheet microscope according to the embodiment of the present invention may further include a third adjusting unit that adjusts the condensing position of the light emitted from the objective lens OB1 in the traveling direction of the light emitted from the objective lens OB1. .. Even when the observation surface and the light sheet are parallel and their relative distance is sufficiently small, if the light sheet is formed at a position far away from the center of the field of view, the thickness of the light sheet in the field of view becomes thick. As a result, the observed image deteriorates. Therefore, the control unit controls the third adjustment unit based on the refractive indexes of the two or more media described above, so that the condensing position of the light emitted from the objective lens OB1 is brought closer to the center of the field of view of the light sheet microscope. You may. As a result, the thickness of the light sheet in the field of view becomes sufficiently thin, so that a good observation image can be obtained.

The specific configuration of the third adjustment unit is not particularly limited. The third adjusting unit may include, for example, a reflecting surface arranged at or near a position optically conjugate with the focal position of the objective lens OB1, and the light incident on the objective lens OB1 by moving the reflecting surface may be included. The converging state may be changed to adjust the focusing position in the traveling direction. Since the reflecting surface may be placed at a position where the light flux diameter is sufficiently small, the vicinity of the position optically conjugate with the focal position may be defined in relation to the light flux diameter. By arranging it at a position where the luminous flux diameter is sufficiently small, there is an advantage that the reflecting surface that functions as a movable part can be made small. Further, the third adjusting unit may include, for example, a varifocal lens, and by changing the focal length of the varifocal lens, the convergent state of the light incident on the objective lens OB1 is changed, and the condensing position is advanced. It may be adjusted in the direction. LCOS® may be used in place of the varifocal lens. Further, the third adjustment unit may include, for example, a single focus lens, and by moving the single focus lens in the optical axis direction of the single focus lens, the converging state of the light incident on the objective lens OB1 is changed to collect the light. The position may be adjusted in the direction of travel.

FIG. 2 shows an example in which a container does not intervene in the optical path from the objective lens OB1 to the objective lens OB2 via the sample, mainly assuming that the light sheet microscope is an upright microscope. The light sheet microscope according to the embodiment may be an upright microscope or an inverted microscope.

The example shown in FIG. 3 mainly assumes an inverted microscope, and the medium M3 is, for example, the bottom surface portion of a container containing a sample. The bottom surface portion (medium M3) of the culture vessel has a constant thickness t. Further, FIG. 3 shows the case where the refractive index of the medium M3> the refractive index of the medium M2> the refractive index of the medium M1.

When the medium M3 exists between the medium M1 and the medium M2, the light shifts in the medium M3 in the direction orthogonal to the traveling direction. Therefore, the light sheet LS3 parallel to the light sheet LS2 formed when the medium M3 does not exist is formed at a position different from that of the light sheet LS2. The difference in position between the light sheet LS2 and the light sheet LS3 depends on the refractive index and the thickness t of the medium M3. Therefore, the distance ΔH in the normal direction between the light sheets and the distance ΔL in the traveling direction between the light sheets can be calculated from the refractive index and the thickness t of the medium M3.

As shown in FIG. 3, when the container is interposed in the optical path, the positional deviation between the observation surface and the light sheet is larger by the amount of ΔH and ΔL than when the container is not interposed. Therefore, especially when the light sheet microscope is an inverted microscope, it is desirable to have the above-mentioned second adjustment unit and third adjustment unit. Further, as described above, both ΔH and ΔL depend on the refractive index and thickness of the container containing the sample. Therefore, the control unit controls the second adjustment unit based on the refractive index and thickness of the container in addition to the refractive indexes of two or more media having different refractive indexes as described above to lighten the observation surface. It is desirable to bring it closer to the surface. In addition, the control unit adjusts the third adjustment unit based on the refractive index of two or more media and the refractive index and thickness of the container to bring the focused position of the illumination light closer to the center of the field of view of the light sheet microscope. Is desirable.

Further, it is desirable that the control unit acquires the refractive index and the thickness of the container when controlling the second adjustment unit and the third adjustment unit. The refractive index and thickness of the container may be obtained based on the operation of the user, or may be obtained using a sensor provided in the light sheet microscope. The sensor may measure the refractive index and thickness of the container by any method, or may read the identification information provided in the container and identify the refractive index and thickness of the container from the identification information.

For example, when observing a clearing-treated sample using a light sheet microscope, the medium M1 shown in FIG. 3 corresponds to the immersion liquid of the objective lens, and the medium M2 corresponds to the clearing solution. By aligning the refractive indexes of the clearing solution and the immersion liquid, it is possible to maintain the parallel relationship between the light sheet and the observation surface. However, many clearing solutions have already been developed and have a refractive index (for example, 1.38 to 1.52) that varies greatly depending on the type of clearing solution. In addition, as for immersion, for example, in drug discovery screening that automatically images a large amount of sample, a large amount of immersion is consumed, so it is necessary to select the immersion to be used from the viewpoint of ease of handling and cost. I don't get it. In view of such circumstances, it is not easy to make the refractive indexes of the clearing solution and the immersion liquid uniform, and it is assumed that there are many cases where the clearing solution and the immersion liquid having different refractive indexes are used. Even in such a case, according to the light sheet microscope according to the embodiment of the present invention, the observation surface and the light sheet can be aligned over the entire field of view, and a good observation image can be obtained. can.

Also, in observation using a light sheet microscope, it is assumed that various clearing solutions will be used properly according to the purpose, and the angle of the observation surface will also differ for each clearing solution. Even in such a case, the light sheet microscope according to the embodiment of the present invention is good because it has a structure for adjusting the angle of the light sheet based on the refractive index of the clearing solution. An observation image can be obtained. Further, as long as the refractive index is specified, even if the refractive index fluctuates due to a temperature change or the like, a good observation image can be obtained by compensating for the fluctuation.

Hereinafter, embodiments of the present invention will be described in more detail.
[First Embodiment]
FIG. 4 is a diagram illustrating the configuration of the light sheet microscope according to the present embodiment. FIG. 5 is a diagram illustrating the configuration of the control device shown in FIG. Hereinafter, the light sheet microscope 1 according to the present embodiment will be described with reference to FIGS. 4 and 5.

The light sheet microscope 1 is a device for observing the sample S housed in the multi-well plate 90, and the sample S is made transparent by the clearing solution (medium M2) stored in the well W. Therefore, the refractive index of the sample S can be regarded as the same as the refractive index of the clearing solution (medium M2).

As shown in FIG. 1, the light sheet microscope 1 is a space between a microscope main body 10 that forms an optical image of sample S, a camera 40 that generates a digital image of sample S, an objective lens 31, and a multi-well plate 90. A water supply device 50 for filling the light sheet microscope 1 with water (medium M1) and a control device 80 for controlling the light sheet microscope 1 are provided.

The microscope main body 10 includes an objective lens 21, an illumination optical system 20 for forming a light sheet on the sample S, and an objective lens 31, and observation optics for forming an optical image of the sample S based on the light from the sample S. System 30 is provided. The objective lens 31 is equipped with a holder 60 for storing the water supplied from the water supply device 50. Further, the microscope main body 10 is provided with a parallel flat plate 11, a galvanometer mirror 12, and a piezo element 13 on the illumination optical path.

The parallel flat plate 11 constitutes the first adjustment unit of the light sheet microscope 1. The rotation axis of the parallel flat plate 11 is oriented in a direction orthogonal to both the optical axis of the objective lens 21 and the optical axis of the objective lens 31. By rotating the parallel flat plate 11, the incident position of the laser beam incident on the objective lens 21 is adjusted in the direction intersecting the optical axis of the objective lens 21.

The galvano mirror 12 constitutes the second adjustment unit of the light sheet microscope 1. The galvano mirror 12 is arranged at a position optically conjugate with the pupil of the objective lens 21, and the galvano mirror 12 changes the angle of the reflection surface of the galvano mirror 12 so that the observation surface and the light sheet surface are relative to each other. Distance is adjusted.

The piezo element 13 constitutes the third adjustment unit of the light sheet microscope 1. The piezo element 13 expands and contracts in the optical axis direction of the lens 26, which will be described later. By moving the reflecting surface of the piezo element 13, the piezo element 13 adjusts the condensing position of the laser light emitted from the objective lens 21 in the traveling direction of the light emitted from the objective lens 21.

The control device 80 is a control unit of the light sheet microscope 1. The control device 80 is a computer that controls the light sheet microscope 1. As shown in FIG. 5, the control device 80 is a portable recording medium driving device that drives a processor 81, a memory 82, an auxiliary storage device 83, an input device 84, an output device 85, and a portable recording medium 89. It includes 86, a communication module 87, and a bus 88. The auxiliary storage device 83 and the portable recording medium 89 are examples of non-transient computer-readable recording media on which programs are recorded.

The processor 81 is an arbitrary processing circuit including, for example, a CPU (Central Processing Unit), a GPU (Graphics Processing Unit), and the like. The processor 81 expands the program stored in the auxiliary storage device 83 or the portable recording medium 89 into the memory 82, and then executes the program to perform the programmed process.

The memory 82 is, for example, an arbitrary semiconductor memory such as a RAM (Random Access Memory). The memory 82 functions as a work memory for storing the program or data stored in the auxiliary storage device 83 or the portable recording medium 89 when the program is executed. The auxiliary storage device 83 is, for example, a non-volatile memory such as a hard disk or a flash memory. The auxiliary storage device 83 is mainly used for storing various data and programs.

The portable recording medium drive device 86 accommodates the portable recording medium 89. The portable recording medium driving device 86 can output the data stored in the memory 82 or the auxiliary storage device 83 to the portable recording medium 89, and reads a program, data, or the like from the portable recording medium 89. Can be done. The portable recording medium 89 is any portable recording medium. The portable recording medium 89 includes, for example, an SD card, a USB (Universal Serial Bus) flash memory, a CD (Compact Disc), a DVD (Digital Versatile Disc), and the like.

The input device 84 is a keyboard, a mouse, or the like. The output device 85 is a display device, a printer, or the like. The communication module 87 is, for example, a wired communication module that communicates with the microscope main body 10 connected via an external port. The communication module 87 may be a wireless communication module. The bus 88 connects the processor 81, the memory 82, the auxiliary storage device 83, and the like to each other so that data can be exchanged.

The configuration shown in FIG. 5 is an example of the hardware configuration of the control device 80. The control device 80 is not limited to this configuration. The control device 80 may be a general-purpose device or a dedicated device. The control device 80 may include, for example, a specially designed electric circuit, for example, an ASIC (Application Specific Integrated Circuit) or the like. Further, the control device 80 may be configured by using an FPGA (Field-Programmable Gate Array). Further, the control device 80 may be integrally configured with the microscope main body 10.

In the light sheet microscope 1, the laser light emitted from a laser (not shown) is collimated by the lens 27 incident through the optical fiber 28 and incident on the polarization beam splitter (PBS) 23. The laser beam reflected from the PBS 23 is focused in the vicinity of the piezo element 13 by the lens 26 incident through the λ / 4 plate 24 and the rectangular diaphragm 25. The laser beam reflected from the reflecting surface provided on the piezo element 13 then enters the PBS 23 via the lens 26, the rectangular diaphragm 25, and the λ / 4 plate 24. The laser beam incident on the PBS 23 from the λ / 4 plate 24 has a polarization direction orthogonal to the light incident on the PBS 23 from the lens 27 by passing through the λ / 4 plate 24 twice after reflecting the PBS 23. Therefore, it permeates PBS23. The laser beam transmitted through the PBS 23 is incident on the movable portion 70 via the parallel flat plate 11, and is incident on the objective lens 21 via the cylindrical lens 22 and the galvanometer mirror 12. The laser beam emitted from the objective lens 21 into the air enters the water (medium M1) stored in the holder 60 through the window 61 provided in the holder 60, and then passes through the bottom surface of the multi-well plate 90 and is transparent. The sample S placed in the chemical solution (medium M2) is irradiated. As a result, a light sheet is formed on the sample S.

In the light sheet microscope 1, the objective lens 21 and the objective lens 31 are arranged so that their optical axes are orthogonal to each other. Further, the direction of the optical axis of the objective lens 21 is different from both the horizontal direction and the vertical direction. As a result, the optical axes of the objective lens 21 and the objective lens 31 are tilted with respect to the bottom surface of the horizontally arranged multi-well plate 90, so that the laser beam is obliquely incident on the bottom surface of the multi-well plate 90. On the other hand, since the window 61 of the holder 60 is provided so as to be orthogonal to the optical axis of the objective lens 21, the laser light emitted from the objective lens 21 is substantially perpendicular to the window 61.

In the light sheet microscope 1, the light generated from the sample S in the clearing solution (medium M2) by the irradiation of the laser beam enters the objective lens 31 via the bottom surface of the multi-well plate 90 and water (medium M1). do. The light incident on the objective lens 31 is then condensed by the imaging lens 32 to form an optical image of the sample S on the imaging surface of the camera 40.

In the light sheet microscope 1, as described above, the optical axes of the objective lens 21 and the objective lens 31 are tilted with respect to the bottom surface of the horizontally arranged multi-well plate 90. Therefore, the light generated from the sample S diagonally crosses the bottom surface of the multi-well plate 90 and enters the objective lens 31.

In the light sheet microscope 1 configured as described above, on the optical path from the objective lens 21 to the objective lens 31, light is emitted from the bottom surface of the multi-well plate 90, the interface between the medium M1 (water), and the multi-well plate 90. It is refracted at the interface between the bottom surface and the medium M2 (clearing solution). Therefore, when light is emitted from the objective lens 21 along the optical axis, the parallel relationship between the light sheet and the observation surface is broken.

Therefore, in the light sheet microscope 1, the control device 80 acquires the refractive indexes of the medium M1 and the medium M2, and rotates and controls the parallel flat plate 11 based on the refractive indexes of the medium M1 and the medium M2. Thereby, the orientation of the light sheet can be adjusted, and the observation surface of the observation optical system 30 and the light sheet formed by the illumination optical system 20 can be made parallel.

Further, in the light sheet microscope 1, the control device 80 further acquires the thickness and the refractive index of the bottom surface of the multi-well plate 90, and is based on the refractive index of the medium M1 and the medium M2 and the thickness and the refractive index of the bottom surface of the multi-well plate 90. Then, the rotation of the galvano mirror 12 is controlled. As a result, the position of the light sheet in the thickness direction can be adjusted, and the light sheet can be brought closer to the observation surface.

Further, in the light sheet microscope 1, the piezo element 13 is moved based on the refractive indexes of the medium M1 and the medium M2 and the thickness and the refractive index of the bottom surface of the multi-well plate 90. As a result, the condensing position of the laser light constituting the light sheet can be adjusted in the traveling direction of the light sheet, and the condensing position of the laser light can be brought closer to the center of the field of view of the light sheet microscope 1.

Therefore, according to the light sheet microscope 1 according to the present embodiment, a good observation image can be obtained even when the multi-well plate 90 is used. Moreover, even when an arbitrary clearing solution is used, a good observation image can be obtained.

Further, in the light sheet microscope 1, the observation surface can be moved with respect to the sample by moving the movable portion 70 to which the objective lens 21 and the objective lens 31 are mounted in the optical axis direction of the objective lens 31. Therefore, three-dimensional information of the sample can be obtained by repeating the movement of the movable portion 70 and the acquisition of the digital image.

[Second Embodiment]
FIG. 6 is a diagram illustrating the configuration of the light sheet microscope according to the present embodiment. Hereinafter, the light sheet microscope 2 according to the present embodiment will be described with reference to FIG.

The light sheet microscope 2 is different from the light sheet microscope 1 in that the microscope main body 100 is provided instead of the microscope main body 10. In other respects, the light sheet microscope 2 is the same as the light sheet microscope 1. The microscope main body 100 is the same as the microscope main body 10 in that it includes a first adjustment unit, a second adjustment unit, and a third adjustment unit. However, the configuration of each adjustment unit is different.

The first adjustment unit of the light sheet microscope 2 includes a mirror 101 that reflects laser light toward the objective lens 21. The first adjusting unit moves the mirror 101 along the optical axis of the objective lens 31 to adjust the incident position of the laser beam incident on the objective lens 21 in a direction intersecting the optical axis of the objective lens 21.

The second adjustment unit of the light sheet microscope 2 includes a piezo element 102 that moves the objective lens 31 in the direction of the optical axis of the objective lens 31. The second adjusting unit adjusts the relative distance between the observation surface and the light sheet surface by moving the objective lens 31 in the optical axis direction by expanding and contracting the piezo element 102.

The third adjustment unit of the light sheet microscope 2 includes a galvano mirror 103 arranged in the vicinity of the focusing position of the laser light in the illumination optical system 20. By moving the galvano mirror 103, the third adjusting unit adjusts the condensing position of the laser light emitted from the objective lens 21 in the traveling direction of the light emitted from the objective lens 21.

Even with the light sheet microscope 2 configured as described above, the control device 80 controls the first adjustment unit, the second adjustment unit, and the third adjustment unit in the same manner as in the case of the light sheet microscope 1, so that good observation can be achieved. You can get an image.

[Third Embodiment]
FIG. 7 is a diagram illustrating the configuration of the light sheet microscope according to the present embodiment. Hereinafter, the light sheet microscope 3 according to the present embodiment will be described with reference to FIG. 7.

The light sheet microscope 3 is different from the light sheet microscope 1 in that the microscope main body 200 is provided instead of the microscope main body 10, the medium holder 220 is provided instead of the holder 60, and the stage 230 that holds the multi-well plate 90 is provided. Is different. In other respects, the light sheet microscope 3 is the same as the light sheet microscope 1.

The microscope main body 200 includes an illumination optical system 210 instead of the illumination optical system 20. The illumination optical system 210 uses the refractive power of the cylindrical lens 22 in a specific direction to linearly condense light to form a light sheet, but instead to form a light sheet, the light condensing position is galvanized. The difference is that a light sheet is formed by moving the mirror 214 at high speed. Specifically, as shown in FIG. 7, the illumination optical system 210 includes a laser 217, a lens 216, an optical fiber 215, a galvano mirror 214, a relay optical system (lens 213, lens 212), a mirror 211, and an objective lens 21. I'm out.

Further, the microscope main body 200 is the same as the microscope main body 10 in that it includes a first adjustment unit, a second adjustment unit, and a third adjustment unit. However, the configuration of each adjustment unit is different.

The first adjusting portion of the light sheet microscope 3 includes a transparent parallel flat plate 201 provided in the movable portion 70. By rotating the parallel flat plate 201, the first adjusting unit adjusts the incident position of the laser beam incident on the objective lens 21 in a direction intersecting the optical axis of the objective lens 21.

The second adjustment unit of the light sheet microscope 3 includes a piezo element 202 that moves the objective lens 31 in the direction of the optical axis of the objective lens 31. The second adjusting unit adjusts the relative distance between the observation surface and the light sheet surface by moving the objective lens 31 in the optical axis direction by expanding and contracting the piezo element 202.

The third adjustment unit of the light sheet microscope 3 includes a lens 203 that collimates the light emitted from the optical fiber 215. By moving the lens 203 along the optical axis of the lens 203, the third adjusting unit adjusts the condensing position of the laser light emitted from the objective lens 21 in the traveling direction of the light emitted from the objective lens 21.

The medium holder 220 is a holder for accommodating water (medium M1) for immersing the multi-well plate 90. Unlike the holder 60 mounted on the objective lens 31, the medium holder 220 is fixed in a state of being separated from the objective lens 315. The medium holder 220 includes a window 221 orthogonal to the optical axis of the objective lens 21 and a window 222 orthogonal to the optical axis of the objective lens 31. Further, the space between the objective lens 31 and the medium holder 220 is filled with the same medium M4 as the medium M1 housed in the medium holder 220, that is, water, using surface tension. In the light sheet microscope 3, the water supply device 50 is a device that supplies the medium M4.

The stage 230 is, for example, an electric stage that holds the multi-well plate 90 from the side surface, and is configured to be movable in the horizontal direction while the multi-well plate 90 is immersed in the medium M1.

Even in the light sheet microscope 3 configured as described above, light is emitted from the bottom surface of the multi-well plate 90 and the interface between the medium M1 (water) and the multi-well plate 90 on the optical path from the objective lens 21 to the objective lens 31. The point of refraction at the interface between the bottom surface of the light sheet and the medium M2 (clearing solution) is the same as that of the light sheet microscope 1.

Therefore, in the light sheet microscope 3 as well as in the light sheet microscope 1, the control device 80 acquires the refractive indexes of the medium M1 and the medium M2. Then, the control device 80 controls the rotation of the parallel flat plate 201 based on their refractive indexes. As a result, the orientation of the light sheet can be adjusted, and the observation surface of the observation optical system 30 and the light sheet formed by the illumination optical system 210 can be made parallel.

Further, also in the light sheet microscope 3, the control device 80 acquires the thickness and the refractive index of the bottom surface of the multi-well plate 90 as in the light sheet microscope 1. Then, the control device 80 controls the piezo element 202 based on the refractive indexes of the medium M1 and the medium M2 and the thickness and the refractive index of the bottom surface of the multi-well plate 90. As a result, the position of the observation surface in the normal direction can be adjusted, and the observation surface can be brought closer to the light sheet.

Further, in the light sheet microscope 3, the control device 80 moves the lens 203 in the optical axis direction of the lens 203 based on the refractive indexes of the medium M1 and the medium M2 and the thickness and the refractive index of the bottom surface of the multiwell plate 90. As a result, the condensing position of the laser light constituting the light sheet can be adjusted in the traveling direction of the light sheet, and the condensing position of the laser light can be brought closer to the center of the field of view of the light sheet microscope 3.

Therefore, even with the light sheet microscope 3 according to the present embodiment, a good observation image can be obtained when the multi-well plate 90 is used. Further, in the light sheet microscope 3, since the observation surface can be moved with respect to the sample by moving the stage 230, which is an electric stage, the movement of the stage 230 and the acquisition of the digital image are repeated to obtain the three-dimensional information of the sample. You may get it. In this method, since the distribution of the medium on the optical path does not change even if the stage 230 is moved, it is possible to maintain a state in which the aberration is well corrected without fluctuation of the aberration.

FIG. 8 is a diagram illustrating the configuration of a varifocal lens. The varifocal lens 204 shown in FIG. 8 is a lens capable of changing the focal length by controlling the interface shape of two media (medium 205, medium 206) in the varifocal lens 204. For example, the interface can be changed. It can be transformed from surface 207 to surface 208. By changing the focal length, it is possible to adjust the condensing position of the illumination light in the traveling direction of the light sheet. Therefore, the third adjustment unit of the light sheet microscope 3 may include the variable focus lens 204, and even if the convergence state of the light incident on the objective lens 21 is changed by changing the focal length of the variable focus lens 204. good.

The above-described embodiments show specific examples for facilitating the understanding of the invention, and the embodiments of the present invention are not limited thereto. Some of the above-described embodiments may be applied to other embodiments. The light sheet microscope, control method, and program can be variously modified and modified without departing from the description of the claims.

In the above-described embodiment, a specific combination of the first adjustment unit, the second adjustment unit, and the third adjustment unit is shown, but the first adjustment unit, the second adjustment unit, and the third adjustment unit The combination is not limited to these. The first adjusting unit, the second adjusting unit, and the third adjusting unit may be incorporated in the light sheet microscope in different combinations.

Further, in the above-described embodiment, although not particularly mentioned, the observation objective lens may include a correction ring. This makes it possible to correct the spherical aberration caused by the refractive index mismatch, so that a better observed image can be obtained.

Further, in the above-described embodiment, the multi-well plate 90 is exemplified as a container for accommodating the sample, but the container is not limited to the multi-well plate. The container is not particularly limited, but may be, for example, a flask, a petridesh, or the like. Further, in the above-described embodiment, a container having a flat bottom surface and a uniform thickness has been illustrated, but the bottom surface of the container may not be flat as in the multi-well plate 91 shown in FIG. 9, for example. It does not have to be uniform in thickness. If the shape of the container is known, the control unit is the first adjustment unit and the second adjustment unit using the shape of the container in addition to the above information (refractive index of the medium, refractive index of the container, thickness of the container). By controlling the third adjustment unit, it is possible to match the observation surface with the light sheet. Although FIG. 9 exemplifies a container having a circular bottom surface, the container is not limited to a circular shape and may have an arbitrary shape such as a parabolic shape or a free curved surface shape.

Further, in the above-described embodiment, the optical axis of the observation objective lens and the optical axis of the illumination objective lens are orthogonal to each other, but the optical axis of the observation objective lens and the optical axis of the illumination objective lens intersect. I just need to be there. Further, the relationship between the optical axis of the observation objective lens and the optical axis of the illumination objective lens may change. In any case, if the angles (angles α, β) of each optical axis with respect to the horizontal plane are known, the light sheet and the observation surface can be matched by controlling the first adjustment unit to the third adjustment unit.

Further, in the above-described embodiment, an example in which there are two media that affect the orientation of the light sheet is shown, but there may be three or more media that affect the orientation of the light sheet.

Further, in the above-described embodiment, an example is shown in which the control parameters required to match the observation surface and the light sheet are calculated based on the refractive index and each adjustment unit is controlled based on the control parameters. The refractive index may be calculated back from the control parameter when the light sheet and the light sheet match. For example, when the refractive index of the medium M2 in the first embodiment is unknown, the refractive index of the medium M1 is acquired, the parallel flat plate 11 is rotated so that a good observation image can be obtained, and the parallel flat plate 11 is rotated. The refractive index of the medium M2 may be calculated back from the angle information of. Further, the refractive index of the medium M1 and the refractive index and thickness of the multi-well plate 90 are acquired, the parallel flat plate 11 is rotated, the galvanometer mirror 12 is rotated, and the piezo element 13 is rotated so that a good observation image can be obtained. After expanding and contracting, the refractive index of the medium M2 may be calculated back from the angle information of the parallel flat plate 11. This makes it possible to use the light sheet microscope as a refractive index calculation device. A good observation image may be specified by a human being confirming the observation image, or may be specified by the control unit comparing a plurality of digital images acquired while changing the angle of the parallel flat plate 11. ..

Further, when the refractive index of the multi-well plate 90 is unknown, the refractive indexes of the medium M1 and the medium M2 and the thickness of the multi-well plate 90 are obtained, and the parallel flat plate 11 is used so that a good observation image can be obtained. You may rotate, rotate the galvano mirror 12, expand and contract the piezo element 13, and then calculate the refractive index of the multiwell plate 90 back from the angle information of the galvano mirror 12 or the expansion and contraction information of the piezo element 13. In this case as well, a good observation image may be specified by a human being confirming the observation image, or may be specified by the control unit comparing a plurality of digital images acquired while adjusting each adjustment unit. ..

That is, by using a light sheet microscope, the refractive index of the medium M1, the refractive index of the medium M2, the refractive index of the container, and the thickness of the container can be determined from the states of the first adjustment unit, the second adjustment unit, and the third adjustment unit. At least one can be calculated back.

Further, in the above-described embodiment, an example is shown in which an immersion objective lens is used as the observation objective lens and a dry objective lens is used as the illumination objective lens, but an immersion objective lens is used as the illumination objective lens. You may. Further, a dry objective lens may be used as the observation objective lens. Further, in the above-described embodiment, water is exemplified as the immersion liquid in contact with the immersion objective lens, but the immersion liquid is not limited to water. For example, silicone oil or the like may be used. In the case of silicone oil, unlike water, it does not evaporate, so a device for supplying the immersion liquid (water supply device 50, etc.) can be omitted.

Further, in the above-described embodiment, the control unit calculates the driving amount of the first adjusting unit (rotation angle of the parallel flat plate 11, translation amount of the mirror 101, etc.) from two or more refractive indexes from the geometrical relationship. However, the control unit may store the drive amount calculated from the refractive index in advance in association with the refractive index. In this case, the control unit may control the first adjustment unit by reading out the corresponding drive amount based on the acquired refractive index.

Further, in the above-described embodiment, the third adjustment unit is used to bring the light sheet closer to the center of the field of view, but the third adjustment unit may be used for other purposes. For example, a digital image having a high resolution may be acquired from the center to the periphery by moving the condensing position by the third adjusting unit according to the rolling shutter of the camera 40.

In this specification, the expression "based on certain information" means "at least using certain information". Therefore, it corresponds to both the case where only certain information is used and the case where certain information and other information are used. For example, in the expression "control based on the refractive index of two or more media", when controlling using only the refractive index of two or more media as an input parameter, the refractive index of two or more media is used. In addition, both are included when controlling using other information such as the focal length of the objective lens as an input parameter.

1-3 Light Sheet Microscope 11, 201 Parallel Flat Plate 12, 103 Galvano Mirror 13, 102, 202 Piezo Element 20, 210 Illumination Optical System 21, 31, OB1, OB2 Objective Lens 22 Cylindrical Lens 30 Observation Optical System 32 Imaging Lens 40 Camera 50 Water supply device 60 Holder 70 Moving part 80 Control device 90, 91 Multi-well plate 101 Mirror 203 Lens 204 Variable focus lens 220 Medium holder 230 Stage F Interface L1, L2 Optical LS0 to LS2 Light sheet M1 to M4 Medium OS Observation surface S sample W well X1, X2 optical axis ΔL, ΔH deviation α, β, γ angle

Claims (16)

An illumination optical system that includes a first objective lens having a first optical axis and forms a light sheet on a sample.
An observation optical system including a second objective lens having a second optical axis intersecting the first optical axis and forming an optical image of the sample based on the light from the sample.
A first adjusting unit that adjusts the incident position of light incident on the first objective lens in a direction intersecting the first optical axis, and
A control unit that makes the observation surface parallel to the light sheet by controlling the first adjustment unit based on the refractive index of two or more media, and the two or more media are the first objective lens. A medium having different refractive indexes, which is arranged on an optical path from the sample to the second objective lens, and has both the first optical axis and the second optical axis on the optical path. A light sheet microscope characterized by including a control unit having non-orthogonal interfaces. In the light sheet microscope according to claim 1, further
A second adjusting unit for adjusting the relative distance between the observation surface and the light sheet is provided.
The control unit is a light sheet microscope characterized in that the observation surface is brought closer to the light sheet by controlling the second adjustment unit based on the refractive index of the two or more media. In the light sheet microscope according to claim 1, further
A second adjusting unit for adjusting the relative distance between the observation surface and the light sheet is provided.
The control unit controls the second adjustment unit based on the refractive index of the two or more media and the refractive index and thickness of the container that houses the sample, thereby making the observation surface into the light sheet. A light sheet microscope characterized by being close to each other. In the light sheet microscope according to claim 2, further
A third adjusting unit for adjusting the condensing position of the light emitted from the first objective lens in the traveling direction of the light emitted from the first objective lens is provided.
The control unit is a light sheet microscope characterized in that the light condensing position is brought closer to the center of the visual field of the light sheet microscope by controlling the third adjustment unit based on the refractive index of the two or more media. In the light sheet microscope according to claim 3, further
A third adjusting unit for adjusting the condensing position of the light emitted from the first objective lens in the traveling direction of the light emitted from the first objective lens is provided.
The control unit controls the third adjustment unit based on the refractive index of the two or more media and the refractive index and thickness of the container to set the light collection position at the center of the visual field of the light sheet microscope. A light sheet microscope characterized by being close to. In the light sheet microscope according to claim 1,
The control unit is a light sheet microscope characterized by acquiring the refractive indexes of the two or more media based on the operation of the user. In the light sheet microscope according to claim 1,
The first adjusting unit includes a reflecting surface that reflects light toward the first objective lens, and shifts the light from the reflecting surface toward the first objective lens by moving the reflecting surface. Light sheet microscope. In the light sheet microscope according to claim 1,
The first adjusting unit includes a transparent parallel plane plate, and by rotating the parallel plane plate, the light directed to the first objective lens changes the shift amount generated by the parallel plane plate. Sheet microscope. In the light sheet microscope according to claim 1,
The second adjusting unit is a light sheet microscope including a light deflector that changes the angle of light incident on the pupil of the first objective lens. In the light sheet microscope according to claim 1,
The second adjusting unit is a light sheet microscope including a driving unit that moves the second objective lens in the direction of the second optical axis. In the light sheet microscope according to claim 1,
The third adjusting unit includes a variable focus lens, and is a light sheet microscope characterized in that the convergent state of light incident on the first objective lens is changed by changing the focal length of the variable focus lens. In the light sheet microscope according to claim 1,
The third adjusting unit includes a reflecting surface arranged at or near a position optically conjugate with the focal position of the first objective lens, and light incident on the first objective lens by moving the reflecting surface. A light sheet microscope characterized by changing the convergence state of an optical lens. In the light sheet microscope according to claim 1, further
A movable part to which the first objective lens and the second objective lens are mounted is provided.
The control unit is a light sheet microscope characterized by moving the movable unit in the direction of the second optical axis. In the light sheet microscope according to claim 1,
A light sheet microscope characterized in that the direction of the first optical axis is different from the horizontal direction and the vertical direction. It is a control method for light sheet microscopes.
The light sheet microscope includes an illumination optical system that forms a light sheet on a sample, including a first objective lens having a first optical axis, and a second objective lens having a second optical axis that intersects the first optical axis. It comprises an observation optical system that forms an optical image of the sample based on the light from the sample.
In the step of acquiring the refractive index of two or more media, the two or more media are arranged on an optical path from the first objective lens to the second objective lens via the sample. A process in which media having different refractive indexes have an interface on the optical path that is not orthogonal to either the first optical axis or the second optical axis.
A step of making the observation surface parallel to the light sheet by adjusting the incident position of the light incident on the first objective lens in a direction intersecting the first optical axis based on the refractive indexes of the two or more media. And, a control method characterized by including. From the sample, including an illumination optical system that forms a light sheet on the sample, including a first objective lens having a first optical axis, and a second objective lens having a second optical axis that intersects the first optical axis. A light sheet microscope comprising an observation optical system that forms an optical image of the sample based on light.
A process for acquiring the refractive index of two or more media, wherein the two or more media are arranged on an optical path from the first objective lens to the second objective lens via the sample. A process in which media having different refractive indexes have an interface on the optical path that is not orthogonal to either the first optical axis or the second optical axis.
A process of making the observation surface parallel to the light sheet by adjusting the incident position of light on the first objective lens in a direction intersecting the first optical axis based on the refractive indexes of the two or more media. A program characterized by executing.

Images