JP2021173960A - Optical sheet microscope and imaging method - Google Patents

Abstract

To provide an optical sheet microscope and an imaging method using the principle which makes a degree of freedom high in alignment between an observation object and an optical system while making a configuration of an optical system comparatively simple, and thereby can observe the whole raw sample.SOLUTION: A raw sample S that is an observation object is stored in a sample container 21 having a bottom surface made of a fluorine resin. The sample container 21 is held in a state in which the bottom surface is immersed in water W stored in the storage container 22. From the bottom surface of the storage container 22, fluorescence is incident on the raw sample S through an incident window 221 orthogonal to exciting light beam Le shaped into a sheet like. The fluorescence emitted from the raw sample S is incident on an observation optical system 41 through an emission window 222 orthogonal to an optical axis of the observation optical system 41.SELECTED DRAWING: Figure 1

Description

The present invention relates to an optical sheet microscope device and an imaging method using the principle thereof, and particularly to a technique suitable for observing a raw sample in a liquid.

An optical sheet microscope is an apparatus suitable for observing and imaging raw samples such as cells and biological tissues. This is done by injecting excitation light shaped into a thin sheet into the raw sample, generating fluorescence from the portion of the raw sample through which the sheet light passes, and performing fluorescence imaging from a direction perpendicular to the sheet surface. A fluorescence image corresponding to a cross section along the sheet surface is obtained. Compared with other imaging technologies such as confocal microscope imaging technology, this technology has less fading and phototoxicity of the raw sample because the excitation light is incident only on the observation target part, and the depth is observable in the fluorescence imaging direction. Is large.

For example, the optical sheet microscope described in Patent Document 1 is a transparent flat-bottomed container in which optical systems for excitation and observation are arranged on the lower surface side of a sample arrangement portion that carries a raw sample together with a liquid inside. Has. Specifically, a sheet-shaped excitation light is incident obliquely upward from the lower surface side of the sample arrangement portion, and an observation optical system having an optical axis orthogonal to the optical path of the excitation light is used through the lower surface of the sample arrangement portion. Fluorescence detection is performed. Further, Patent Document 1 also discloses a configuration example using a so-called immersion lens in which the space between the objective lens of the observation optical system and the lower surface of the sample arrangement portion is in a liquid-tight state.

Japanese Unexamined Patent Publication No. 2014-202967

The size of the raw sample to be observed and the position in the container vary, and in order to observe the desired position, it is necessary to match the optical path of the excitation light and the focal position of the observation optical system to the desired positions. be. That is, it is necessary to align the observation target position in the raw sample, the optical path of the excitation light by the excitation optical system, and the focal position of the observation optical system. In the technique described in Patent Document 1, this is realized by changing the relative position between the optical system for excitation and observation and the sample arrangement portion. However, since the positional relationship between the sample placement portion and the excitation and observation optical systems is fixed, for example, for a raw sample that is separated from the bottom surface of the container and is above (floating) or a sample that is large in the vertical direction. Is difficult to image the whole image.

Further, in the configuration using the immersion lens, since the air layer does not intervene between the sample arrangement portion and the lens, it is possible to suppress the aberration caused by refraction and obtain better image quality. However, in this configuration, it is difficult to greatly change the distance between the lens and the sample placement portion while maintaining the liquidtight state, and the degree of freedom regarding alignment is low. As a result, at least a portion of the raw sample may be out of the observable range. In addition, when removing the sample arrangement portion for exchanging a raw sample or the like, it is necessary to take measures to prevent the liquid filling the space between the lens and the lens from flowing out. As described above, the configuration using the immersion lens also has a problem that the configuration of the optical system becomes complicated and the cost tends to be high.

The present invention has been made in view of the above problems, and in the optical sheet microscope and the imaging method using the principle thereof, the raw sample and the optical system, which are the objects to be observed, while making the structure of the optical system relatively simple. It is an object of the present invention to provide a technique capable of observing the entire raw sample by increasing the degree of freedom in the alignment of the raw sample.

One aspect of the present invention is an optical sheet microscope, wherein, in order to achieve the above object, a storage container for storing a first liquid and a sample container for carrying a second liquid containing a raw sample are described. A holding portion that holds at least the bottom surface of the sample container immersed in the first liquid in the storage container and that holds the sample container so as to be relatively movable with respect to the storage container, and is arranged below the storage container. Then, the sheet light as the excitation light is emitted diagonally upward, and the sheet light is incident on the sample container through the bottom surface of the storage container and the bottom surface of the sample container, and below the storage container. Arranged, the optical axes are perpendicular to the optical path of the sheet light and intersect within the sample container, the intersection is focused, and the fluorescence excited from the raw sample is transferred to the bottom surface of the sample container and the storage. It is equipped with an observation optical system that receives light through the bottom surface of the container. Here, the bottom surface of the sample container is formed of a light-transmitting fluororesin, the first liquid has a refractive index substantially equal to that of the fluororesin, and at least a part of the bottom surface of the storage container is. An incident window that protrudes downward, has a flat incident surface that intersects the optical path of the excitation light perpendicularly to the excitation light, and is light-transmitting to the excitation light, and the optical axis of the observation optical system. An exit window having a flat exit surface perpendicular to the light and having light transmission to the fluorescence is provided.

Further, another aspect of the present invention is an imaging method in which a raw sample is fluorescently imaged, and in order to achieve the above object, a first liquid is stored in a storage container, and the raw sample is illuminated by light on the bottom surface. The first liquid is supported together with the second liquid in a sample container formed of a permeable fluororesin, and the first liquid and the fluororesin have substantially the same refractive index and are stored in the storage container. At least the bottom surface of the sample container is immersed in the liquid of the above, and the sheet light as excitation light is emitted diagonally upward from below the storage container to expose the bottom surface of the storage container and the bottom surface of the sample container. The sheet light is incident on the sample container through the sample container, and is arranged below the storage container so that the optical axis intersects the optical path of the sheet light and intersects in the sample container, and the intersection is focused. Then, a fluorescence image is imaged using an observation optical system that receives the fluorescence excited from the raw sample through the sample container and the bottom surface of the storage container. Here, at least a part of the bottom surface of the storage container protrudes downward, and the protruding portion has a flat incident surface perpendicularly intersecting the optical path of the excitation light to provide light transmission to the excitation light. An incident window having an entrance window and an exit window having a flat exit surface perpendicular to the optical axis of the observation optical system and having light transmission to the fluorescence are provided.

In the invention configured as described above, the sheet-shaped excitation light is incident on the first liquid in the storage container through the incident window provided on the bottom surface of the storage container, and further presses the bottom surface of the sample container made of fluororesin. It enters the sample container through the sample container. On the other hand, the fluorescence excited in the raw sample in the sample container is incident on the observation optical system through the bottom surface of the sample container and the exit window.

Here, the entrance window and the exit window are arranged at the protruding portion provided on the bottom surface of the storage container, and the excitation light is vertically incident on the incident surface of the entrance window, and the fluorescence generated by the excitation is emitted from the exit window. It is configured to emit vertically from the exit surface. Therefore, it is possible to minimize the deviation of the optical path and the increase in aberration caused by the refraction that occurs at the interface between the atmosphere and the bottom surface of the storage container.

Further, the bottom surface of the sample container carrying the liquid containing the raw sample is made of fluororesin, and is immersed in the first liquid having the same refractive index as the fluororesin stored in the storage container. be. Therefore, the refraction at the interface between the sample container and the first liquid can be almost ignored. That is, it can be considered that there is substantially no optical interface that causes refraction between the bottom surface of the storage container and the raw sample. Therefore, the problem of image quality deterioration due to refraction due to this interface can be eliminated.

Further, by moving the storage container and the sample container relative to each other, the relative position of the raw sample as seen from the storage container can be arbitrarily changed. As described above, since the bottom surface of the sample container in the storage container does not act as an optical interface, the raw sample can apparently move freely in the liquid in the storage container without affecting the optical path of excitation light and fluorescence. Can be made to. Therefore, any part of the raw sample can be positioned at the intersection of the path of the excitation light and the optical axis of the observation optical system and used as an observation target. If the sample container has a degree of freedom to move in various directions with respect to the storage container, the degree of freedom in positioning the raw sample is also high.

As described above, in the present invention, although the configuration of the optical system is relatively simple, the degree of freedom in positioning the raw sample as an observation object and the optical system is high, so that the entire raw sample is observed. It is possible.

It is a figure which shows the schematic structure of one Embodiment of the optical sheet microscope which concerns on this invention. It is a flowchart which shows the fluorescence imaging process. It is a figure which shows the relationship between the position of a sample container, and the position of the cross section to be imaged. It is a perspective view which shows the appearance shape of a storage container. It is a perspective view which shows the appearance shape of the modified example of a storage container. It is sectional drawing which shows the other shape example of a storage container.

FIG. 1 is a diagram showing a schematic configuration of an embodiment of an optical sheet microscope according to the present invention. This optical sheet microscope 1 has a function of acquiring an image of a raw sample such as a cell or tissue specimen. For example, a specimen into which a fluorescent reagent has been introduced can be irradiated with excitation light to capture a fluorescent image. It is possible. In this respect, it is a suitable device for carrying out the imaging method according to the present invention. Since the principle of the optical sheet microscope is known, it will not be described here. In order to show the directions in a unified manner in the following description, the XYZ Cartesian coordinate system is set as shown in FIG. Here, the XY plane is a horizontal plane, and the Z direction represents a vertical direction. More specifically, the (-Z) direction represents vertically downward.

The optical sheet microscope 1 includes a sample holding unit 20, an irradiation unit 30, a fluorescence imaging unit 40, an optical observation unit 50, and a control unit 90. The sample holding unit 20 has a function of holding the raw sample S, which is the object to be imaged, and positioning the raw sample S at an appropriate imaging position. The irradiation unit 30 irradiates the raw sample S with excitation light for exciting fluorescence. The fluorescence imaging unit 40 receives the fluorescence emitted by the raw sample S and images the fluorescence image of the raw sample S. The optical observation unit 50 is used when the user visually observes the raw sample S.

The control unit 90 has a function of controlling each of the above units. For this purpose, the control unit 90 includes a CPU 91 that executes various arithmetic processes, a memory 92 that temporarily stores data, a storage 93 that stores data obtained by a control program or imaging, and an operation from a user. A user interface unit 94 or the like that controls input reception and output of processing results is provided. Although not shown, the user interface unit 94 is provided with an input device that accepts operation input from the user and an output device that displays and outputs a message indicating the progress of processing, a processing result, and the like.

By operating these and dedicated hardware provided as needed based on a predetermined control program, the control unit 90 includes a light source control unit 95, a positioning control unit 96, a focus control unit 97, an image processing unit 98, and the like. Functional blocks are realized.

The sample holding unit 20 includes a sample container 21 that holds the raw sample S in the liquid Lq. As the liquid Lq, an appropriate liquid mainly composed of water (for example, a culture solution) can be used, and for example, a liquid Lq gelled by an appropriate gelling agent may be used. The sample container 21 is, for example, a flat-bottomed container having an open top surface, and at least the bottom surface 211 thereof is made of a fluororesin material having light transmission. Of course, the entire sample container 21 may be made of fluororesin, and in this case, in addition to the light entering and exiting through the bottom surface, which will be described later, the entrance and exit from the side surface is also permitted. Further, the shape of the sample container 21 is not limited to a flat bottom shape, and the opening may be provided as long as the raw sample S can be taken in and out. For example, the opening may be provided with a lid. When the raw sample S is formed by culturing, the raw sample S may be cultivated using the sample container 21 as a culture container, or is transferred from the culture container to the sample container 21 at the time of imaging. May be good.

The sample holding unit 20 also includes a storage container 22 having an upper surface that opens sufficiently larger than the plane size of the sample container 21 and stores water W inside. The storage container 22 has a flat bottom shape as a whole, but a protruding portion 220 that protrudes downward is provided at the center of the bottom surface 225. The protruding portion 220 is formed of a transparent material such as a polycarbonate resin or PET (polyethylene terephthalate) resin or a fluororesin material having light transmittance, and as will be described later, from the excitation light incident on the raw sample S and the raw sample. It functions as an optical window that transmits the emitted fluorescence. The storage container 22 is fixed to a device housing (not shown) by a support portion 23. For convenience of water W injection, replacement, etc., it is desirable that the storage container 22 is removable from the support portion 23.

The sample container 21 is detachably held by the holding hand 24. The holding hand 24 holds the sample container 21 in a state where the bottom surface 211 of the sample container 21 is immersed in the water W stored in the storage container 22. The holding hand 24 is connected to the positioning mechanism 25, and the positioning mechanism 25 moves the sample container 21 in the X direction, the Y direction, and the Z direction in response to a control command from the positioning control unit 96 provided in the control unit 90. Let me.

An irradiation unit 30, a fluorescence imaging unit 40, and an optical observation unit 50 are arranged below the storage container 22. More specifically, the optical observation unit 50 is arranged directly below the protrusion 220, the irradiation unit 30 is on the (+ Y) side, and the fluorescence imaging unit 40 is on the (-Y) side so as to sandwich the optical observation unit 50 in the Y direction. Are placed respectively.

The irradiation unit 30 irradiates the raw sample S with excitation light shaped into a sheet diagonally upward from below the storage container 22. Specifically, the irradiation unit 30 has a laser light source 31 that emits light having a wavelength capable of exciting the fluorescent reagent introduced into the raw sample S. A cylindrical lens 32, a collimator lens 33, and an objective lens 34 are arranged in this order along the optical path. The laser light source 31 operates in response to a control command from the light source control unit 95 of the control unit 90.

The cylindrical lens 32 changes the laser light emitted from the laser light source 31 into a sheet-like light beam that is thinly shaped in one direction orthogonal to the optical path. That is, the cylindrical lens 32 converges the laser light in one direction (in this example, the direction orthogonal to the traveling direction and the X direction of the laser light), so that the vicinity of the focal position of the cylindrical lens 32 is narrowed in the one direction. A sheet-like light beam (sheet light) having a wide flat beam cross section in the X direction is formed.

The optical path of the excitation light beam Le emitted from the objective lens 34, specifically, the path of the optical center thereof is indicated by the alternate long and short dash line L1. The excitation light beam Le is incident on the liquid Lq through the incident window 221 provided on the projecting portion 220 and transparent to the excitation light, the water W in the storage container 22, and the bottom surface 211 of the sample container 21, and is a collimator. It converges in a sheet shape at a position conjugate with the focal point of the cylindrical lens 32 with respect to the illumination optical system including the lens 33 and the objective lens 34.

The observation optical system 41 of the fluorescence imaging unit 40 is arranged so as to focus on this convergence position. More specifically, the observation optical system 41 includes an objective lens 411, a fluorescence observation filter 412, and an imaging lens 413. Then, at the above convergence position, the optical axis of the observation optical system 41 indicated by the alternate long and short dash line L2 is orthogonal to the optical path of the excitation light beam indicated by the alternate long and short dash line L1 and orthogonal to the sheet surface of the beam. It is set.

In each of the following figures, the alternate long and short dash line L1 represents the optical path of the excitation light beam Le, and the alternate long and short dash line L2 represents the optical axis of the observation optical system 41.

The focus control unit 97 of the control unit 90 can adjust the focal position of the observation optical system 41 by displacing the objective lens 411 along the optical axis direction thereof. The focus control unit 97 adjusts the observation optical system 41 so as to focus on the intersection of the optical axis of the observation optical system 41 (dashed-dotted line L2) and the optical path of the excitation light beam Le (dashed-dotted line L1).

By irradiating the raw sample S with the excitation light beam Le, fluorescence is excited from the region of the raw sample S through which the light beam Le has passed. Of the generated fluorescence, a part of the generated fluorescence transmitted below the storage container 22 through the exit window 222 provided on the protruding portion 220 and having transparency to the fluorescence and incident on the objective lens 411 was captured by the camera via the observation optical system 41. It is received by 42. That is, the camera 42 can take a fluorescence image of the raw sample S.

The excitation light beam Le is shaped into a sheet, and the optical axis of the observation optical system 41 is arranged so as to be orthogonal to the sheet surface of the excitation light beam Le and to be in focus on the sheet surface. Therefore, a fluorescence image of one cross section of the raw sample S having the sheet surface as the cross section is imaged by the camera 42. The fluorescence observation filter 412 can selectively receive only the fluorescence of a specific wavelength component among the light emitted from the raw sample S.

In this way, the optical sheet microscope 1 can acquire a fluorescence image of one cross section of the raw sample S. Then, by synthesizing the fluorescence images of different cross sections by image processing, it is possible to create a three-dimensional fluorescence image of the raw sample S. Such image processing is executed by the image processing unit 98 of the control unit 90.

In order to realize the above-mentioned imaging, it is necessary to arrange the raw sample S at the intersection of the optical path of the excitation light beam Le and the optical axis of the observation optical system 41. Further, in order to acquire fluorescence images having different cross sections and create a three-dimensional image, it is necessary to perform a plurality of imagings by sequentially changing the passing position of the excitation light beam Le with respect to the raw sample S.

In order to perform good imaging, it is necessary to align the optical path of the excitation light beam Le, the optical axis of the observation optical system 41, and the raw sample S relative to each other. Specifically, the optical path of the excitation light beam Le and the optical axis of the observation optical system 41 are vertically intersected, and the observation optical system 41 is focused on the intersection, and the intersection and the raw sample S are aligned with each other. It is necessary that the relative positional relationship can be set arbitrarily. Hereinafter, the configuration of the present embodiment for responding to this request will be described.

In this embodiment, the excitation light beam Le is incident on the storage container 22 through the incident window 221 on the bottom surface of the storage container 22. The outer main surface of the incident window 221 which is the interface with the external atmosphere is a flat incident surface 221a, and the optical path of the excitation light beam Le is such that the excitation light beam Le is incident perpendicularly to the incident surface 221a. And the inclination of the incident surface 221a are set. With such a structure, it is possible to prevent the optical path of the excitation light beam Le from being bent by refraction at the incident surface 221a. The inclination of the incident surface 221a with respect to the horizontal plane can be, for example, 45 degrees.

Similarly, the outer main surface of the exit window 222, which is an interface with the external atmosphere, is a flat exit surface 222a. Since the optical path of the excitation light beam Le and the optical axis of the observation optical system 41 are orthogonal to each other, it is desirable that the entrance surface 221a and the emission surface 222a are perpendicular to each other. In this example, the inclination of the exit surface 222a with respect to the horizontal plane may be 45 degrees. The optical axis of the observation optical system 41 is set to intersect the exit surface 222a perpendicularly. As a result, the aberration of the observation optical system 41 in the image pickup through the exit window 222 can be suppressed to a small value, and the image pickup can be performed with good image quality.

As described above, since the excitation light beam Le is incident perpendicularly to the incident surface 221a and the optical axis of the observation optical system 41 is orthogonal to the exit surface 222a, the optical path due to refraction on the incident surface 221a and the exit surface 222a Increased deviation and aberration are avoided. Therefore, it is possible to ensure that the optical path of the excitation light beam Le and the optical axis of the observation optical system 41 are orthogonal to each other, and the adjustment work for that purpose is also performed as compared with the case where the bottom surface of the container and the optical axis are obliquely crossed. It's relatively easy.

As the optical characteristics required for the entrance window 221 and the exit window 222, it is required to have at least transparency to the excitation light and the fluorescence emitted from the raw sample S. From this point, various transparent materials can be used. In particular, if a material having a refractive index equivalent to that of water is used, the difference in the refractive index at the interface with the water W stored in the storage container 22 can be reduced to a level that can be almost ignored. For example, a fluororesin can be preferably applied.

It should be noted that there is no particular requirement regarding the optical characteristics of each part of the storage container 22 other than the protruding portion 220 provided with the incident window 221 and the exit window 222. Therefore, only the protruding portion 220 may be made of the material having the above-mentioned optical characteristics, and the other parts may be made of other appropriate materials. Since the storage container 22 is required to have a size sufficient to accommodate the sample container 21 and a mechanical strength capable of storing an amount of water W commensurate with the size, there are a portion required to have predetermined optical characteristics and a portion other than that. By separating the above, the degree of freedom in selecting each material can be increased.

Further, the raw sample S is supported on the sample container 21 whose bottom surface is immersed in the water W in the storage container 22, and the excitation light beam Le is incident on the raw sample S and the fluorescence generated from the raw sample S is received. Is performed via the bottom surface 211 of the sample container 21. The bottom surface 211 of the sample container 21 is made of fluororesin, and both sides thereof are covered with water or a liquid mainly composed of water. Since the bottom surface 211 is made of a fluororesin material having a refractive index (about 1.33) substantially equal to that of water, the existence of an optical interface between the bottom surface 211 and the liquid can be almost ignored. That is, it can be considered that the bottom surface of the storage container 22 and the raw sample S are optically filled with a substantially uniform medium having no interface. Therefore, the excitation light beam Le incident on the sample S through the incident window 221 and the fluorescent optical path emitted from the sample S through the exit window 222 are not bent by refraction in the storage container 22.

In order to reduce the refraction of the surface 221b of the incident window 221 on the side in contact with the water W, it is desirable that the surface 221b is orthogonal to the optical path of the excitation light beam Le, that is, parallel to the incident surface 221a. Similarly, in order to reduce refraction on the surface 222b of the exit window 222 on the side in contact with the water W, this surface 222b must be orthogonal to the optical axis of the observation optical system 41, that is, parallel to the exit surface 222a. desirable. However, when the entrance window 221 and the exit window 222 are made of a material having a refractive index substantially equal to that of water (for example, fluororesin), refraction on these surfaces 221b and 222b is not a problem.

When the entrance window 221 and the exit window 222 are made of fluororesin, it is not necessary that the entire storage container 22 is made of fluororesin. For example, the entire protruding portion 220 including the incident window 221 and the exit window 222 may be integrally formed of a fluororesin material, and may be fitted into an opening provided in the center of the bottom surface of the main body of the storage container 22. By doing so, it is possible to satisfy predetermined optical characteristics of the incident window 221 and the exit window 222 through which light is transmitted while maintaining the strength of the storage container 22 as a whole. Further, by using different materials for the part that requires mechanical strength and the part that should give priority to optical characteristics, it is possible to optimize the cost allocation of the device. Further, the number of parts can be reduced by integrally forming the protruding portion 220 including the incident window 221 and the exit window 222.

Next, the alignment of the optical path and the raw sample S is realized by moving the sample container 21 by the positioning mechanism 25. As described above, the existence of the sample container 21 is optically negligible, but mechanically, the raw sample S can be moved to the storage container 22 while being held inside. There is. Specifically, the operation of the positioning mechanism 25 moves the holding hand 24 that holds the sample container 21, so that the sample container 21 moves in various directions while the bottom surface 211 is immersed in the water W of the storage container 22. do. At this time, the raw sample S moves in the storage container 22 together with the sample container 21.

That is, by changing the position of the sample container 21 in the storage container 22, the position of the raw sample S with respect to the optical path of the excitation light beam Le and the fluorescence can be arbitrarily changed. Is. Therefore, by positioning the sample container 21 so that the imaged portion of the raw sample S is located at the intersection of the optical path of the excitation light beam Le and the optical axis of the observation optical system 41, the space between the optical path and the raw sample S is reached. It is possible to align the.

The optical observation unit 50 can be used in order for the user to smoothly perform such alignment. The optical observation unit 50 has an objective lens 51, an imaging lens 52, and a camera 53 arranged directly below the storage container 22. Specifically, the objective lens 51 has a substantially vertical optical axis, and is provided so as to face a substantially horizontal and transparent observation window 223 provided at the lower end of the protruding portion 220 at the lower part of the storage container 22. The imaging lens 52 forms an image of the light focused by the objective lens 51 on the light receiving surface of the camera 53. Therefore, the image captured by the camera 53 allows the user to visually observe the position of the raw sample S in the sample container 21.

While confirming the image captured by the camera 53, the user gives an instruction input for locating a desired position in the imaging range to the positioning control unit 96 via the UI unit 94. In response to this, the positioning control unit 96 operates the positioning mechanism 25 to move the sample container 21, and as a result, the raw sample S moves according to the user's instruction. In this way, the alignment of the raw sample S can be performed.

According to the above configuration, the sample container 21 containing the raw sample S moves with respect to the storage container 22 for alignment with the raw sample S, while the irradiation unit 30 and the fluorescence imaging unit 40 No need to move. Since the object to be moved is only the small and lightweight sample container 21 and it is not necessary to move the heavy object, the mechanism for alignment can be compactly configured.

Further, the positional relationship between the storage container 22, the irradiation unit 30, and the fluorescence imaging unit 40 is unchanged. Therefore, when changing the imaging position, it is not necessary to align between them, and the alignment work becomes simple. Further, the positional relationship between the irradiation unit 30 and the incident window 221 and the positional relationship between the observation optical system 41 and the exit window 222 are also unchanged. That is, the incident position of the excitation light beam Le with respect to the incident window 221 and the position where the optical axis of the observation optical system 41 intersects with the exit window 222 are also constant regardless of the position of the raw sample S. Therefore, the positions and sizes of the entrance window 221 and the exit window 222 can be uniquely determined regardless of the size of the movable range of the sample container 21 with respect to the storage container 22. As a result, it is not necessary to take these areas larger than necessary, and it is possible to suppress the increase in size of the storage container 22.

In the sample container 21, the raw sample S can exist at various positions in the horizontal direction and the vertical direction. Even in such a case, the raw sample S is positioned at the intersection of the optical path of the excitation light beam Le and the optical axis of the observation optical system 41 as long as the movable range of the sample container 21 in the storage container 22 allows. It is possible to make an image. Further, even when a plurality of raw samples S as imaging objects are contained in the sample container 21, by moving the sample container 21, they can be sequentially moved to the imaging range for imaging.

Further, the excitation light beam Le incident on the storage container 22 is vertically incident on the incident window 221 and the optical path of fluorescence emitted from the storage container 22 and incident on the observation optical system 41 is perpendicular to the optical axis of the observation optical system 41. be. Therefore, it is possible to suppress an increase in optical path deviation and aberration caused by a difference in refractive index between the bottom surface of the storage container 22 and the external space (atmosphere), and to perform imaging with good image quality. Therefore, maintenance of the irradiation unit 30 and the fluorescence imaging unit 40 is easy.

Next, the fluorescence imaging process using the optical sheet microscope 1 configured as described above will be described. This fluorescence imaging process is a process of fluorescently imaging a prepared raw sample S and creating a three-dimensional fluorescence image thereof based on the imaging data. The main part of this processing is realized by the CPU 91 of the control unit 90 executing a control program prepared in advance and causing each part of the device to perform a predetermined operation. It is assumed that the fluorescent reagent has been introduced into the raw sample S in advance.

FIG. 2 is a flowchart showing a fluorescence imaging process. First, the raw sample S, which is the object to be imaged, is housed in the sample container 21 (step S101). The sample container 21 is detachable from the support hand 24, and the work for accommodating the raw sample S can be performed with the sample container 21 removed from the support hand 24. The raw sample S may be prepared externally and stored in the sample container 21, or may be cultured in the sample container 21 as a culture container.

The sample container 21 for accommodating the raw sample S is attached to the support hand 24, and the support hand 24 holds the sample container 21 in a state where the bottom surface of the sample container 21 is immersed in the storage container 22 filled with water W ( Step S102). Next, the position of the sample container 21 is adjusted (step S103).

Specifically, the user confirms the image captured by the camera 53 of the optical observation unit 50, and instructs the control unit 90 via the UI unit 94 so that the desired portion of the raw sample S falls within the imaging range. Give input. In response to this, the positioning control unit 96 operates the positioning mechanism 25 to move the sample container 21. In this way, the user positions the raw sample S.

After positioning, imaging is performed (step S104). Specifically, the excitation light beam Le converged in a sheet shape from the irradiation unit 30 is irradiated to the raw sample S, and the fluorescence excited in the raw sample S is received by the camera 42 via the observation optical system 41. .. As a result, a fluorescence image of the raw sample S having the sheet surface of the excitation light beam Le as a cross section is acquired. When a plurality of fluorescence reagents having different emission wavelengths are introduced and these fluorescence images are individually imaged, the wavelength of the excitation light and the fluorescence observation filter 412 are exchanged according to the fluorescence reagent to be imaged. Imaging may be performed each time.

In order to create a three-dimensional fluorescence image, it is necessary to take images of a plurality of cross sections that are different from each other. In this embodiment, the positioning mechanism 25 moves the sample container 21 to move the raw sample S along the optical axis direction of the observation optical system 41. Specifically, after imaging one cross section, it is determined whether or not imaging of the next cross section is necessary (step S105), and if necessary (YES in step S105), the position of the sample container 21 is determined. It is moved by a predetermined distance in the optical axis direction (step S106). As a result, only the cross section to be imaged moves one step in the optical axis direction while the focal point of the observation optical system 41 remains in focus on the sheet surface of the excitation light beam Le.

By performing imaging again in this state (step S104), a fluorescence image of the sample S in a new cross section is acquired. This process is repeated until a fluorescence image having a cross section necessary and sufficient to form a three-dimensional image is obtained (NO in step S105).

FIG. 3 is a diagram showing the relationship between the position of the sample container and the position of the cross section to be imaged. As shown in FIG. 3A, when the raw sample S is positioned at an appropriate position, the optical path of the excitation light beam Le (dashed line L1) and the optical axis of the observation optical system 41 (dashed line L2) Imaging is performed on one cross section S1 represented by a thick line, which includes the intersection of the above and is perpendicular to the optical axis. After that, the sample container 21 is moved by a predetermined distance along the direction Dm parallel to the optical axis of the observation optical system 41. As a result, as shown in FIG. 3B, the position of the raw sample S that the excitation light beam Le crosses moves by a predetermined distance along the optical axis of the observation optical system 41. Therefore, in the next imaging, a fluorescence image of another cross section S2 that is separated from the previously imaged cross section S1 by a predetermined distance in the optical axis direction is imaged.

Looking at the movement of the raw sample S from the raw sample S, the incident position of the excitation light beam Le with respect to the raw sample S and the focal position of the observation optical system 41 sequentially move along the optical axis direction of the observation optical system 41. I will continue to do it.

When the position of the sample container 21 is further moved by a predetermined distance along the direction Dm, as shown in FIG. 3C, in the next imaging, the cross section S2 previously imaged is predetermined in the optical axis direction. A fluorescence image of another cross section S3 separated by a distance will be imaged. In this way, a plurality of cross-sectional images having different depths along the optical axis direction are acquired. The sample container S is not limited to the movement along the direction Dm parallel to the optical axis of the observation optical system 41, and can move in any direction in the XYZ direction. For example, after imaging at each position, it is possible to create a three-dimensional fluorescence image of the raw sample S from a plurality of fluorescence images based on their moving directions and moving distances. This makes it possible to arbitrarily select and observe the portion of the raw sample S to be observed.

Since the excitation light is shaped into a sheet, the excitation light is incident on the raw sample S in one imaging only in a thin region along the sheet surface of the excitation light. In a plurality of imagings, the position of the sheet surface passing through the raw sample S moves in sequence, so that the same location in the raw sample S is not repeatedly irradiated with the excitation light. Therefore, deterioration of the raw sample S due to fading of the fluorescent reagent and phototoxicity can be minimized. Further, since fluorescence is generated only from a limited region, that is, a cross section to be imaged in one imaging, it is possible to suppress noise caused by mixing of fluorescence from other than the cross section and obtain a clear image.

Returning to FIG. 2, the description of the imaging process will be continued. When the raw sample S contained in one sample container 21 is continuously imaged at another position (YES in step S107), the process returns to step S103, the position adjustment by the user is accepted again, and the above-mentioned imaging is performed. conduct.

When all the necessary imaging is completed (NO in step S107), the image processing unit 98 creates a three-dimensional image of the raw sample S based on the imaging data obtained in each imaging so far (step S108). Since image processing for reconstructing a three-dimensional image from image data of a cross-sectional image is known, description thereof will be omitted here. As described above, the cross section and the three-dimensional fluorescence image of the raw sample S in the sample container 21 can be obtained.

Next, the structure of the storage container 22, particularly the shape of the protrusion 220 will be described in more detail. The storage container 22 can store the liquid in the internal space where the upper part opens. The plane size is such that the sample container 21 housed in the internal space can move within a predetermined movable range. Further, the bottom surface 225 is provided with a protruding portion 220 that projects downward and whose side surfaces serve as an incident window 221 and an exit window 222. The cross-sectional shape of the storage container 22 having a plane orthogonal to the X-axis as a cut surface is as shown in FIG.

FIG. 4 is a perspective view showing the external shape of the storage container. More specifically, FIG. 4A shows the internal structure when the storage container 22 is viewed from diagonally above, and FIG. 4B shows the internal structure when the storage container 22 is viewed from diagonally below. There is. The central portion of the bottom surface 225 of the storage container 22 projects downward to form a protruding portion 220. The lower surface of the projecting portion 220 serves as the observation window 223, while the side surface on the (-Y) side having an inclination of 45 degrees constitutes the exit window 222 and the side surface on the (+ Y) side constitutes the incident window 221. ing.

As shown in FIG. 4B, the outer surface 221a of the incident window 221 when viewed from the inside of the storage container 22 constitutes a substantially flat incident surface. On the other hand, the outer surface 222a of the exit window 222 when viewed from the inside of the storage container 22 constitutes a substantially flat exit surface. As described above, since the incident position of the excitation light beam Le with respect to the incident window 221 and the optical axis position of the observation optical system 41 with respect to the exit window 222 are basically unchanged, the incident surface 221a is present at these positions and around them. And the exit surface 222a may be flat.

FIG. 5 is a perspective view showing an external shape of a modified example of the storage container. More specifically, FIG. 5A shows the internal structure of the storage container 22A of this modified example when viewed from diagonally above, and FIG. 5B shows the storage container 22A when viewed from diagonally below. Shows the internal structure. In this modification, the protruding portion 220A provided on the bottom surface of the storage container 22A extends from one end to the other end of the storage container 22A in the X direction in the X direction. That is, the cross-sectional shape of the storage container 22A on the cut surface orthogonal to the X axis is constant except for both ends in the X direction.

In such a structure, the incident window 221A and the exit window 222A are wider in the X direction as compared with the structure shown in FIG. However, as described above, since the incident position of the excitation light beam Le and the optical axis position of the observation optical system 41 are unchanged, it is necessary to expand the incident window 221A and the exit window 222A in this way from the viewpoint of imaging technology. Is not always. For example, such a shape may be adopted when it is advantageous in terms of manufacturing. For example, when the protruding portion 220A and the other storage container 22A are formed as separate bodies, the shape of FIG. 5 is advantageous in that each portion can be formed by continuous extrusion molding. ..

FIG. 6 is a cross-sectional view showing another shape example of the storage container. The storage container 22 shown in FIGS. 1 and 4 has an observation window 223 horizontal to the protrusion 220. The observation window 223 can be expanded in function by arranging an optical system for visual observation or an optical system of an imaging device based on another imaging principle, for example, an optical coherence tomography (OCT) imaging device. However, it is not an indispensable configuration for performing optical sheet microscope imaging. Therefore, for example, as in the storage container 22B shown in FIG. 6A, the observation window may be omitted, and the projecting portion 220B may be formed by the incident window 221B and the exit window 222B.

Further, since the incident position of the excitation light beam Le with respect to the storage container and the optical axis position of the observation optical system 41 are constant, the incident window and the exit window may be made smaller. For example, as in the storage container 22C shown in FIG. 6B, the protruding portion 220Ca corresponding to the small-sized incident window 221C and the protruding portion 220Cb corresponding to the exit window 222C are individually provided on the bottom surface 225C of the storage container 22C. It may be provided. In this case, the sizes of the two protrusions 220Ca and 220Cb do not have to be the same. Further, the horizontal portion between the two protrusions 220Ca and 220Cb can be used as the observation window 223C.

Further, in the storage container 22 of the above embodiment, both the incident surface 221a of the incident window 221 and the exit surface 222a of the exit window 222 have an inclination of 45 degrees with respect to the horizontal plane. However, the incident surface and the exit surface need not be the same as long as they are perpendicular to each other. For example, as in the storage container 22D shown in FIG. 6C, the angle θ1 formed by the incident surface 221Da and the angle θ2 formed by the exit surface 222Da with respect to the horizontal plane may be different from each other. In such a configuration, the areas of the entrance window 221D and the exit window 222D can be made different from each other. For example, when it is necessary to make the size (diameter) of one of the objective lens 34 of the irradiation unit 30 and the objective lens 411 of the observation optical system 41 larger than the other due to the optical characteristics, the shape of the protrusion 220D is set as this. Such a shape can be adopted.

As shown in these modifications, various shapes of protrusions formed at the bottom of the storage container for providing the entrance window and the exit window can be considered, and the above shows some examples thereof. Is. It is conceivable to dent the bottom of the storage container upward and provide an incident window and an exit window there, but as in the above example, the movable range of the sample container can be increased without interfering with the sample container. A structure that projects downward is more advantageous.

As a technical idea similar to the above, it is also conceivable to consider a configuration in which an incident window and an exit window are provided on the bottom surface of the sample container that supports the raw sample, instead of adopting the double structure of the sample container and the storage container as described above. Be done. Even in this case, the imaging position can be changed by moving the sample container. However, due to this movement, the distance between the incident window of the sample container and the objective lens of the irradiation unit and the distance between the exit window of both samples and the objective lens of the observation optical system are different from the distance assumed in the optical design. This causes aberrations to occur. Further, depending on the position of the raw sample in the sample container, it may be impossible to position the raw sample at the intersection of the excitation light and the optical axis of the observation optical system. In order to enable positioning with a high degree of freedom while suppressing the occurrence of aberrations, it is effective to have a double structure as in this embodiment.

Further, in the above embodiment, the entrance surface 221a of the entrance window 221 intersects the excitation light beam Le, and the emission surface 222a of the emission window 222 intersects the optical axis of the observation optical system 41 perpendicularly to each other. Of these, the exit surface 222a is strongly required to be perpendicular to the optical axis of the observation optical system 41 in order to minimize the deterioration of image quality due to lens aberration. On the other hand, with respect to the incident surface 221a, it is sufficient that the light beam converged in a sheet shape can be incident on a predetermined position in the storage container 22 from a predetermined direction, and it is considered that the light beam does not necessarily have to be perpendicular to the optical path of the light beam. ..

In particular, in the above embodiment, the incident position and direction of the excitation light beam Le with respect to the incident surface 221a do not change. Therefore, it is possible to design an optical system in which the excitation light beam Le is incident at a predetermined position in the storage container 22 at a predetermined angle, taking into consideration the influence of refraction in the incident window 221. In this sense, the incident surface may not be perpendicular to the optical path of the light beam, but unless there are special circumstances, it is considered that there is no advantage in doing so.

As described above, in the optical sheet microscope 1 of the above embodiment, the storage container 22 and the sample container 21 function as the “storage container” and the “sample container” of the present invention, respectively. Then, the holding hand 24 and the positioning mechanism 25 integrally function as the "holding portion" of the present invention. Further, the irradiation unit 30 functions as the "irradiation unit" of the present invention. Further, the observation optical system 41 functions as the "observation optical system" of the present invention, while the camera 42 functions as the "imaging unit" of the present invention. Further, the protruding portion 220 in the present embodiment corresponds to the "protruding portion" of the present invention. Further, in the above embodiment, the water W corresponds to the "first liquid" of the present invention, and the liquid Lq corresponds to the "second liquid" of the present invention.

The present invention is not limited to the above-described embodiment, and various modifications other than those described above can be made without departing from the spirit of the present invention. For example, in the above description, the sample container 21 is treated as a part of the apparatus configuration of the optical sheet microscope 1. However, the sample container may be treated as an independent product separate from the optical sheet microscope. For example, if there is a commercially available culture container or sample container whose bottom surface is made of fluororesin, it can be used as the sample container of the present embodiment.

Further, in the above embodiment, the raw sample S is directly contained in the liquid Lq in the sample container 21, but instead, the raw sample is supported on a carrier such as a sheet, a tube, or a mesh. It may be contained in a sample container in a state. In this case, in order to suppress the influence of these carriers on imaging, it is desirable that the carriers are made of a material having a refractive index substantially equal to that of the liquid in the sample container. For example, a fluororesin material is suitable.

In such a case, the raw sample S may exist above the bottom surface of the sample container 21. In the present embodiment, the position of the raw sample S can be greatly moved by changing the position of the sample container 21 in the storage container 22, so that the raw sample S is appropriate even in such a case. It is possible to perform imaging by positioning it at the imaging position, that is, at the intersection of the excitation light and the optical axis of the observation optical system.

Further, the irradiation unit 30 of the above embodiment converges the laser beam with the cylindrical lens 32 and shapes it into a sheet shape. On the other hand, as a light source for generating sheet light for an optical sheet microscope, a light source that scans a laser beam in one direction to form a sheet is also known. It is possible to apply such a light source also in this embodiment.

Further, in the above embodiment, the alignment of the raw sample S with respect to the imaging system is realized by moving the sample container 21 with respect to the storage container 22. However, the alignment of the optical system and the raw sample S is also possible by integrally moving the storage container 22, the irradiation unit 30, and the fluorescence imaging unit 40 without moving the sample container 21. .. However, it is clearly advantageous as an apparatus configuration to move the sample container 21 and fix the other as in the above embodiment.

Further, for example, in order to change the position of the intersection of the optical path of the excitation light beam Le and the optical axis of the observation optical system 41, at least one of the irradiation unit 30 and the fluorescence imaging unit 40 is moved with respect to the storage container 22. An adjustment mechanism may be added. In this way, for example, it becomes possible to deal with a sample container that could not be used until now due to its shape. Also in this case, the excitation light beam Le is required to be vertically incident on the incident window 221 and the optical axis of the observation optical system 41 to intersect the exit window perpendicularly.

Further, in the above embodiment, the liquid in the storage container 22 is water, but this liquid is not limited to water. That is, according to the technical idea of the present invention, it suffices if it can be considered that there is substantially no optical interface between the bottom surface of the storage container 22 and the raw sample S, and various liquids satisfying this can be provided in the storage container 22. Can be stored. For example, a liquid having substantially the same composition as the liquid in the sample container may be used. As described above, if the liquid in the sample container 21 and the liquid in the culture container 22 are mainly water and have light transmission to excitation light and refraction, these liquids and a fluororesin are used. The refractive index is almost the same as that of the bottom surface 211 of the sample container, and it can be considered that there is virtually no interface between them.

As described above by exemplifying a specific embodiment, the optical sheet microscope according to the present invention may be configured such that the entrance surface and the emission surface are orthogonal to each other. According to such a configuration, the optical path of the sheet light incident perpendicularly to the incident surface and the optical axis of the observation optical system perpendicularly intersecting the exit surface intersect each other perpendicularly, and from the principle of the optical sheet microscope, It is possible to obtain good image quality.

Further, for example, the entrance window and the exit window may be formed of a fluororesin having light transmission. According to such a configuration, the refractive index can be made substantially the same between the incident window and the exit window and the water stored in the storage container, and the influence of refraction at these interfaces can be almost eliminated. can.

Further, for example, the first liquid may be a liquid containing water as a main component and having light transmission to excitation light and fluorescence. According to such a configuration, since the refractive index is substantially equal between the first liquid and the fluororesin bottom surface of the sample container, it can be considered that there is no interface optically. On the other hand, with respect to the second liquid, it is the highest priority to support the raw sample S in a good state, but it is desirable that the second liquid has the same conditions as the first liquid described above.

Further, for example, the optical sheet microscope and the imaging method according to the present invention may be configured to move the sample container relative to the storage container to change the focusing position of the observation optical system with respect to the raw sample in multiple stages. .. According to such a configuration, fluorescence images having different cross sections can be obtained in the focused state. Such a fluorescence image of each cross section can be used to create a three-dimensional fluorescence image of a raw sample. That is, in the optical sheet microscope and the imaging method according to the present invention, it is possible to create a three-dimensional fluorescence image of a raw sample from a plurality of fluorescence images having different focusing positions.

Further, the sample container may be detachable from the holding portion. According to such a configuration, various raw samples can be observed by replacing the raw sample together with the sample container. It is also possible to use a culture container for culturing cells and the like as a sample container of the present invention.

INDUSTRIAL APPLICABILITY The present invention can be suitably used for the purpose of fluorescence observation and imaging of raw samples such as cell and tissue specimens, and is particularly suitable for the use of creating a three-dimensional fluorescence image of those raw samples.

1 Optical sheet microscope 21 Sample container 22 Storage container 24 Holding hand (holding part)
25 Positioning mechanism (holding part)
30 Irradiation unit (irradiation part)
41 Observation optical system 42 Camera (imaging unit)
98 Image processing unit 220 Protruding part (protruding part)
221 Incident window 221a Incident surface 222 Exit window 222a Exit surface Le Excitation light beam (sheet light)
Lq liquid (second liquid)
W water (first liquid)

Claims (10)

A storage container for storing the first liquid,
A sample container carrying a second liquid containing a raw sample is placed in a state where at least the bottom surface of the sample container is immersed in the first liquid in the storage container, and the sample container is placed in the storage container. A holding part that holds it so that it can be moved relative to each other,
An irradiation unit that is arranged below the storage container, emits sheet light as excitation light diagonally upward, and causes the sheet light to enter the sample container via the bottom surface of the storage container and the bottom surface of the sample container. ,
Arranged below the storage vessel, the optical axis is perpendicular to the optical path of the sheet light and intersects within the sample vessel, and the intersection is focused on the fluorescence excited from the raw sample. It is provided with an observation optical system that receives light through the bottom surface of the container and the bottom surface of the storage container.
The bottom surface of the sample container is formed of a fluororesin having light transmittance, and the first liquid has a refractive index substantially equal to that of the fluororesin.
At least a part of the bottom surface of the storage container protrudes downward, and an incident window having a flat incident surface perpendicular to the optical path of the excitation light and having light transmission to the excitation light is provided at the protruding portion. And an emission window having a flat emission surface perpendicular to the optical axis of the observation optical system and having light transmission to the fluorescence.
Optical sheet microscope. The optical sheet microscope according to claim 1, wherein the entrance surface and the exit surface are orthogonal to each other. The optical sheet microscope according to claim 1 or 2, wherein the incident window and the exit window are formed of a fluororesin having light transmittance. The optical sheet microscope according to any one of claims 1 to 3, wherein the first liquid is a liquid containing water as a main component and having light transmission to the excitation light and the fluorescence. The optical sheet according to any one of claims 1 to 4, wherein the holding portion moves the sample container relative to the storage container to change the focusing position of the observation optical system with respect to the raw sample in multiple stages. microscope. An imaging unit that captures a plurality of fluorescence images having different focusing positions, and an imaging unit.
The optical sheet microscope according to claim 5, further comprising an image processing unit that creates a three-dimensional fluorescence image of the raw sample from the plurality of fluorescence images. The optical sheet microscope according to any one of claims 1 to 6, wherein the sample container is removable from the holding portion. This is an imaging method in which a raw sample is fluorescently imaged.
The first liquid is stored in the storage container,
The raw sample is supported together with the second liquid in a sample container whose bottom surface is made of a fluororesin having light transmission, and the first liquid and the fluororesin have substantially the same refractive index.
At least the bottom surface of the sample container is held in a state of being immersed in the first liquid stored in the storage container.
Sheet light as excitation light is emitted obliquely upward from below the storage container, and the sheet light is incident on the sample container via the bottom surface of the storage container and the bottom surface of the sample container.
Arranged below the storage vessel, the optical axis is perpendicular to the optical path of the sheet light and intersects within the sample vessel, and the intersection is focused on the fluorescence excited from the raw sample. A fluorescence image is imaged using an observation optical system that receives light through the container and the bottom surface of the storage container.
At least a part of the bottom surface of the storage container protrudes downward, and an incident window having a flat incident surface perpendicularly intersecting the optical path of the excitation light and having light transmission to the excitation light is provided at the protruding portion. And an emission window having a flat emission surface perpendicular to the optical axis of the observation optical system and having light transmission to the fluorescence.
Imaging method. The imaging method according to claim 8, wherein the sample container is relatively moved with respect to the storage container, the focusing position of the observation optical system with respect to the raw sample is changed in multiple stages, and imaging is performed each time. The imaging method according to claim 9, wherein a three-dimensional fluorescence image of the raw sample is created from a plurality of fluorescence images having different focusing positions.

Images