This application is based on and claims priority to Japanese Patent Application No. 2016-238447 filed on Dec. 8, 2016, the entire content of which is incorporated herein by reference.
The present invention relates to a microplate and a microscope system.
There is a well known light-sheet microscope that can acquire images of a plurality of samples successively in order to enhance the throughput for image acquisition in a case where the images are used in drug development screening or in vitro diagnosis (refer to, for example, PTL 1).
In this light-sheet microscope, a deflector for introducing sheet-shaped illumination light and an objective lens for collecting fluorescence generated in the samples are inserted, from above, into a container that has an opening at the top and that accommodates a plurality of samples.
U.S. Unexamined Patent Application Publication No. 2016/153892
An aspect of the present invention provides a microplate including: a plurality of container sections having wells that open at a first plane and that accommodate samples; and a connection section connecting a plurality of rows of the container sections so as to be arrayed in a manner in which the rows are spaced from each other in a direction along the first plane, wherein each of the container sections includes: at least one side wall section that is optically transparent at at least a portion thereof; and a bottom surface section that is disposed on a second plane on a side opposite from the first plane and that is optically transparent at at least a portion thereof, and wherein a recessed section is formed between neighboring rows of the container sections, and the recessed section allows an optical member to be inserted thereinto crossing the second plane, wherein the said optical member introduces sheet-shaped illumination light into the well via the side wall section in a direction substantially parallel to the first plane.
In addition, another aspect of the present invention provides a microscope system including: the above-described microplate; and a microscope which acquires an observation image of the sample accommodated in the well of the microplate, wherein the microscope includes: a movable stage which supports the microplate so as to be movable at least in a horizontal direction; the optical member that is inserted from a lower side into the recessed section formed between the neighboring rows of the container sections of the microplate, that introduces illumination light coming from a light source and from a lower side of the microplate, and that bends the illumination light to enter the well via the side wall section as the sheet-shaped illumination light extending in a horizontal direction; an objective lens that collects, via the bottom surface section, light generated in the sample as a result of being irradiated with the illumination light; and an image acquisition unit which acquires an image of the light collected by the objective lens.
In addition, another aspect of the present invention provides a microscope system including: the above-described microplate; and a microscope which acquires an observation image of the sample accommodated in the well of the microplate, wherein the microscope includes: a movable stage which supports the microplate so as to be movable at least in a horizontal direction; two of the optical members that are inserted from a lower side into the recessed sections formed between the neighboring rows of the container sections of the microplate, that introduce illumination light from light sources and from a lower side of the microplate, and that bend the illumination light to enter the well via the two side wall sections as the sheet-shaped illumination light extending in a horizontal direction; an objective lens that collects, via the bottom surface section, light generated in the sample as a result of being irradiated with the illumination light; and an image acquisition unit which acquires an image of the light collected by the objective lens.
In addition, another aspect of the present invention provides a microscope system including: the above-described microplate; and a microscope which acquires an observation image of the sample accommodated in the well of the microplate, wherein the microscope includes: a movable stage which supports the microplate so as to be movable at least in a horizontal direction; the optical member that is inserted from a lower side into the recessed section formed between the neighboring rows of the container sections of the microplate, that introduces illumination light coming from a light source and from a lower side of the microplate, and that bends the illumination light to enter the well via the side wall section as the sheet-shaped illumination light extending in a horizontal direction; an objective lens that collects, via the bottom surface section, light generated in the sample as a result of being irradiated with the illumination light; and an image acquisition unit which acquires an image of the light collected by the objective lens, and wherein the movable stage rotates the microplate about the axial line.
A microscope system 1 and a microplate 2 according to an embodiment of the present invention will now be described with reference to the drawings.
As shown in
As shown in
In this embodiment, each row of the container sections 5 has the common planar side wall section 7 extending in a direction orthogonal to the connection section 6. In addition, the bottom surface sections 8 are disposed orthogonal to the side wall section 7. With this configuration, when the connection section 6 is disposed substantially horizontally with the openings in the wells 4 oriented upward, the side wall sections 7 of the container sections 5 are disposed in a plane extending in a substantially vertical direction, and the bottom surface sections 8 are disposed so as to extend in a substantially horizontal direction.
As shown in
The movable stage 9 is configured to be capable of moving the mounted microplate 2 three-dimensionally.
The illumination optical system 11 includes: a collimator lens 14 for converting excitation light coming from the light source 10 into substantially collimated light; a cylindrical lens 15 for focusing the excitation light converted into collimated light in one direction; an optical member 16 that bends, through deflection, the sheet-shaped excitation light focused by this cylindrical lens 15 and introduces the sheet-shaped excitation light to the well 4 via the side wall section 7 of a container section 5.
The optical member 16 includes two mirrors 17 each of which deflects the excitation light by substantial 90°. The horizontal position of the optical member 16 relative to the objective lens 12 is set so that the focal position of the sheet-shaped excitation light intersects the optical axis of the objective lens 12.
The objective lens 12 includes a focusing mechanism, which is not shown in the figure, for moving this objective lens 12 up/down along the optical axis thereof.
The image acquisition element 13 is a two-dimensional sensor, such as a CCD or a CMOS imaging device.
As shown in
The operation of the microscope system 1 according to this embodiment with the above-described structure will be described below.
In order to observe samples X using the microscope system 1 according to this embodiment, a predetermined medium is stored in each of the wells 4, and then the microplate 2 accommodating the samples X immersed in these media is placed on the movable stage 9.
Subsequently, the microplate 2 is moved by operating the movable stage 9 so that the objective lens 12 is disposed vertically below the bottom surface section 8 of the container section 5 accommodating the sample X to be observed. By doing so, the objective lens 12 is disposed in a manner spaced apart from, and vertically below, the bottom surface section 8 of one of the container sections 5 of the microplate 2, and the optical member 16 is disposed such that the top end thereof is inserted in the recessed section D formed in the lower part of the microplate 2.
When the light source 10 emits excitation light in the horizontal direction in this state, the excitation light emitted from the light source 10 is converted by the collimator lens 14 into substantially collimated light and is then focused by the cylindrical lens 15 in one direction, thus generating sheet-shaped excitation light in which the thickness of the light beam is reduced gradually to the focal position. The sheet-shaped excitation light is bent in a crank manner by the two mirrors 17 of the optical member 16, passes through the side wall section 7 of the container section 5, and is then incident upon the sample X in the well 4.
Since its focal position is located in the sample X in the well 4, the sheet-shaped excitation light is radiated onto a thin region along a plane horizontally intersecting the sample X, which generates fluorescence in the irradiated region. Part of the generated fluorescence goes downward through the bottom surface section 8 of the container section 5, is collected by the objective lens 12 disposed below the bottom surface section 8, and is then imaged by the image acquisition element 13. By doing so, a fluorescence image of the sample X along a plane extending in the focal plane of the objective lens 12 can be acquired.
Thereafter, when the sample X is to be observed at a different position in the optical-axis direction of the objective lens 12, the focal plane in the sample X can be changed by moving the movable stage 9 up/down, without changing the relationship between the focal position of the objective lens 12 and the plane position at which the sheet-shaped excitation light is disposed. By doing so, image information of the sample X can be acquired in a three-dimensional manner. The focusing mechanism of the objective lens 12 can be used and operated for adjustment purposes in a case where the focal plane of the objective lens 12 and the plane in which the excitation light is disposed are shifted.
In addition, also when the sample X in the well 4 of a container section 5 disposed in a different row of the microplate 2 is to be observed, observation can be performed easily by operating the movable stage 9 to change the bottom surface section 8 facing the objective lens 12 and the recessed section D in which the optical member 16 is inserted.
In this case, the microscope system 1 according to this embodiment affords an advantage in that, because the optical member 16 is inserted from the lower side into a recessed section D formed between the rows of the container sections 5, and sheet-shaped excitation light is made incident upon a sample X by causing the excitation light to pass through the transparent portion of the side wall section 7, the sample X can be reliably irradiated with the sheet-shaped excitation light even if the sample X is disposed in the vicinity of the bottom surface section 8 in the well 4. In other words, because the optical member 16, unlike the conventional way, is not inserted from above into a container section accommodating a sample X, the problem that the sample X cannot be observed at a position near the bottom surface due to interference between the optical member 16 and the bottom surface does not occur. Note that the index of refraction of the bottom surface section 8 and the index of refraction of the medium in the well 4 are preferably identical to each other in order to prevent part of the excitation light from refracting at the bottom surface section 8. In addition, it is more preferable that the bottom surface section 8 be manufactured such that the index of refraction of the material of the bottom surface section 8 is adjusted to the index of refraction of the medium assumed to be used.
In addition, according to the microscope system 1 of this embodiment, the samples X accommodated in the four wells 4 arrayed in one row can be successively observed merely by moving the movable stage 9 along the horizontal direction in which the wells 4 are arrayed. In this case, an advantage is afforded in that because the optical member 16 does not come into contact with the media in the wells 4, observation can be efficiently performed while preventing the occurrence of contamination between different wells 4 and a change in the state of the sample X resulting from each of the media being agitated.
In addition, according to the microplate 2 of this embodiment, because the side wall section 7 of each of the container sections 5 is configured to extend in a substantially vertical direction, the refraction, at the side wall section 7, of sheet-shaped illumination light introduced in the horizontal direction is reduced, thereby allowing the sheet-shaped illumination light to be incident upon the focal plane of the objective lens 12 more simply and more accurately. By doing so, a sharp image can be easily acquired by minimizing the burden involved with focal position alignment work using the focusing mechanism.
In the microscope system 1 according to this embodiment, two of the side wall sections 7 facing each other with the well 4 interposed therebetween may be provided with optically transparent portions in each of the container sections 5, thereby allowing the sample X in one container section 5 to be irradiated with sheet-shaped excitation light via the two side wall sections 7 in two directions, as shown in
This embodiment has been described by way of an example where the container section 5 in each of the rows has the planar, common side wall section 7. Instead of this, this embodiment may be configured so that the container sections 5 have individual cylindrical side wall sections 7, as shown in
This embodiment has been described by way of an example of the optical member 16 having two mirrors 17. Instead of this, the optical member 16 having a mirror 17 and a prism 18 or two prisms 18 may be employed, as shown in
This embodiment has been described by way of an example of the microplate 2 in which the wells 4 are arrayed in a linear shape. Instead of this, as shown in
In this case, the movable stage 9 may be provided with a motor 19 for rotating the microplate 2 about the central axis of the array of the wells 4.
By doing so, once the optical member 16 and the objective lens 12 have been positioned relative to the microplate 2 mounted on the movable stage 9, the samples X in the neighboring wells 4 in the same row can be observed in sequence by rotating the microplate 2 about the central axis through the operation of the motor 19.
In addition, in this embodiment, the frame constituting the connection section 6 and the side wall sections 7 may be formed through resin injection molding, and only the bottom surface sections 8 may be formed of glass plates or a resin film, and then the frame and the bottom surface section 8 may be adhered or fixed through thermal bonding, as shown in
In this case, a support member 20 for supporting the glass plate of each of the bottom surface sections 8 may be provided on the frame side, as shown in
In addition, as shown in
In addition, as shown in
In addition, the inner shape of each of the bottom surface sections 8 may be, for example, hemispheric, and the interior may be subjected to non-cellular adhesive surface treatment. By doing so, a cell culture environment suitable for formation of a spheroid through cell culture can be provided. In contrast, a culture environment suitable for forming a cell sheet, in which cells are layered in a sheet shape, can be provided by forming the inner shape of a bottom surface section 8 to be planar and then applying cell adhesive surface treatment. The above-described variations can be set depending on the purpose. For example, variations with different inner shapes and different surface treatment may be connected to the connection section 6. In addition, with these variations, for example, the configuration of the microplate with a barcode added to the top surface of the connection section 6 can be recognized. Furthermore, the configuration may be changed freely by employing a set-in method, instead of a bonding method, for connection.
In addition, as shown in,
In addition, as shown in
In addition, flow channels 23 connecting between neighboring wells 4 of the plurality of wells 4 arrayed in one row may be provided as shown in,
In addition, as shown in
In this case, a second medium B having an index of refraction equivalent to that of a first medium A stored in the wells 4 of the microplate 2 is stored in the medium container 25, and the microplate 2 is disposed such that the side wall sections 7 and the bottom surface sections 8, which transmit excitation light and light from the sample, are immersed into the second medium B.
The medium container 25 is disposed at a set position in the horizontal direction relative to the objective lens 12 and the optical members 16.
In addition, the outer periphery of the microplate 2 may be subjected to hydrophobic surface treatment so that the media do not adhere to the outer periphery of the microplate 2 when the microplate 2 is extracted from the medium container 25 after image acquisition is finished.
Employing the immersion objective lens allows an increase in the NA of fluorescence to be collected, thereby making it possible to acquire a high-resolution fluorescence image. In addition, by disposing the medium container 25 at a set position in the horizontal direction relative to the objective lens 12, even when the microplate 2 is horizontally swiveled in order to change the observed sample X, it is not necessary to relatively move the medium container 25 and the objective lens 12, thereby making it possible to reliably hold the liquid immersion medium.
In addition, there is an advantage in that because the first medium A stored in the wells 4 and the second medium B stored in the medium container 25 are formed of media having the same index of refraction, even when the proportion between the first medium A and the second medium B disposed in a direction along the optical axis of the objective lens 12 is changed as a result of moving the microplate 2 up/down through the operation of the movable stage 9, it is not necessary to change the focal plane of the objective lens 12.
In addition, although this embodiment has been described by way of an example of the microplate 2 having a plurality of wells 4 arrayed in a ring shape about the central axis and that are also arranged in a plurality of rows in a manner spaced apart from each other in the radial direction, this embodiment may be configured of a microplate formed by arraying a plurality of rows in a square shape. As shown in
The inventors have arrived at the following aspects of the present invention.
An aspect of the present invention provides a microplate including: a plurality of container sections having wells that open at a first plane and that accommodate samples; and a connection section connecting a plurality of rows of the container sections so as to be arrayed in a manner in which the rows are spaced from each other in a direction along the first plane, wherein each of the container sections includes: at least one side wall section that is optically transparent at at least a portion thereof; and a bottom surface section that is disposed on a second plane on a side opposite from the first plane and that is optically transparent at at least a portion thereof, and wherein a recessed section is formed between neighboring rows of the container sections, and the recessed section allows an optical member to be inserted thereinto crossing the second plane, wherein the said optical member introduces sheet-shaped illumination light into the well via the side wall section in a direction substantially parallel to the first plane.
According to this aspect, the microplate is disposed with the first plane oriented upward, and each of the plurality of wells that open in the first plane accommodates a sample. In this state, the optical member is inserted into the recessed section formed between neighboring rows of the container sections from the second plane side, which is a lower side disposed on the opposite side from the first plane. Thereafter, by causing the optical member to introduce, substantially parallel to the first plane, sheet-shaped illumination light to the side wall section of the container section, the illumination light that has passed through the optically transparent portion of the side wall section is radiated onto the sample in the well. On the other hand, part of the light generated in the sample passes through the optically transparent portion of the bottom surface and then can be detected below the bottom surface.
In this case, because the optical member is inserted between the container sections from the bottom surface side, instead of inserting the optical member from above into the well, the optical member can be disposed sufficiently upward relative to the bottom surface of the well, thereby making it possible to observe even a sample located on the bottom surface. In addition, the samples in the plurality of container sections can be efficiently observed merely by relatively moving the optical member and the microplate in a direction along the rows of the container sections.
In the above-described aspect, the side wall section may be disposed orthogonally to the bottom surface section.
By doing so, the illumination light can be introduced in a direction orthogonal to the side wall section, thereby making it possible to prevent refraction of the illumination light at the side wall section.
In addition, in the above-described aspect, each of the container sections may have two of the side wall sections disposed parallel to each other with the well interposed therebetween.
By doing so, the illumination light can be introduced via the two side wall sections, and thereby high-intensity illumination light can be radiated evenly across the whole of a relatively large sample by introducing the illumination light from both sides in a horizontal direction of the sample in the well.
In addition, in the above-described aspect, the side wall sections of the container sections in each of the rows may be formed of a single continuous member.
In addition, in the above-described aspect, the bottom surface sections of the container sections in each of the rows may be formed of a single continuous member.
In addition, in the above-described aspect, the container sections may be formed of a material different from that of the connection section.
In addition, in the above-described aspect, the side wall sections may be formed of a material different from that of the connection sections.
In addition, in the above-described aspect, the bottom surface sections may be formed of a material different from that of the connection section.
The side wall sections and the bottom surface sections of the container sections are required to have characteristics such that they transmit illumination light or light from the samples, and hence there is no choice but to use a relatively costly material; therefore, by forming them of a material different from that of the connection section, the microplate can be configured less costly.
In addition, in the above-described aspect, the side wall section and the bottom surface section of each of the container sections may be formed of a single moldable material.
By doing so, the side wall section and the bottom surface section, which are required to have light-transmitting characteristics, can be easily configured by molding.
In addition, in the above-described aspect, the container sections in each of the rows may be detachably attached to the connection section.
By doing so, the microplate can be configured such that the container sections, which are required to have light-transmitting characteristics, are manufactured independently of, and then combined with, the connection section.
In addition, in the above-described aspect, the container sections may be arrayed in a ring shape about a predetermined axial line and may be disposed in a plurality of rows in a manner where the rows are spaced from each other in a radial direction.
By doing so, the samples in different container sections can be successively irradiated with illumination light by horizontally swiveling the microplate about the predetermined axial in a state where the optical member is disposed at a position that allows illumination light to be introduced to the side wall section of one of the container sections, thereby allowing efficient observation.
In addition, another aspect of the present invention provides a microscope system including: the above-described microplate; and a microscope which acquires an observation image of the sample accommodated in the well of the microplate, wherein the microscope includes: a movable stage which supports the microplate so as to be movable at least in a horizontal direction; the optical member that is inserted from a lower side into the recessed section formed between the neighboring rows of the container sections of the microplate, that introduces illumination light coming from a light source and from a lower side of the microplate, and that bends the illumination light to enter the well via the side wall section as the sheet-shaped illumination light extending in a horizontal direction; an objective lens that collects, via the bottom surface section, light generated in the sample as a result of being irradiated with the illumination light; and an image acquisition unit which acquires an image of the light collected by the objective lens.
According to this aspect, samples are accommodated in a plurality of wells that open in the first plane, and the microplate is placed on the movable stage of the microscope with the openings oriented upward. In this state, the optical member is inserted into the recessed section formed between neighboring rows of the container sections and from a lower side of the microplate.
Thereafter, the sheet-shaped illumination light, which is formed by causing the optical member to introduce illumination light generated by the light source from below the microplate and to bend the illumination light, is made incident on the side wall section of the container section substantially horizontally and is thereby radiated onto the sample in the well after passing through the optically transparent portion of the side wall section. On the other hand, part of the light generated in the sample passes through the optically transparent portion of the bottom surface, is collected by the objective lens disposed below the bottom surface, and is imaged by the image acquisition unit.
An image spanning a wide area of the sample can be acquired all at once by causing the focal position of the objective lens to be aligned with the incident plane of the sheet-shaped illumination light.
In addition, another aspect of the present invention provides a microscope system including: the above-described microplate; and a microscope which acquires an observation image of the sample accommodated in the well of the microplate, wherein the microscope includes: a movable stage which supports the microplate so as to be movable at least in a horizontal direction; two of the optical members that are inserted from a lower side into the recessed sections formed between the neighboring rows of the container sections of the microplate, that introduce illumination light from light sources and from a lower side of the microplate, and that bend the illumination light to enter the well via the two side wall sections as the sheet-shaped illumination light extending in a horizontal direction; an objective lens that collects, via the bottom surface section, light generated in the sample as a result of being irradiated with the illumination light; and an image acquisition unit which acquires an image of the light collected by the objective lens.
According to this aspect, illumination light can be introduced via the two side wall sections, and high-intensity illumination light can be evenly radiated across the whole of a relatively large sample by introducing the illumination light from both sides of the sample in the well along the horizontal direction.
In addition, another aspect of the present invention provides a microscope system including: the above-described microplate; and a microscope which acquires an observation image of the sample accommodated in the well of the microplate, wherein the microscope includes: a movable stage which supports the microplate so as to be movable at least in a horizontal direction; the optical member that is inserted from a lower side into the recessed section formed between the neighboring rows of the container sections of the microplate, that introduces illumination light coming from a light source and from a lower side of the microplate, and that bends the illumination light to enter the well via the side wall section as the sheet-shaped illumination light extending in a horizontal direction; an objective lens that collects, via the bottom surface section, light generated in the sample as a result of being irradiated with the illumination light; and an image acquisition unit which acquires an image of the light collected by the objective lens, and wherein the movable stage rotates the microplate about the axial line.
According to this aspect, the samples in different container sections can be sequentially irradiated with illumination light to perform efficient observation by horizontally swiveling the microplate about the predetermined axial line through the operation of the movable stage in a state where the optical member is disposed at a position that allows the illumination light to enter the side wall section of one of the container sections.
In the above-described aspect, a first medium into which the samples are immersed may be stored in the wells, a medium container that stores a second medium into which at least portions of the side wall sections and the bottom surface sections of the microplate are immersed and which has a recessed section formed between the neighboring rows of the container sections of the microplate such that the optical member can be inserted into the recessed section from a lower side may be provided, and a refraction index of the first medium is substantially equivalent to a refraction index of the second medium.
By doing so, even if the proportion between the first medium and the second medium disposed between the incident plane of the sheet-shaped illumination light and the objective lens is changed as a result of the microplate being moved in the medium container, the state in which the focal position of the objective lens is aligned with the incident plane of the illumination light can be maintained. In addition, observation with a high-resolution image can be performed by employing an immersion objective lens as the objective lens.
The above-described aspect affords an advantage in that not only is it possible to observe a sample located on the bottom surface, but also, a plurality of samples can be efficiently observed.
Number | Date | Country | Kind |
---|---|---|---|
2016-238447 | Dec 2016 | JP | national |