JP6479368B2 - Culture container, multiphoton excitation microscope, and observation method - Google Patents

Description

  The present invention relates to a culture vessel for culturing a biological specimen, a multiphoton excitation microscope, and an observation method.

  A multiphoton excitation microscope is known as a means for observing a biological specimen to the deep part. In the multiphoton excitation microscope, fluorescence is generated from one point in the specimen where the excitation light is collected, but is scattered within the biological specimen and diffused over a wide range. For this reason, in order to efficiently detect the generated fluorescence, it is effective to use an objective lens having a high numerical aperture and a wide detection field.

  However, even if such an objective lens is used, only a part of the fluorescence emitted from the specimen in all directions (360 degrees) can be captured by the objective lens. Therefore, techniques for realizing further improvement in detection efficiency have been proposed.

  In Patent Document 1, in addition to the objective lens, an additional optical system that is arranged outside the objective lens in the radial direction and captures fluorescence from the specimen is provided, and fluorescence from the specimen collected by the objective lens and the additional optical system is collected. A fluorescence observation apparatus configured to detect with a photodetector is described.

JP 2011-196852 A

  By the way, although a biological specimen may be observed in the state accommodated in the culture container, this culture container may prevent the detection of fluorescence. In particular, when a container provided with a plurality of accommodating portions for accommodating a specimen, such as a microplate, is used, light having a large NA exceeding the NA (Numerical Aperture) of the objective lens It is easily blocked by the upper wall and side wall.

  For this reason, when observing a specimen contained in a culture vessel, it is sufficient to simply widen the angle range (detection solid angle) in which fluorescence can be captured, as in the apparatus described in Patent Document 1. It is difficult to obtain high detection efficiency.

  In light of the above circumstances, an object of the present invention is to provide a technique for brightly observing a specimen stored in a culture vessel.

  A first aspect of the present invention is a culture container capable of storing a biological specimen immersed in a culture solution, and the light from the biological specimen incident on the inner surface of the culture container is directed above or below the culture container. A culture vessel comprising a light guiding means for guiding is provided.

The second aspect of the present invention provides the culture container according to the first aspect, wherein the light from the biological specimen is fluorescence generated by multiphoton excitation.
A third aspect of the present invention provides the culture container according to the first aspect or the second aspect, wherein the culture container is a microplate having a structure for individually accommodating a plurality of biological specimens. To do.
According to a fourth aspect of the present invention, in the culture container according to any one of the first to third aspects , the culture container is the biological specimen that has passed through the inner surface as the light guide means. A culture vessel having a reflecting surface for reflecting light from the top or bottom is provided.

According to a fifth aspect of the present invention, in the culture container according to any one of the first to third aspects , the culture container is formed on the inner surface as the light guiding means. A culture vessel having a diffraction surface for diffracting light from a biological specimen is provided.

According to a sixth aspect of the present invention, in the culture container according to any one of the first to third aspects , the culture container is formed on the inner surface as the light guiding means. A culture vessel having a reflective surface for reflecting light from a biological specimen is provided.

According to a seventh aspect of the present invention, in the culture container according to any one of the first to sixth aspects, the biological specimen further includes a bottom surface through which light is transmitted and the inner side surface. A container body that can be immersed in the culture medium, and a lid that is placed over the container body, and has a reflective surface that reflects light from the biological specimen formed toward the bottom surface side And a culture vessel comprising a lid.

According to an eighth aspect of the present invention, in the culture container according to the seventh aspect, the lid has an opening or a transmission part through which light from the biological specimen is transmitted, and the reflection formed on the lid. The surface provides a culture vessel formed around the opening or the permeable part.

A ninth aspect of the present invention provides the culture container according to the seventh aspect or the eighth aspect, wherein the reflective surface formed on the lid has a concave shape.

According to a tenth aspect of the present invention, there is provided the culture container according to any one of the first to ninth aspects, a stage on which the culture container is disposed, and the biological specimen. An objective lens for irradiating excitation light; and a first detection means for detecting fluorescence generated from the biological specimen through the objective lens, and the objective lens disposed on the opposite side of the stage has a capacitor, and a second detecting means for detecting fluorescence generated from the biological specimen via the front Symbol capacitor, from the culture vessel a fluorescence generated from the biological specimen of the fluorescence emitted on the stage side A multi-photon excitation microscope is provided that includes third detection means for detecting fluorescence guided to the outside of the objective lens.

An eleventh aspect of the present invention is an observation method for observing a biological specimen housed in the culture container according to any one of the first to ninth aspects, which is disposed on a stage of a multiphoton excitation microscope. The biological specimen is irradiated with excitation light through the objective lens of the multiphoton excitation microscope, and is guided above or below the culture vessel by the light guide means by the detection means of the multiphoton excitation microscope. An observation method for detecting fluorescence emitted from the biological specimen and emitted from the culture vessel is provided.

  ADVANTAGE OF THE INVENTION According to this invention, the technique which observes the sample accommodated in the culture container brightly can be provided.

It is the figure which illustrated the composition of the microscope concerning Example 1 of the present invention. It is a figure for demonstrating the structure of the culture container which concerns on Example 1 of this invention. It is a figure for demonstrating the structure of the culture container which concerns on Example 2 of this invention. It is a figure for demonstrating the structure of the culture container which concerns on Example 3 of this invention. It is a figure for demonstrating the structure of the culture container which concerns on Example 4 of this invention. It is a figure for demonstrating the structure of the culture container which concerns on Example 5 of this invention. It is a figure for demonstrating the structure of the culture container which concerns on Example 6 of this invention. It is a figure for demonstrating the structure of the conventional culture container. It is a figure for demonstrating the structure of the culture container which concerns on Example 7 of this invention. It is a figure for demonstrating the structure of the culture container which concerns on Example 8 of this invention, and a mode that the lid | cover and a culture solution are not contacting is shown. It is a figure for demonstrating the structure of the culture container which concerns on Example 8 of this invention, and a mode that the cover and the culture solution are contacting is shown. It is a figure for demonstrating the structure of the culture container which concerns on Example 9 of this invention.

  FIG. 1 is a diagram illustrating the configuration of the microscope 1 according to the present embodiment. The microscope 1 is a two-photon excitation microscope that detects fluorescence from the biological specimen 101 accommodated in the culture vessel 100.

  The culture vessel 100 is a microplate (also referred to as a multiwell plate) having a structure that individually accommodates a plurality of biological specimens 101. The biological specimen 101 is accommodated in the culture vessel 100 while being immersed in the culture solution 102. The detailed configuration of the culture vessel 100 will be described later.

  The microscope 1 includes a stage 2 on which the culture container 100 is disposed, and the culture container 100 is held on the stage 2 by a container holding unit 2a. The stage 2 is an XY stage configured to be movable in the XY directions orthogonal to the optical axis of the objective lens 7. A user of the microscope 1 operates the stage 2 so that the biological specimen 101 that is an observation target selected from the plurality of biological specimens 101 is positioned on the optical axis of the objective lens 7.

  The microscope 1 further includes a laser 3, an XY scanner 4, a relay optical system 5, a dichroic mirror 6, and an objective lens 7. In the microscope 1, these function as illumination means for irradiating the biological specimen 101 accommodated in the culture vessel 100 with excitation light from below.

  The laser 3 is, for example, a titanium sapphire laser. The laser 3 emits laser light having an infrared wavelength. The XY scanner 4 is a galvanometer mirror, for example. Laser light emitted from the laser 3 is deflected by the XY scanner 4. The relay optical system 5 is an optical system that projects the pupil of the objective lens 7 onto the XY scanner 4. When the XY scanner 4 changes the deflection direction of the laser light, the incident angle of the laser light incident on the pupil position of the objective lens 7 changes, and thereby the condensing position of the laser light emitted from the objective lens 7 is changed. It moves in the XY direction orthogonal to the optical axis of the objective lens 7. The dichroic mirror 6 is means for branching the illumination optical path and the detection optical path. The dichroic mirror 6 has a wavelength characteristic that transmits laser light as excitation light and reflects fluorescence generated from the biological specimen 101. The objective lens 7 is an objective lens that irradiates the biological specimen 101 with laser light, and has a high transmittance with respect to light from the visible region to the infrared region.

  In the microscope 1, the biological specimen 101 accommodated in the culture vessel 100 is excited by the illumination means, and fluorescence is emitted from the biological specimen 101.

  The microscope 1 further includes a relay lens 8, an infrared (IR) cut filter 9, and a photomultiplier tube (PMT) 10 on the optical path of the fluorescence reflected by the dichroic mirror 6. In the microscope 1, these together with the objective lens 7 and the dichroic mirror 6 constitute first detection means for detecting fluorescence. The first detection means is epi-illumination detection means for detecting fluorescence via the objective lens 7 that irradiates the biological specimen 101 with laser light.

  The relay lens 8 is an optical system that projects the pupil of the objective lens 7 onto the PMT 10. The fluorescence emitted from the biological specimen 101 and taken into the objective lens 7 is reflected by the dichroic mirror 6 and guided to the PMT 10 by the relay lens 8. The IR cut filter 9 is an optical filter that blocks light having a wavelength in the infrared region, and is means for separating the fluorescence from the laser light. The laser light incident on the IR cut filter 9 together with the fluorescence is blocked by the IR cut filter 9. The PMT 10 is a photodetector that detects fluorescence. Fluorescence separated from the laser light by the IR cut filter 9 enters the PMT 10 and is detected.

  In the microscope 1, fluorescence reflected by the objective lens 7 among the fluorescence emitted from the biological specimen 101 is detected by the epi-illumination detection means.

  The microscope 1 further includes a condenser 11, a relay lens 12, a mirror 13, an IR cut filter 14, and a PMT 15 on an optical path (transmission detection optical path) opposite to the objective lens 7 across the culture vessel 100. It has. In the microscope 1, these constitute a second detection means for detecting fluorescence. The second detection means is a transmission detection means for detecting fluorescence via a capacitor 11 disposed on the opposite side of the objective lens 7 with the stage 2 interposed therebetween.

  The relay lens 12 is an optical system that projects the pupil of the condenser 11 onto the PMT 15. The fluorescence emitted from the biological specimen 101 and taken into the capacitor 11 is guided to the PMT 15 by the relay lens 12 via the mirror 13 and the IR cut filter 14. The IR cut filter 14 is an optical filter that blocks light having a wavelength in the infrared region, and is a means for separating fluorescence from laser light. The laser light incident on the IR cut filter 14 together with the fluorescence is blocked by the IR cut filter 14. The PMT 15 is a photodetector that detects fluorescence. Fluorescence separated from the laser light by the IR cut filter 14 enters the PMT 15 and is detected.

  In the microscope 1, the fluorescence captured by the capacitor 11 among the fluorescence emitted from the biological specimen 101 is detected by the transmission detecting means.

  The microscope 1 includes a plurality of optical systems 16 disposed outside the objective lens 7 so as to surround the objective lens 7 and a plurality of optical fibers 17. These together with the IR cut filter 9 and the PMT 10 constitute a third detection means.

  The optical system 16 is an optical system that takes in the fluorescence guided to the outside of the objective lens 7 by the culture vessel 100. The fluorescence taken into the optical system 16 is guided to the PMT 10 via the optical fiber 17 and the IR cut filter 9.

  In the microscope 1, the fluorescence guided to the outside of the objective lens 7 among the fluorescence emitted from the culture vessel 100 to the stage 2 side is detected by the third detection means.

  The microscope 1 can realize high fluorescence detection efficiency by including three detection units that detect fluorescence emitted from the culture vessel 100 in different directions. Thereby, the microscope 1 can observe the biological specimen 101 accommodated in the culture container 100 brightly. Furthermore, the microscope 1 can achieve higher detection efficiency by using a culture vessel 100 having a configuration to be described later.

  Hereinafter, the configuration of the culture vessel 100 will be described in detail with reference to FIG. FIG. 2 shows a configuration for one cycle of the culture vessel 100 having a periodic structure. This also applies to FIGS. 3 to 12 referred to later.

  The culture container 100 includes a container body 110 disposed on the stage 2 and a lid 120 disposed on the container body 110. In the culture container 100, combinations of the container main body 110 and the lid 120 shown in FIG. 2 are periodically arranged. The container main body 110 is connected to an adjacent container main body 110 by a structure (not shown), and a plurality of periodically arranged container main bodies 110 constitute one large container main body. Further, as shown in FIG. 1, the lid 120 has a support member 122, which will be described later, connected to the support member 122 of the adjacent lid 120, and a single large lid for the plurality of periodically arranged lids 120. Is configured.

  In the container body 110, a well composed of a bottom surface 111 and a side surface 112 is formed. The biological specimen 101 is accommodated in the well of the container main body 110 while being immersed in the culture solution 102. The container main body 110 is configured such that the laser light emitted from the objective lens 7 passes through the bottom surface 111 and is irradiated on the biological specimen 101, and the fluorescence generated from the biological specimen 101 passes through the bottom surface 111 and passes through the objective lens 7. It is made of a material that transmits light so that it enters the light.

  The outer side surface of the container main body 110 (hereinafter referred to as the outer side surface and distinguished from the side surface 112 which is the inner side surface) is a slope inclined with respect to the optical axis at the lower part of the container main body 110, and the fluorescence transmitted through the side surface 112. Is a reflection surface 113 that reflects the light downward. Specifically, the reflecting surface 113 which is the outer surface is a total reflection surface using a difference in refractive index between the container main body 110 and air, and is inclined so that fluorescence incident on the side surface 112 at a shallow angle is totally reflected. Yes. The inclination angle of the reflection surface 113 is such that, for example, the incident angle range of the fluorescence incident on the reflection surface 113 is specified based on the maximum height of the reflection surface 113, and the fluorescence in the specified incident angle range is totally reflected. Then, you just have to decide.

  The lid 120 includes a lid member 121 formed so as to cover at least a part of the opening of the container main body 110 when disposed on the container main body 110, and a support member 122 that supports the lid member 121.

  The lid member 121 is a member having a dome shape in which an opening 123 is formed at the center, and the lid member 121 has a reflective surface 124 directed toward the bottom surface 111 side. The reflection surface 124 formed around the opening 123 may be configured to reflect at least fluorescence that is light from the biological specimen 101. The reflection surface 124 is formed by coating the inner surface of the lid member 121 to increase the reflectance of light (fluorescence) from the biological specimen 101, for example. The opening 123 is formed so as not to block the fluorescence L1 incident on the capacitor 11. Instead of the opening 123, a transmission part through which light (fluorescence) from the biological specimen 101 passes may be formed. In that case, it is desirable that the permeation part is made of a material that allows gas to permeate.

  In the microscope 1, the fluorescence L 1 that has passed through the opening 123 among the fluorescence emitted upward from the biological specimen 101 accommodated in the culture vessel 100 enters the capacitor 11 and is detected by the transmission detection means. Of the fluorescence emitted upward from the biological specimen 101, the fluorescence L2 emitted toward the outside of the opening 123 is reflected by the reflecting surface 124, and then the objective lens 7 together with the fluorescence emitted downward from the biological specimen 101. And is detected by the epi-illumination detection means. Further, the fluorescence L3 radiated from the biological specimen 101 to the side and transmitted through the side surface 112 is reflected by the reflecting surface 113 and detected by the third detecting means.

  In this way, by accommodating the biological specimen 101 in the culture vessel 100 and observing it, it becomes possible to detect the fluorescence generated from the biological specimen 101 with high detection efficiency. For this reason, according to the culture container 100, the accommodated biological specimen 101 can be observed brightly.

  2 illustrates an example in which the lid member 121 has a dome shape, the shape of the lid member 121 is not limited to the dome shape. As long as the light reflected by the reflecting surface 124 formed on the lid member 121 is concave so that the light is guided to the objective lens 7, the shape of the lid member 121 may be any shape. Further, when used in a microscope having no transmission detecting means, the reflection surface 124 may be formed on the entire surface of the lid member 121 without forming the opening 123.

  FIG. 3 is a diagram for explaining the configuration of the culture vessel 200 according to the present embodiment. The culture container 200 shown in FIG. 3 is different from the culture container 100 shown in FIG. 2 in that a container main body 210 is provided instead of the container main body 110. In FIG. 3, the lid 120 is not shown.

  The container body 210 is different from the container body 110 in that a diffraction surface 212 that diffracts the light from the biological specimen 101 is formed on the inner surface. The diffractive surface 212 causes the fluorescence L4 incident at different angles from the bottom surface 111 to be incident on the outer surface 213 at an angle equal to or greater than the total reflection angle, so that the grating period varies depending on the height from the bottom surface 111. have. Specifically, the diffractive surface 212 is configured such that the grating period gradually decreases from the bottom surface 111 side toward the opening side.

  Most of the fluorescence L4 incident on the diffraction surface 212 from the biological specimen 101 is diffracted by the diffraction surface 212. Thereafter, the light is totally reflected by the outer surface 213 and emitted downward from the culture vessel 200. Thereby, it is detected by the epi-illumination detection means or the third detection means described above. Further, among the fluorescence L5 that is the 0th-order diffracted light that travels straight on the diffraction surface 212, the light incident on the capacitor 11 is detected by the transmission detecting means.

  According to the culture container 200 configured as described above, light incident on the side surface (diffraction surface 212) of the container body 210 can be guided to the detection means. Therefore, similarly to the culture container 100, high detection efficiency is realized, and the stored biological specimen 101 can be observed brightly.

  FIG. 4 is a diagram for explaining the configuration of the culture vessel 300 according to the present embodiment. The microscope according to this example is the same as the microscope 1 shown in FIG. However, the transmission detecting means may be omitted.

  The culture container 300 includes a container body 310 disposed on the stage 2 and a lid 320 disposed on the container body 310. The container body 310 is the same as the container body 110 shown in FIG. 2 in that a well composed of a bottom surface 111 and a side surface 112 is formed. The same applies to the point that the container body 310 is made of a material that transmits light.

  In the container main body 310, a reflection surface 313 that reflects light (fluorescence) from the biological specimen 101 that has passed through the side surface 112 is formed on the outer surface of the container main body 310. The reflective surface 313 is formed, for example, by coating the outer surface of the container body 310 to increase the reflectance of light (fluorescence) from the biological specimen 101.

  The lid 320 is formed with a reflection surface 321 directed toward the bottom surface 111 side. The lid 320 is configured such that the reflective surface 321 covers the entire opening of the container body 310 in a state where the lid 320 is disposed on the container body 310. The reflective surface 321 has a concave shape. The reflective surface 321 may be formed in a size that matches the outer diameter of the container body 310, and may be formed so that, for example, the end of the reflective surface 321 is positioned on the extension line of the reflective surface 313. With such a configuration, it is possible to prevent the fluorescence generated from the biological specimen 101 from being emitted from the side surface or the upper surface of the culture vessel 300.

  In the microscope according to the present embodiment, the fluorescence (fluorescence L6 and fluorescence L7) radiated upward or sideward from the biological specimen 101 is reflected by the reflective surface 313 and / or the reflective surface 321 and from below the culture vessel 300. It is emitted and detected by the epi-illumination detection means or the third detection means. Further, the fluorescence L8 emitted downward from the biological specimen 101 is emitted downward from the culture vessel 300 and detected by the epi-illumination detection means.

  Therefore, according to the culture container 300, high detection efficiency is implement | achieved similarly to the culture container 100, and the accommodated biological specimen 101 can be observed brightly.

  FIG. 5 is a diagram for explaining the configuration of the culture vessel 400 according to the present embodiment. The culture vessel 400 corresponds to a unit in which the container body 110 and the lid 120 of the culture vessel 100 shown in FIG. The microscope according to the present embodiment is different from the microscope 1 shown in FIG. 1 in that a mirror 18 is disposed behind the capacitor 11.

  In the culture container 400, the vicinity of the opening is curved inward, a reflecting surface 415 is formed on the bottom surface 111 side of the curved portion, and a part of the side surface 112 is formed as a lens surface 414. However, it is different from the container body 110 of the culture container 100. The reflective surface 415 corresponds to the reflective surface 124 of the lid 120 shown in FIG. The reflective surface 415 is the same as the reflective surface 124 in that it is formed around the opening toward the bottom surface 111, and is configured to reflect at least fluorescence that is light from the biological specimen 101. The opening is formed so that the reflection surface 415 does not block the fluorescence L1 incident on the capacitor 11.

  In the microscope according to the present embodiment, the fluorescence L1 that has passed through the opening of the culture vessel 400 out of the fluorescence emitted upward from the biological specimen 101 is collimated by the capacitor 11 and enters the mirror 18. Thereafter, the light is reflected by the mirror 18 and is incident on the objective lens 7 through the condenser 11, and is detected by the epi-illumination detecting means. Of the fluorescence emitted upward from the biological specimen 101, the fluorescence L9 emitted toward the outside of the opening is reflected by the reflecting surface 415, and then is reflected on the objective lens 7 together with the fluorescence emitted downward from the biological specimen 101. Incident light is detected by the epi-illumination detection means. Further, the fluorescence L10 emitted from the biological specimen 101 to the side and transmitted through the lens surface 414 is reflected by the reflecting surface 113 and detected by the third detecting means. The lens surface 414 has an effect of suppressing the spread of the light beam incident on the optical fiber 17 via the optical system 16.

  Therefore, according to the culture container 400, high detection efficiency is implement | achieved similarly to the culture container 100, and the accommodated biological specimen 101 can be observed brightly.

  FIG. 6 is a diagram for explaining the configuration of the culture vessel 500 according to the present embodiment. The microscope according to this example is the same as the microscope 1 shown in FIG. However, the third detection means may be omitted.

  The culture vessel 500 is different from the vessel main body 310 of the culture vessel 300 shown in FIG. 4 in that a reflection surface 512 is formed on the inner side instead of the outer side. Other points are the same as those of the container body 310. The reflection surface 512 is formed, for example, by coating the inner surface of the culture vessel 500 to increase the reflectance of light (fluorescence) from the biological specimen 101.

  In the microscope according to the present embodiment, the fluorescence (fluorescence L1 and fluorescence L11) emitted upward from the biological specimen 101 is incident on the capacitor 11 directly or after being reflected by the reflection surface 512, and is transmitted by the transmission detecting means. Detected. Further, the fluorescence emitted downward from the biological specimen 101 enters the objective lens 7 and is detected by the epi-illumination detection means.

  Therefore, according to the culture container 500, the light incident on the side surface (reflection surface 512) of the culture container 500 can be guided to the detection means. Therefore, similarly to the culture container 100, high detection efficiency is realized, and the stored biological specimen 101 can be observed brightly.

  FIG. 7 is a diagram for explaining the configuration of the culture vessel 600 according to the present embodiment. The microscope according to the present embodiment is different from the microscope 1 shown in FIG. 1 in that a mirror 18 is disposed behind the capacitor 11. Further, the third detection means may be omitted.

  The culture vessel 600 has a tapered shape in which a well formed in the vessel spreads from the bottom surface 111 side toward the opening side, and the point that the reflection surface 612 is inclined with respect to the optical axis of the objective lens 7 is the culture vessel. It is different from 500. Other points are the same as those of the culture vessel 500. The reflection surface 612 is formed, for example, by coating the inner surface of the culture vessel 600 to increase the reflectance of fluorescence that is light from the biological specimen 101.

  Therefore, as with the culture vessel 500, the culture vessel 600 also achieves high detection efficiency, so that the stored biological specimen 101 can be observed brightly. In the culture vessel 600, the fluorescence L13 emitted from the biological specimen 101 in a substantially horizontal direction is also reflected upward and detected by the detection means via the capacitor 11, the mirror 18, and the objective lens 7. For this reason, detection efficiency higher than that of the culture vessel 500 can be realized.

  The culture containers according to Examples 1 to 6 described above are common in that they include light guide means for guiding light from the biological specimen 101 incident on the inner surface of the culture container to the upper side or the lower side of the culture container. Since this light guide means contributes to improvement in detection efficiency, the stored biological specimen 101 can be observed brightly by using these culture vessels.

  In Example 1, the reflecting surface 113 is used, in Example 2, the diffractive surface 212 is used, in Example 3, the reflecting surface 313 is used, in Example 4, the reflecting surface 113 and the reflecting surface 415 are used, and in Example 5, the reflecting surface 512 is used. In Example 6, the reflective surface 612 functions as a light guide means.

  FIG. 8 is a diagram for explaining the configuration of a conventional culture vessel 700. FIG. 9 is a diagram for explaining the configuration of the culture vessel 800 according to the present embodiment.

  As shown in FIG. 8, in a conventional culture vessel 700 that does not have a lid (or the lid does not contact the culture medium 102), the interface between the culture medium 102 and air (hereinafter referred to as the gas-liquid interface) has a concave surface. It has a shape facing upward. This is due to the influence of the interfacial tension between the culture medium 102 and the side surface 112.

  The culture solution 102 having the gas-liquid interface having such a shape acts as a concave lens on the fluorescence emitted from the biological specimen 101, and reduces the amount of fluorescence that can be captured by the capacitor 11. In other words, due to the presence of the gas-liquid interface (culture solution 102), the NA of the fluorescence that can be taken up by the capacitor 11 is lower than the NA of the capacitor 11. The culture container 800 according to the present embodiment is an improvement on this point.

  A culture container 800 shown in FIG. 9 includes a container main body 810 that can store the biological specimen 101 by immersing it in the culture solution 102, and a lid 820 arranged on the container main body 810. The container body 810 is the same as the culture container 700 shown in FIG. 8 and has a bottom surface 111 and side surfaces 112 through which light passes.

  The lid 820 is made of a material that transmits at least light (fluorescence) from the biological specimen 101, and has a convex portion having the same diameter as the diameter of the opening of the container body 810. The lid 820 is disposed so as to overlap the container body 810 in a state where the position of the convex portion is matched with the position of the opening of the container body 810. Thereby, a convex part is inserted in opening and the front-end | tip of a convex part contacts the culture solution 102. FIG. Since the tip of the convex portion is constituted by a surface 821 having a planar shape, the interface between the surface 821 and the culture medium 102 (hereinafter referred to as a solid-liquid interface) has a planar shape. Thereby, it can prevent that the culture solution 102 acts as a concave lens.

  Therefore, according to the culture vessel 800, the amount of fluorescent light that can be captured by the capacitor 11 can be increased compared to the conventional culture vessel 700. Thereby, since high detection efficiency is implement | achieved, the accommodated biological specimen 101 can be observed brightly.

  FIG.10 and FIG.11 is a figure for demonstrating the structure of the culture container 900 which concerns on a present Example. FIG. 10 shows a state where the lid 920 and the culture medium 102 are not in contact with each other, and FIG. 11 shows a state where the lid 920 and the culture medium 102 are in contact with each other. The culture vessel 900 is different from the culture vessel 800 in that a lid 920 is provided instead of the lid 820. Other points are the same as those of the culture vessel 800.

  The lid 920 is the same as the lid 820 in that the lid 920 is overlaid on the container main body 810, and is made of a material that transmits at least light from the biological specimen 101. The lid 920 has a convex portion that protrudes up and down. The convex portion has a convex surface 921 directed to the bottom surface 111 side and a convex surface 922 directed in the opposite direction to the convex surface 921, each functioning as a lens surface. As a result, the lid 920 functions as a positive lens for fluorescence that is light from the biological specimen 101 generated from the biological specimen 101 in a state where the lid 920 is placed on the container body 810.

  As shown in FIG. 10, the culture vessel 900 can condense fluorescence spread at the gas-liquid interface having a concave shape with the lid 920, and thus can be taken in by the capacitor 11 compared to the conventional culture vessel 700. The amount of fluorescent light can be increased. Further, as shown in FIG. 11, the surface 921 of the lid 920 is brought into contact with the culture solution 102 to eliminate the gas-liquid interface, thereby preventing the refraction of fluorescence at the gas-liquid interface. Thereby, more fluorescence can be taken into the capacitor 11. Thus, according to the culture container 900, since the high detection efficiency is implement | achieved, the accommodated biological specimen 101 can be observed brightly.

  FIG. 12 is a diagram for explaining the configuration of the culture vessel 1000 according to the present embodiment. The culture vessel 1000 is different from the culture vessel 800 in that the culture vessel 1000 includes a lid 1020 instead of the lid 820. Other points are the same as those of the culture vessel 800.

  The lid 1020 is made of a material that transmits at least light from the biological specimen 101, and has a convex portion having the same diameter as the opening of the container body 810, similar to the lid 820. The lid 1020 is different from the lid 820 in that a Fresnel lens 1021 is disposed at the tip of the convex portion. The Fresnel lens 1021 has a Fresnel lens surface 1022 directed toward the bottom surface 111 side. The Fresnel lens surface 1022 has a positive power. For this reason, the lid 1020 functions as a positive lens with respect to fluorescence that is visible light generated from the biological specimen 101 in a state where the lid 1020 is placed on the container body 810.

  In the culture vessel 1000 as well as the culture vessel 900, since the fluorescence spread at the gas-liquid interface having a concave shape can be converged by the lid 1020, it can be taken in by the capacitor 11 compared to the conventional culture vessel 700. The amount of fluorescent light can be increased. Therefore, according to the culture container 1000, since high detection efficiency is implement | achieved, the accommodated biological specimen 101 can be observed brightly. Further, by employing the Fresnel lens structure, the thickness of the lid 1020 functioning as a positive lens can be reduced.

  The above-described embodiments are specific examples for facilitating understanding of the invention, and the present invention is not limited to these embodiments. The culture vessel and the multiphoton excitation microscope can be variously modified and changed without departing from the concept of the present invention defined by the claims. For example, several features in the context of the individual embodiments described in this specification may be combined into a single embodiment.

  For example, the culture containers according to Examples 1 to 4 have lids, but these lids may be omitted. Moreover, although the microscope according to each embodiment is configured as a two-photon excitation microscope, it may be another multi-photon excitation microscope in which fluorescence is generated from one point in the specimen.

  Although FIG. 1 shows an example in which the optical system 16 is arranged around the objective lens 7 as the third detecting means, the photoelectric conversion element itself may be arranged around the objective lens 7 instead of the optical system 16. Good. The third detection unit may be configured by arranging the optical system 16 around the capacitor 11. In this case, for example, the culture vessel 100 shown in FIG. 2 may be configured to have a reflection surface that reflects the fluorescence upward instead of the reflection surface 113 that reflects the fluorescence downward.

DESCRIPTION OF SYMBOLS 1 Microscope 2 Stage 2a Container holding part 3 Laser 4 XY scanner 5 Relay optical system 6 Dichroic mirror 7 Objective lens 8, 12 Relay lens 9, 14 IR cut filter 10, 15 PMT
11 Condenser 13, 18 Mirror 16 Optical system 17 Optical fiber 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 Culture vessel 101 Biological specimen 102 Culture solution 110, 210, 310, 810 Container body 111 Bottom 112 Side surfaces 113, 124, 313, 321, 415, 512, 612 Reflective surfaces 120, 320, 820, 920, 1020 Lid 121 Lid member 122 Support member 123 Opening 212 Diffraction surface 213 Outer surface 414 Lens surfaces 821, 921, 922 Surface 1021 Fresnel lens 1022 Fresnel lens surface

Claims (11)

A culture container that can contain a biological specimen immersed in a culture solution,
A culture vessel comprising light guide means for guiding light from the biological specimen incident on the inner surface of the culture vessel to the upper side or the lower side of the culture vessel. In the culture container according to claim 1,
The culture vessel, wherein the light from the biological specimen is fluorescence generated by multiphoton excitation. In the culture container according to claim 1 or 2,
The culture container is a microplate having a structure for individually storing a plurality of biological specimens. In the culture container according to any one of claims 1 to 3,
The culture container, as the light guide means, has a reflective surface that reflects light from the biological specimen transmitted through the inner surface upward or downward. In the culture container according to any one of claims 1 to 3,
The culture vessel has a diffracting surface formed on the inner surface as the light guide means for diffracting light from the biological specimen. In the culture container according to any one of claims 1 to 3,
The culture vessel has a reflective surface that reflects light from the biological specimen formed on the inner surface as the light guide means. The culture container according to any one of claims 1 to 6, further comprising:
A container main body having a bottom surface through which light is transmitted and the inner side surface, the biological specimen being able to be immersed in the culture solution
A culture container, comprising: a lid that is placed over the container body, the lid having a reflective surface that reflects light from the biological specimen formed toward the bottom surface side. The culture container according to claim 7,
The lid has an opening or a transmission part through which light from the biological specimen is transmitted,
The culture container according to claim 1, wherein the reflection surface formed on the lid is formed around the opening or the transmission part. In the culture container according to claim 7 or claim 8,
The culture vessel, wherein the reflection surface formed on the lid has a concave shape. The culture container according to any one of claims 1 to 9, wherein a biological specimen is accommodated,
A stage on which the culture vessel is disposed;
An objective lens that irradiates the biological specimen with excitation light; and first detection means that detects fluorescence generated from the biological specimen via the objective lens;
Has a capacitor disposed on the opposite side to the objective lens across the stage, a second detecting means for detecting fluorescence generated from the biological specimen via the front Symbol capacitor,
And third detection means for detecting the fluorescence generated from the biological specimen and emitted from the culture vessel to the stage side and guided to the outside of the objective lens. Multiphoton excitation microscope. An observation method for observing a biological specimen housed in a culture container according to any one of claims 1 to 9 disposed on a stage of a multiphoton excitation microscope,
Irradiating the biological specimen with excitation light through the objective lens of the multiphoton excitation microscope,
An observation characterized in that the detection means of the multi-photon excitation microscope detects fluorescence generated from the biological specimen, which is guided upward or downward of the culture container by the light guide means and emitted from the culture container. Method.