JP6832735B2 - microscope - Google Patents

Description

The present invention relates to a microscope.

Conventionally, a microscope has been known in which excitation light is incident on a sample from a direction intersecting the detection optical axis of the detection optical system to acquire a three-dimensional stereoscopic image of the sample based on fluorescence from the sample detected by the detection optical system. (See, for example, Patent Document 1 and Patent Document 2). Since the microscopes described in Patent Document 1 and Patent Document 2 are not irradiated with excitation light other than the imaging plane, fading of fluorescence can be suppressed and a good three-dimensional stereoscopic image can be obtained.

In recent years, this technique has been aimed not only at obtaining stereoscopic images of organisms such as zebrafish labeled with fluorescent proteins on target molecules, but also such as spheroids and organoids (artificial organs or parts thereof). It is also attracting attention for the purpose of obtaining a three-dimensional stereoscopic image of three-dimensional cultured cells and evaluating the drug efficacy by using image analysis technology, so-called drug discovery screening, and is applied to a wide range of applications. Is expected. Further, in this observation method, finer and higher resolution observation is required due to the strong needs of researchers who want to observe individual cells at a recognizable level of resolution.

In this observation method, the microscopes described in Patent Documents 1 and 2 use an immersion objective lens. However. In the microscope described in Patent Document 1, when the container is moved with respect to the objective lens to change the observation position, the amount of the immersion medium held between the objective lens and the container decreases, so that the immersion medium is reduced. Need to be replenished. In particular, the longer the relative movement distance between the objective lens and the container, such as when the container is composed of a plurality of arrays, the more frequently it is necessary to replenish the immersion medium, and a large amount of immersion medium is prepared. There is an inconvenience that it must be kept. Further, since it takes time to replenish the immersion medium, there is an inconvenience that the total observation time becomes longer as the replenishment frequency increases.

On the other hand, in the microscope described in Patent Document 2, the sample is housed in a cuvette filled with an immersion medium such as a clearing solution, and the cuvette is further housed in a chamber filled with the immersion medium. It is placed on the XYZ stage. Further, the tip of the objective lens for observation is immersed in the immersion medium in the chamber via a sealing member for preventing liquid leakage. According to the configuration of the microscope of Patent Document 2, the immersion medium does not decrease even if the XYZ stage is moved, so that the above-mentioned problem of the microscope described in Patent Document 1 can be solved.

Japanese Patent No. 4443832 International Publication No. 2015/184124

However, in the microscope described in Patent Document 2 in which the objective lens cannot be moved, when the focal position of the objective lens shifts according to the refractive index distribution in the sample due to the change in the observation position, the excitation light is emitted by the scanner. The deviation of the focal position is corrected by adjusting the irradiation position of the lens to the focal position of the objective lens, and there is a problem that a complicated and expensive adjustment mechanism is required to perform such correction.

The present invention has been made in view of the above circumstances, and has a simple and inexpensive configuration, reduces the amount of the immersion medium and the frequency of replenishment thereof, prevents the immersion medium from running out, and is reliable. It is an object of the present invention to provide a microscope capable of realizing high observation.

In order to achieve the above object, the present invention provides the following means.
One aspect of the present invention is a medium container for storing a second immersion medium having a refractive index equivalent to that of the first immersion medium, in which the sample container for accommodating the first immersion medium is immersed together with the sample. An objective lens that is arranged outside the medium container and collects the light emitted from the sample, an imaging unit that captures the light collected by the objective lens, and a direction in which the objective lens is along the detection optical axis. The specimen container and the medium container include a movable stage that movably supports the sample container in the medium container in a direction along at least the detection optical axis, and the sample container and the medium container are the same from the sample. A microscope having a light transmitting portion capable of transmitting light, and the objective lens is arranged so as to face the light transmitting portion of the sample container via the light transmitting portion of the medium container.

According to this aspect, the specimen is housed in the specimen container together with the first immersion medium, and the specimen container is immersed in the second immersion medium in the medium container. Then, the light emitted from the sample is transmitted through the first immersion medium, the light transmitting portion of the sample container, the second immersion medium having the same refractive index as the first immersion medium, and the light transmission portion of the medium container. It is focused by the objective lens and photographed by the imaging unit. Therefore, by moving the sample container in the medium container in the direction along the detection optical axis of the objective lens by the movable stage, it is possible to acquire a tomographic image of the sample intersecting the detection optical axis of the objective lens.

In this case, even if the sample container is moved in the medium container by the movable stage to change the observation position, the relative position between the objective lens and the medium container does not change, so that the tip of the objective lens and the light transmitting portion of the medium container Even if the immersion medium is held between the two, the amount of the immersion medium does not change due to the movement of the sample container. Therefore, it is not necessary to prepare a large amount of immersion medium between the tip of the objective lens and the light transmitting portion of the medium container or to replenish the immersion medium frequently, and the immersion medium does not run out.

Further, when the observation position of the sample is changed, even if the focal position of the objective lens shifts according to the refractive index distribution in the sample, the aiming portion determines the position in the direction along the detection optical axis of the objective lens. By making fine adjustments, the deviation of the focal position can be eliminated.
Therefore, it is possible to construct a microscope that realizes highly reliable observation by preventing the immersion medium from running out while reducing the amount of the immersion medium and the replenishment frequency thereof with a simple and inexpensive configuration.

In the above aspect, the objective lens may be arranged at a distance from the light transmitting portion of the medium container.
With this configuration, it is possible to switch the objective lens to one with a different magnification.

In the above aspect, the objective lens may be an immersion objective lens, and the immersion objective lens may be arranged between the immersion objective lens and the light transmitting portion of the medium container via a third immersion medium.
With this configuration, by adopting a third immersion medium having a refractive index larger than that of air, the numerical aperture of the immersion objective lens can be increased and higher resolution can be obtained. Further, since the amount of the third immersion medium does not change due to the movement of the specimen container, it is not necessary to prepare a large amount of the third immersion medium or replenish it frequently, and the third immersion medium runs out of liquid. There is no such thing.

In the above aspect, the immersion objective lens is arranged with the detection optical axis oriented in a direction intersecting the vertical direction, and the third immersion medium is the light transmission of the immersion objective lens and the medium container. It may be held by surface tension between the parts.
With such a configuration, a mechanism for holding the third immersion medium between the tip of the immersion objective lens and the light transmitting portion of the medium container is not required, and a simple configuration can be achieved.

In the above embodiment, the sample container and the medium container have the light transmitting portion on the side wall portion, and the immersion objective lens is arranged with the detection optical axis directed in a direction substantially orthogonal to the vertical direction. May be.
With this configuration, the immersion objective lens can be arranged on the side of the medium container so as to be close to the light transmitting portion of the side wall portion via the third immersion medium.

In the above aspect, the third immersion medium may have a refractive index equivalent to that of the second immersion medium.
With this configuration, when the observation position of the sample is changed, the focal position of the immersion objective lens shifts according to the refractive index distribution in the sample, and the detection optical axis of the immersion objective lens is caused by the aiming portion. Even if the focus is finely adjusted in the direction along the above, the occurrence of spherical aberration can be suppressed.

In the above aspect, the light transmitting portion of the specimen container may have a refractive index equivalent to that of the second immersion medium.
With this configuration, it is possible to suppress the occurrence of spherical aberration even when the thickness of the light transmitting portion of the specimen container varies due to manufacturing errors.

In the above aspect, the light transmitting portion of the medium container may have a refractive index equivalent to that of the second immersion medium.
With this configuration, it is possible to suppress the occurrence of spherical aberration even when the thickness of the light transmitting portion of the medium container varies due to manufacturing errors.

In the above aspect, the movable stage may movably support the specimen container in a direction intersecting the detection optical axis.
With this configuration, the observation position of the sample can be changed in the direction intersecting the detection optical axis of the objective lens.

In the above aspect, the imaging unit may capture the light emitted by the specimen by itself.
With such a configuration, the light emitting microscope can be configured, and the configuration can be as simple and inexpensive as the light source and the illumination optical system are not required.

In the above embodiment, the sample is provided with an illumination optical system that irradiates the sample with excitation light from a direction intersecting the detection optical axis, and the sample container and the medium container transmit the excitation light from the illumination optical system to the sample. It may have each of the excitation light transmitting portions which are directed and transmitted.
With this configuration, the illumination optical system is arranged on the side of the medium container, and the excitation light emitted from the illumination optical system is sampled from the side of the medium container and the sample container via each excitation light transmitting portion. Can be irradiated.

In the above aspect, the medium container has the excitation light transmitting portion at the bottom, the illumination optical system includes a reflection mirror arranged in the medium container, and the excitation light is used as the excitation light at the bottom. It may be incident on the medium container from the transmitting portion and reflected toward the sample by the reflection mirror.

With this configuration, the illumination optical system excluding the reflection mirror can be arranged below the medium container. This makes it possible to avoid mechanical interference between the movable stage and the sample container and the illumination optical system, and the excitation light is sampled not only from one direction but also from multiple directions intersecting the detection optical axis of the objective lens. It is possible to easily construct an illumination optical system capable of incident.

In the above aspect, the medium container has the excitation light transmitting portion on the side wall portion, and the illumination optical system causes the excitation light to enter the medium container from the excitation light transmitting portion of the side wall portion. May be.
With this configuration, the illumination optical system can be arranged on the side of the medium container. As a result, it can be configured by simply adding a medium container, a movable stage, and an illumination optical system to an ordinary inverted microscope provided with an objective lens, an aiming unit, and an imaging unit.

In the above aspect, the illumination optical system may include a lens having positive power arranged in the medium container.
In order to reduce the thickness of the sheet illumination and increase the resolution, it is necessary to set a large injection NA of the lens. With this configuration, the distance from the lens to the sample can be shortened as compared with the case where the lens is arranged outside the medium container, and the injection NA of the lens can be set large. As a result, the resolution can be improved with a simple configuration in which a lens having positive power is simply arranged in the medium container.

In the above aspect, the lens having positive power may be a cylindrical lens having positive power in one direction intersecting the illumination optical axis of the illumination optical system.
With this configuration, the cylindrical lens can collect the excitation light in a plane along the plane intersecting the detection optical axis of the detection optical system and make it incident on the sample. As a result, by matching the focal plane of the objective lens with the incident plane of the excitation light, the light generated in a wide range along the focal plane is focused at once by the objective lens, and a higher resolution image is acquired. can do.

In the above aspect, the illumination optical system includes a microlens array in which a plurality of microlenses are two-dimensionally arranged in a direction intersecting the photographing optical axis of the imaging unit, and the illumination optical system emits the excitation light having a substantially parallel luminous flux. It may be incident on the sample.

With this configuration, by matching the focal plane of the objective lens with the incident range of the excitation light, the light generated in a wide range along the focal plane can be focused at once by the objective lens. Then, by photographing the image projected by the microlens array by the imaging unit, it is possible to acquire a plurality of image information having different parallax at one time.

In the above aspect, the movable stage may support the plurality of specimen containers so as to be movable in the medium container around an axis parallel to the detection optical axis.
With this configuration, the specimen containers arranged on the detection optical axis can be switched by simply moving a plurality of specimen containers around the axis parallel to the detection optical axis by the movable stage. Therefore, the excitation light can be sequentially incident on the sample of each sample container by the illumination optical system, and the light from the sample of each sample container can be collected in order by the objective lens and continuously photographed by the imaging unit. As a result, images of a large number of specimens can be acquired efficiently and at high speed.

In the above aspect, the specimen container has a plurality of specimen accommodating portions arranged in one direction intersecting the illumination optical axis of the illumination optical system, and the movable stage is arranged on the detection optical axis. The specimen housing may be supported in a switchable manner.
With this configuration, a plurality of samples can be observed in order by simply switching the sample accommodating portion arranged on the detection optical axis of the objective lens by the movable stage.

In the above aspect, the movable stage may rotatably support the specimen container in the medium container around the detection optical axis.
With this configuration, the excitation light can be incident on the same observation position on the specimen from different directions simply by rotating the specimen container around the detection optical axis by the movable stage. As a result, the depth of incidence of the excitation light from each direction on the sample can be made shallow to suppress the influence of scattering on the sample, and a clear image can be obtained.

In the above aspect, the illumination optical system may make the planar excitation light incident on the sample along a plane intersecting the detection optical axis of the objective lens.
With this configuration, by aligning the focal plane of the objective lens with the incident plane of the excitation light, the fluorescence generated in a wide range along the focal plane of the objective lens is condensed at once by the objective lens. A light sheet microscope capable of acquiring a higher resolution image can be constructed.

According to the microscope according to the present invention, it is possible to realize highly reliable observation by preventing the immersion medium from running out while reducing the amount of the immersion medium and the frequency of replenishment thereof with a simple and inexpensive configuration. It has the effect of being able to do it.

It is a schematic block diagram which shows the microscope which concerns on 1st Embodiment of this invention. It is a top view which looked at the cuvette and the prism of FIG. 1 in the direction along the detection optical axis. It is a schematic block diagram which shows the microscope which concerns on the modification of 1st Embodiment of this invention. It is a schematic block diagram which shows the microscope which concerns on 2nd Embodiment of this invention. It is a schematic block diagram which shows the microscope which concerns on 3rd Embodiment of this invention. It is a schematic block diagram which shows the microscope which concerns on 4th Embodiment of this invention. It is a perspective view which shows the chamber and the cuvette of FIG. It is a perspective view which shows the cuvette of FIG. It is a perspective view which shows the chamber of FIG. It is a schematic block diagram which looked at the microscope which concerns on 5th Embodiment of this invention in the vertical direction. It is a schematic block diagram which looked at the microscope of FIG. 10 in the direction along the detection optical axis of the immersion objective lens. It is a schematic block diagram which shows the microscope which concerns on 6th Embodiment of this invention.

[First Embodiment]
The microscope according to the first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the microscope 1 according to the present embodiment includes a cuvette (specimen container) 3 for accommodating a sample (specimen) S, a chamber (medium container) 5 capable of accommodating the cuvette 3, and a cuvette 3. A movable stage 7 to support, an illumination optical system 9 for irradiating sample S with laser light (excitation light), an immersion objective lens (objective lens) 11 for condensing fluorescence emitted from sample S, and an immersion objective lens. An aiming portion 12 capable of moving the 11 in the direction along the detection optical axis P, and an imaging optical system 13 for acquiring an image of the sample S based on the fluorescence collected by the immersion objective lens 11 are provided.

As shown in FIG. 2, the cuvette 3 has an array structure in which three accommodating portions (sample accommodating portions) 3a for accommodating the sample S are arranged in one direction. Each accommodating portion 3a is filled with a cuvette solution (first immersion medium) W1 such as a clearing solution, and the sample S is immersed in the cuvette solution W1. Each sample S is made transparent by being immersed in the cuvette solution W1. Further, as shown in FIG. 1, the cuvette 3 has transparent portions (excitation light transmitting portion and light transmitting portion) 3b capable of transmitting laser light and fluorescence on the side wall portion and the bottom portion, respectively.

The chamber 5 has an opening 5a at the top and a transparent portion (excitation light transmitting portion, light transmitting portion) 5b capable of transmitting laser light and fluorescence at the bottom. The transparent portion 5b is formed over a wide range of the bottom portion including the illumination optical axis Q of the illumination optical system 9 and the detection optical axis P of the immersion objective lens 11. A chamber solution (second immersion medium) W2 having a refractive index substantially equal to that of the cuvette solution W1 is stored in the chamber 5, and the cuvette 3 is immersed in the chamber solution W2.

The movable stage 7 includes an arm portion 7a for holding the cuvette 3 and a support portion 7b for supporting the arm portion 7a. In this movable stage 7, the cuvette 3 is immersed in the chamber solution W2 through the opening 5a of the chamber 5 by the arm portion 7a and the support portion 7b, and the transparent portion 3b of the side wall portion is located on the illumination optical axis Q. As such, it has come to support the cuvette 3.

Further, the movable stage 7 moves the holding cuvette 3 in the vertical direction (hereinafter referred to as the Z direction) and the two-dimensional directions intersecting the vertical direction and orthogonal to each other (hereinafter referred to as the X and Y directions). It is designed to be able to. As a result, the movable stage 7 can switch the accommodating portion 3a of the cuvette 3 arranged on the detection optical axis P, and can change the observation position of the sample S within the same accommodating portion 3a. There is.

The immersion objective lens 11 is configured by combining a large number of lenses (not shown). The immersion objective lens 11 is provided below the chamber 5 in the vicinity of the transparent portion 5b at the bottom, and is arranged vertically upward facing the transparent portion 5b. An immersion solution (third immersion medium) W3 such as pure water is injected into the gap between the upper surface 11a of the state-of-the-art lens of the immersion objective lens 11 and the transparent portion 5b at the bottom of the chamber 5, and the immersion solution is injected. W3 is held in the gap by surface tension.

The aiming portion 12 uses the immersion objective lens 11 within a range in which the surface tension of the immersion solution W3 acts in the gap between the upper surface 11a of the state-of-the-art lens of the immersion objective lens 11 and the transparent portion 5b at the bottom of the chamber 5. By finely moving in the Z direction, the focal position of the immersion objective lens 11 can be finely adjusted in the direction along the detection optical axis P.

The illumination optical system 9 converts the laser light source 15 that generates laser light, the optical fiber 17 that guides the laser light emitted from the laser light source 15, and the laser light that has been guided by the optical fiber 17 into parallel light beams. Convex lenses 19A and 19B, cylindrical lenses (lenses) 21A and 21B that collect the laser light condensed into parallel light beams by the convex lenses 19A and 19B, and laser light focused by the cylindrical lenses 21A and 21B are directed toward the sample S. It is provided with prisms 23A and 23B having mirror-coated reflective surfaces (reflecting mirrors) 24A and 24B that reflect light.

The optical fiber 17 has two tip portions 18A and 18B branched from an intermediate position in the longitudinal direction. These tip portions 18A and 18B are provided at intervals in a direction intersecting the detection optical axis P with the immersion objective lens 11 interposed therebetween, and are arranged vertically upward facing the transparent portion 5b at the bottom of the chamber 5. There is.

The laser light emitted from one tip 18A of the optical fiber 17 is reflected toward the sample S by the reflecting surface 24A of the prism 23A via the convex lens 19A and the cylindrical lens 21A, and is emitted from the other tip 18B. The laser light is reflected toward the sample S by the reflecting surface 24B of the prism 23B via the convex lens 19B and the cylindrical lens 21B.

The convex lenses 19A and 19B and the cylindrical lenses 21A and 21B are arranged outside the chamber 5 with an interval in the direction intersecting the detection optical axis P, and the prisms 23A and 23B are the detection lights inside the chamber 5. The shaft P is arranged at intervals in the direction intersecting the shaft P, and is fixed to the inner bottom portion.

The cylindrical lenses 21A and 21B have power in one direction orthogonal to the illumination optical axis Q. The cylindrical lenses 21A and 21B focus laser light composed of substantially parallel light flux on a substantially detection optical axis P of the immersion objective lens 11 by condensing it in a plane having the same predetermined width dimension as the luminous flux diameter dimension thereof. It is designed to connect.

The prisms 23A and 23B reflect each laser beam focused in a plane by the cylindrical lenses 21A and 21B by the reflecting surfaces 24A and 24B, and the cuvette is along the same incident plane extending in the direction perpendicular to the detection optical axis P. The laser beam is incident on the sample S through the transparent portion 3b of the side wall portion of the sample 3.

The convex lenses 19A and 19B, the cylindrical lenses 21A and 21B, and the reflecting surfaces 24A and 24B of the prisms 23A and 23B are preliminarily adjusted so that the focal positions of the respective laser beams match.

The imaging optical system 13 includes a mirror 25 that reflects the fluorescence focused by the immersion objective lens 11, an emission filter 27 that removes laser light and the like from the fluorescence reflected by the mirror 25, and fluorescence that has passed through the emission filter 27. It is provided with an imaging lens 29 for forming an image of the image, and a camera (imaging unit) 31 for photographing the fluorescence imaged by the imaging lens 29.

The cuvette solution W1 in the cuvette 3, the chamber solution W2 in the chamber 5, the immersion solution W3 between the immersion objective lens 11 and the chamber 5, the transparent portion 3b of the cuvette 3 and the transparent portion 5b of the chamber 5 are substantially equivalent to each other. Has a refractive index of.

The operation of the microscope 1 configured in this way will be described.
In order to observe the sample S with the microscope 1 according to the present embodiment, first, the movable stage 7 supports the cuvette 3 containing the sample S and the cuvette solution W1 and is immersed in the chamber 5 at a target observation position. Move to. In the example shown in FIG. 2, the accommodating portion 3a in the center of the cuvette 3 is arranged on the detection optical axis P of the immersion objective lens 11.

Subsequently, the laser light is generated from the laser light source 15. The laser light emitted from the laser light source 15 is guided by the optical fiber 17 and branched in the middle, and is emitted from the two tip portions 18A and 18B. Then, the laser light is made into a parallel light flux by the convex lenses 19A and 19B, respectively, is condensed in a plane by the cylindrical lenses 21A and 21B, and is incident on the transparent portion 5b at the bottom of the chamber 5.

The laser beam incident on the chamber 5 from the transparent portion 5b at the bottom enters the prisms 23A and 23B, respectively, and is reflected by the reflecting surfaces 24A and 24B. Then, the laser beam is incident on the sample S from two opposite directions intersecting the detection optical axis P via the chamber solution W2, the transparent portion 3b of the cuvette 3, and the cuvette solution W1.

Since the transparent portion 5b of the chamber 5, the chamber solution W2, the transparent portion 3b of the cuvette 3 and the cuvette solution W1 have substantially the same refractive index, the laser beam emitted by the illumination optical system 9 is incident on the sample S without being refracted. Can be made to.

When a planar laser beam is incident on the sample S, the fluorescent substance in the sample S is excited along the incident plane of the laser beam to generate fluorescence. Among the fluorescence generated in the sample S, the fluorescence radiated in the direction along the detection optical axis P is the cuvette solution W1, the transparent portion 3b at the bottom of the cuvette 3, the chamber solution W2, the transparent portion 5b at the bottom of the chamber 5, and the liquid. The light is collected by the immersion objective lens 11 via the immersion solution W3.

Also in this case, the cubic solution W1, the transparent portion 3b at the bottom of the cuvette 3, the chamber solution W2, the transparent portion 5b at the bottom of the chamber 5, and the immersion solution W3 have substantially the same refractive index, so that the fluorescence from the sample S is fluorescent. Can be focused by the immersion objective lens 11 without refracting.

The fluorescence collected by the immersion objective lens 11 is reflected by the mirror 25, passes through the emission filter 27, and is imaged on the imaging surface of the camera 31 by the imaging lens 29. As a result, a tomographic image of sample S is obtained in the camera 31.

By injecting a planar laser beam along the incident plane extending in the direction perpendicular to the detection optical axis P of the immersion objective lens 11 onto the sample S, the focal positions of the cylindrical lenses 21A and 21B and the immersion objective lens 11 can be detected. By matching the optical axis P and the focal plane of the immersion objective lens 11 with the incident plane of the laser beam, the immersion objective lens can generate fluorescence in a wide range along the focal plane of the immersion objective lens 11. The lens can be focused at one time by 11 and photographed by the camera 31. As a result, a clear fluorescence image of the observation region in the sample S can be obtained. Further, since the laser beam is not emitted to the plane other than the imaging plane of the camera 31, fading of fluorescence can be suppressed and a good three-dimensional stereoscopic image can be obtained.

In this case, even if the cuvette 3 is moved in the chamber 5 by the movable stage 7 to change the observation position of the sample S, the amount of the immersion solution W3 arranged in the gap between the immersion objective lens 11 and the chamber 5 Does not change, so that it is not necessary to prepare a large amount of the immersion solution W3 or replenish it frequently, and it is possible to prevent the immersion solution W3 from running out.

Further, when the observation position of the sample S is changed, even if the focal position of the immersion objective lens 11 shifts according to the refractive index distribution in the sample S, the immersion objective lens 11 is detected by the aiming unit 12. By finely adjusting the position in the direction along the optical axis P, the deviation of the focal position can be eliminated. As a result, the desired observation position in the sample S can be observed with high accuracy.

Since the immersion solution W3 has a refractive index substantially equal to that of the chamber solution W2, the spherical surface is formed when the aiming portion 12 finely adjusts the focus in the direction along the detection optical axis P of the immersion objective lens 11. The occurrence of aberration can be suppressed. Further, since the transparent portion 3b of the cuvette 3 and the transparent portion 5b of the chamber 5 have a refractive index substantially equal to that of the chamber solution W2, the thickness of the transparent portion 3b of the cuvette 3 and the thickness of the transparent portion 5b of the chamber 5 due to manufacturing errors. Even when there are variations, the occurrence of spherical aberration can be suppressed.

As described above, according to the microscope 1 according to the present embodiment, even if the focal position of the immersion objective lens 11 shifts when the observation position of the sample S is changed, the immersion objective lens 11 is displaced by the aiming portion 12. The deviation of the focal position can be eliminated only by making fine adjustments in the direction along the detection optical axis P. Therefore, unlike a conventional microscope, it is not necessary to use a complicated and expensive adjustment mechanism for correcting the deviation of the focal position of the immersion objective lens by a scanner, and the amount of the immersion solution W3 is simple and inexpensive. It is possible to prevent the immersion solution W3 from running out while reducing the frequency of replenishment thereof, and to realize highly reliable observation.

Further, the illumination optical system 9 allows the laser light to enter the chamber 5 from the transparent portion 5b at the bottom of the chamber 5, so that the illumination optical system 9 excluding the prisms 23A and 23B can be arranged below the chamber 5. .. As a result, mechanical interference between the movable stage 7 and the cuvette 3 and the illumination optical system 9 can be avoided, and laser light is incident on the sample S from two directions intersecting the detection optical axis P of the immersion objective lens 11. It is possible to easily configure the illumination optical system 9 to be used.

In the present embodiment, the illumination optical system 9 allows the laser light to be incident on the sample S from two different directions at the same time, but instead of this, a single illumination in which the laser light is incident on the sample S from only one direction is used. There may be. In many cases, the sample S absorbs or scatters light, so that it is advantageous to illuminate with a plurality of illumination luminous fluxes in terms of illumination uniformity.

Further, the movable stage 7 may rotatably support the cuvette 3 in the chamber 5 around the detection optical axis P. By doing so, the laser beam can be incident on the same observation position in the sample S from different directions simply by rotating the cuvette 3 around the detection optical axis P by the movable stage 7. As a result, the depth of incidence of the laser beam from each direction on the sample S can be made shallow to suppress the influence of scattering in the sample S, and a clear fluorescent image can be obtained. It is more effective in the case of single lighting.

This embodiment can be modified as follows.
In the present embodiment, the cylindrical lenses 21A and 21B are arranged outside the chamber 5, but instead, for example, as shown in FIG. 3, the cylindrical lenses 21A and 21B are arranged inside the chamber 5. May be good. In this case, for example, the cylindrical lenses 21A and 21B may be attached to the injection ends of the prisms 23A and 23B.

In order to increase the resolution, it is necessary to set a large emission NA of the cylindrical lenses 21A and 21B to reduce the thickness of the planar laser beam. According to this modification, the distance from the cylindrical lenses 21A and 21B to the sample S can be shortened as compared with the case where the cylindrical lenses 21A and 21B are arranged outside the chamber 5. Therefore, the injection NA of the cylindrical lenses 21A and 21B can be set large to reduce the thickness of the planar laser beam. As a result, the resolution can be improved with a simple configuration in which the cylindrical lenses 21A and 21B are arranged in the chamber 5.

[Second Embodiment]
Next, the microscope according to the second embodiment of the present invention will be described.
In the microscope 41 according to the present embodiment, as shown in FIG. 4, the illumination optical system 9 further includes a cylindrical lens 43 having a negative power and shutters 45A and 45B, and the shutters 45A and 45B are switched by switching the shutters 45A and 45B. It differs from the first embodiment in that the function as a light field microscope and the function as a light sheet microscope can be switched.
Hereinafter, the parts having the same configuration as the microscope 1 according to the first embodiment are designated by the same reference numerals and the description thereof will be omitted.

The cylindrical lens 43 having a negative power is provided, for example, on the optical path of the laser beam emitted from the tip portion 18A of the optical fiber 17, and is attached to the ejection end of the prism 23A. The cylindrical lens 43 makes it possible to form a laser beam having a rectangular cross section having a thickness corresponding to the observation depth in the direction along the detection optical axis P of the immersion objective lens 11.

The shutters 45A and 45B are detachably provided on both illumination optical axes Q of the illumination optical system 9, and are arranged between the tip portions 18A and 18B of the optical fiber 17 and the convex lenses 19A and 19B. ..

The imaging optical system 13 includes a microlens array 47 composed of a plurality of microlenses 48 arranged in front of the camera 31. The microlenses 48 are two-dimensionally arranged in a direction intersecting the photographing optical axis of the camera 31.

Further, the imaging optical system 13 includes an imaging lens 29A for forming an image on the microlens array 47, an imaging lens 29B for forming an image on the imaging surface of the camera 31, and the imaging lens 29A and the imaging lens 29B. It is provided with an imaging lens turret 49 to be held.

The microlens array 47 is designed to project an image on the imaging surface of the camera 31. As a result, the camera 31 can acquire a plurality of image information having different parallax at one time.

The imaging lens turret 49 is rotatably provided around the rotation axis 49a so that the imaging lens 29A and the imaging lens 29B can be selectively arranged on the fluorescence optical path.

The laser light source 15, the optical fiber 17, the convex lens 19A, the cylindrical lens 21A, the prism 23A and the cylindrical lens 43 of the illumination optical system 9, the immersion objective lens 11, the mirror 25 of the imaging optical system 13, the emission filter 27, and the imaging. The lens 29A, the microlens array 47, and the camera 31 function as a light field microscope. Further, the laser light source 15 of the illumination optical system 9, the optical fiber 17, the convex lens 19B, the cylindrical lens 21B and the prism 23B, the immersion objective lens 11, the mirror 25 of the imaging optical system 13, the emission filter 27, the imaging lens 29B and the like. With the camera 31, it functions as a light sheet microscope.

The operation of the microscope 41 configured in this way will be described.
When observing the sample S with the microscope 41 according to the present embodiment, the functions as a light field microscope and the function as a light sheet microscope are switched by the shutters 45A and 45B.

When observing the sample S as a light field microscope, the shutter 45A is detached from the optical path of the laser beam emitted from the tip 18A of the optical fiber 17, and the shutter is placed on the optical path of the laser beam emitted from the tip 18B. Insert 45B. Further, the imaging lens 29A is inserted on the photographing optical axis of the camera 31 by the imaging lens turret 49.

In this state, the laser light emitted from the tip 18A of the optical fiber 17 passes through the convex lens 19A and the cylindrical lens 21A having positive power, is reflected by the reflecting surface 24A of the prism 23A, and then applies negative power. The cylindrical lens 43 is converted into a parallel light beam having a thickness corresponding to the observation depth in the direction along the detection optical axis P of the immersion objective lens 11 and incident on the sample S. By aligning the focal plane of the immersion objective lens 11 with the incident range of the laser light, the fluorescence generated in a wide range along the focal plane can be focused at once by the immersion objective lens 11.

The fluorescence from the sample S focused by the immersion objective lens 11 is imaged on the microlens array 47 by the imaging lens 29A via the mirror 25 and the emission filter 27, and is imaged by the camera 31 by each microlens 48. Projected on the surface. As a result, it is possible to acquire a plurality of image information having different parallax at a time and construct three-dimensional image data from one image.

On the other hand, when observing the sample S as a light sheet microscope, the shutter 45A is inserted into the optical path of the laser beam emitted from the tip 18A of the optical fiber 17, and the shutter 45A is inserted from the optical path of the laser beam emitted from the tip 18B. The shutter 45B is detached. Further, the imaging lens 29B is inserted on the photographing optical axis of the camera 31 by the imaging lens turret 49. Hereinafter, the case of observing as a light sheet microscope is the same as that of the first embodiment, and thus the description thereof will be omitted.

As described above, according to the microscope 41 according to the present embodiment, different observation methods can be selected with one inverted microscope. In this case, even if the observation position of the sample S is changed in the chamber 5 by moving the movable stage 7, the aiming portion 12 finely adjusts the position of the immersion objective lens 11 in the direction along the detection optical axis P. As a result, the deviation of the focal position can be eliminated, and the desired observation position in the sample S can be observed with high accuracy.

In this embodiment, it can be modified as follows.
As a first modification, a variable diaphragm may be provided on the optical path of the laser beam emitted from the tip portion 18A of the optical fiber 17. By changing the thickness of the illumination luminous flux of the laser beam with the variable diaphragm, it is possible to avoid irradiating the depth obtained by the function as a light field microscope with unnecessary laser beam.

As a second modification, in the function as a light field microscope and the function as a light sheet microscope, instead of moving the sample S in the Z direction by the movable stage 7, a scanner is adopted to swing the laser beam in the Z direction. You may do so. In this case, since the sample S is not moved in the Z direction, it is not necessary to give the sample S a stimulus due to the movement when observing the ecology. In particular, in the case of imaging changes in calcium in a living body, it is possible to perform more accurate measurement without stimulating the sample S.

[Third Embodiment]
Next, the microscope according to the third embodiment of the present invention will be described.
As shown in FIG. 5, the microscope 51 according to the present embodiment is different from the first embodiment in that the illumination optical system 9 transmits the side wall portion of the chamber 5 and causes the laser beam to enter the sample S.
Hereinafter, the parts having the same configuration as the microscope 1 according to the first embodiment are designated by the same reference numerals and the description thereof will be omitted.

The microscope 51 according to the present embodiment includes an inverted microscope component 53, a light sheet illumination module 55, a laser light source 15, and an optical fiber 17.
The inverted microscope component 53 includes an immersion objective lens 11, a mirror 25, an emission filter 27, an imaging lens 29, and a camera 31.

The light sheet illumination module 55 includes a cuvette 3, a chamber 5, a movable stage 7, an aiming portion 12, a convex lens 19A, and a cylindrical lens 21A.
Both the cuvette 3 and the chamber 5 have transparent portions 3b and 5b on the detection optical axis P on the bottom surface and on the illumination optical axis Q on the side wall. There is an advantage that it can be additionally configured (add-on) simply by mounting the light sheet illumination module 55 on the inverted microscope component 53, which is an ordinary inverted microscope.

The injection end of the optical fiber 17 is detachably connected to the light sheet lighting module 55 by a fiber connector 57 such as an FPC (Flexible Printed Circuit).

When observing the sample S with the microscope 51 configured in this way, the laser light emitted from the laser light source 15 is guided by the optical fiber 17 and incident on the light sheet illumination module 55 via the fiber connector 57. .. The laser light incident on the light sheet illumination module 55 is converted into a parallel light flux by the convex lens 19A, passes through the transparent portion 5b of the side wall portion of the chamber 5, and is focused by the cylindrical lens 21A. The laser beam focused by the cylindrical lens 21A passes through the transparent portion 3b of the side wall portion of the cuvette 3 and is incident on the sample S.

Even when observing the sample S with the microscope 51 according to the present embodiment, the immersion objective lens 11 is detected by the aiming unit 12 even if the observation position of the sample S is changed in the chamber 5 by moving the movable stage 7. By finely adjusting the position in the direction along the optical axis P, the deviation of the focal position can be eliminated. Therefore, the desired observation position in the sample S can be observed with high accuracy.

As described above, according to the microscope 51 according to the present embodiment, the illumination optical system 9 can be arranged on the side of the chamber 5. Further, the chamber 5, the movable stage 7, the aiming unit 12, and the illumination optical system 9 are mounted on the inverted microscope component 53, which is an ordinary inverted microscope provided with the immersion objective lens 11, the aiming unit 12, and the camera 31. It can be configured only by adding the light sheet lighting module 55 provided.
In the present embodiment, the light sheet microscope is used, but a lighting module for a light field microscope may be used.

[Fourth Embodiment]
Next, the microscope according to the fourth embodiment of the present invention will be described.
In the microscope 61 according to the present embodiment, as shown in FIGS. 6 and 7, the cuvette 3 and the chamber 5 have an annular shape, and the movable stage 7 rotates the cuvette 3 around the axis parallel to the detection optical axis P. It differs from the first embodiment in that it is rotatably supported.
Hereinafter, the parts having the same configuration as the microscope 1 according to the first embodiment are designated by the same reference numerals and the description thereof will be omitted.

The cuvette 3 is, for example, as shown in FIG. 8, a microplate in which a plurality of accommodating portions 3a accommodating the sample S are arranged in the circumferential direction. The cuvette 3 has a transparent portion 3b for each accommodating portion 3a on the side wall portion and the bottom portion. In the example shown in FIG. 8, the cuvette 3 has a side wall portion and a bottom portion formed by a transparent portion 3b over the entire circumferential direction.

The chamber 5 has an opening 5a having an inner diameter smaller than the inner diameter of the cuvette 3 and an outer diameter larger than the outer diameter of the cuvette 3. As shown in FIGS. 6 and 9, the chamber 5 has a transparent portion 5b on the illumination optical axis Q of the illumination optical system 9 on the side wall portion and on the detection optical axis P of the immersion objective lens 11 at the bottom portion, respectively. ing. The chamber 5 may also have a side wall portion and a bottom portion formed by a transparent portion 5b over the entire circumferential direction.
The illumination optical system 9 is adapted to inject the laser beam through the transparent portion 5b of the side wall portion of the chamber 5.

The operation of the microscope 61 configured in this way will be described.
When observing the sample S with the microscope 61 according to the present embodiment, the cuvette 3 is rotated around the axis parallel to the detection optical axis P by the movable stage 7, and one of the accommodating portions 3a is arranged on the detection optical axis P. ..

The laser light emitted from the laser light source 15 in this state is guided by the optical fiber 17 to be made into a parallel light beam by the convex lens 19A, and then is condensed in a plane by the cylindrical lens 21A to make the side wall of the chamber 5 transparent. It penetrates the part 5b. The laser beam incident on the chamber 5 passes through the transparent portion 3b of the side wall portion of the cuvette 3 and is incident on the sample S. As a result, it is possible to acquire a fluorescence image of the sample S of the accommodating portion 3a arranged on the detection optical axis P.

Subsequently, the movable stage 7 rotates the cuvette 3 around the axis parallel to the detection optical axis P, and the adjacent next accommodating portion 3a is arranged on the detection optical axis P. Then, a laser beam is incident on the sample S of the next accommodating portion 3a arranged on the detection optical axis P in the same manner to acquire a fluorescence image. In this way, the accommodating portion 3a of the cuvette 3 arranged on the detection optical axis P is switched, and the fluorescence images of the sample S accommodated in each accommodating portion 3a are sequentially acquired.

As described above, according to the microscope 61 according to the present embodiment, the sample S arranged on the detection optical axis P is simply moved around the axis parallel to the detection optical axis P by the movable stage 7. You can switch. Therefore, the laser beam is sequentially incident on the sample S of each accommodating portion 3a by the illumination optical system 9, and the fluorescence from the sample S of each accommodating portion 3a is sequentially condensed by the immersion objective lens 11 and continuously by the camera 31. Can be taken. As a result, a large number of images of the sample S can be acquired efficiently and at high speed.

In the present embodiment, the microplate has been illustrated as the cuvette 3, but instead, for example, a plurality of cuvettes 3 may be arranged in an array in the circumferential direction. In this case, the movable stage 7 may support each cuvette 3 so as to be movable around an axis parallel to the detection optical axis P, and switch the cuvette 3 arranged on the detection optical axis P.
Further, in the present embodiment, the light sheet microscope has been described as an example, but the light field microscope may be applied.

[Fifth Embodiment]
Next, the microscope according to the fifth embodiment of the present invention will be described.
In the microscope 71 according to the present embodiment, as shown in FIGS. 10 and 11, the illumination optical system 9 transmits the side wall portion of the chamber 5 through the side wall portion of the chamber 5 and causes the laser beam to enter the sample S to be immersed in the sample S. The objective lens 11 collects the fluorescence transmitted from the sample S through the side wall portion of the chamber 5, and the movable stage 7 can move the cuvette 3 in the chamber 5 in the X, Y, and Z directions, and illuminates the cuvette 3. It differs from the first to fourth embodiments in that it rotatably supports around a predetermined rotation axis orthogonal to the optical axis Q and the detection optical axis P.
Hereinafter, the parts having the same configuration as the microscopes 1, 41, 51, 61 according to the first to fourth embodiments are designated by the same reference numerals and the description thereof will be omitted.

The microscope 71 includes a cuvette 3, a chamber 5, a movable stage 7, an illumination optical system 9, a plurality of immersion objective lenses 11 having different magnifications, and a revolver 73 that supports the plurality of immersion objective lenses 11. The aiming unit 12 that moves the immersion objective lens 11 supported by the revolver 73 in the direction along the detection optical axis P, the imaging optical system 13, the water replenisher 75 that replenishes the immersion solution W3, and the movable stage 7 It is provided with a control device 77 for controlling the above. In FIG. 11, reference numeral 79 indicates a drain tank.

In the present embodiment, the cuvette 3 is one storage container filled with the cuvette solution W1, and the sample S is immersed in the cuvette solution W1. Further, as shown in FIG. 10, the cuvette 3 has transparent portions (excitation light transmitting portion and light transmitting portion) 3b on all side wall portions in the circumferential direction.
The chamber 5 has a transparent portion (excitation light transmitting portion, light transmitting portion) 5b on two side wall portions adjacent to each other.

The illumination optical system 9 includes a laser light source 15, an optical fiber 17, a convex lens 19 that converts the laser light guided by the optical fiber 17 into a parallel light beam, and a cylindrical lens 21 having the same configuration as the cylindrical lenses 21A and 21B. It is equipped with a variable aperture 81.

The variable diaphragm 81 is arranged between the cylindrical lens 21 and the transparent portion 5b of the side wall portion of the chamber 5. By changing the luminous flux diameter of the laser beam with the variable diaphragm 81, the thickness of the laser beam focused in a plane by the cylindrical lens 21 can be changed. This change is made according to the immersion objective lens 11 inserted in the optical path.

The tip portion 18, the convex lens 19, the cylindrical lens 21, and the variable aperture 81 of the optical fiber 17 are arranged so as to face the transparent portion 5b of one side wall portion of the chamber 5, and emit the laser light emitted from the laser light source 15. It is adapted to enter the sample S through the transparent portion 5b of one side wall portion of the chamber 5 and the transparent portion 3b of any side wall portion of the cuvette 3.

As shown in FIG. 10, the immersion objective lens 11 is arranged outside the chamber 5 so as to face the transparent portion 5b of the other side wall portion with its detection optical axis P orthogonal to the illumination optical axis Q. .. An immersion solution W3 such as pure water is injected into the gap between the upper surface 11a of the state-of-the-art lens of the immersion objective lens 11 and the transparent portion 5b on the other side wall of the chamber 5, and the immersion solution W3 has a surface tension. It is designed to be held in the gap.

The revolver 73 can be selectively arranged on the fluorescent optical path for detecting the plurality of immersion objective lenses 11. Thereby, for example, the immersion objective lens 11 to be used can be switched according to the purpose of observation.

The water replenisher 75 has a nozzle 75a at the tip thereof, and when switching the immersion objective lens 11, the upper surface 11a of the most advanced lens of the immersion objective lens 11 and the transparent portion 5b of the side wall portion of the chamber 5 The immersion solution W3 can be replenished from the nozzle 75a in the gap between the lens and the lens.

The imaging optical system 13 includes a mirror 25, an imaging lens 29 for forming an image of fluorescence reflected by the mirror 25, and a camera 31.

The control device 77 controls the movement of the cuvette 3 in the X, Y, Z directions and the rotation around a predetermined rotation axis by the movable stage 7. Further, the control device 77 controls the laser light source 15 and the camera 31, adjusts the light beam diameter of the laser light by the variable diaphragm 81, switches the immersion objective lens 11 by the revolver 73, and the immersion objective lens 11 by the aiming unit 12. The position of the immersion solution W3 is finely adjusted along the detection optical axis P, and the replenishment of the immersion solution W3 by the water replenisher 75 is controlled.

The operation of the microscope 71 configured in this way will be described.
In order to observe the sample S with the microscope 71 according to the present embodiment, first, the control device 77 supports the cuvette 3 containing the sample S and the cuvette solution W1 by the movable stage 7, and the chamber solution W2 in the chamber 5 is supported. Is immersed in, and laser light is generated from the laser light source 15.

The laser light emitted from the laser light source 15 is guided by the optical fiber 17 to be made into a parallel light beam by the convex lens 19, then is condensed in a plane by the cylindrical lens 21 and passes through the variable aperture 81, and is passed through the variable aperture 81 of the chamber 5. It passes through the transparent portion 5b of the side wall portion and is incident into the chamber 5.

The laser beam incident on the chamber 5 is incident on the sample S from a direction orthogonal to the detection optical axis P via the chamber solution W2, the transparent portion 3b of the side wall portion of the cuvette 3, and the cuvette solution W1. When a planar laser beam is incident on the sample S, the fluorescent substance in the sample S is excited along the incident plane of the laser beam to generate fluorescence.

Among the fluorescence generated in the sample S, the fluorescence radiated in the direction along the detection optical axis P is the transparent portion 3b of the side wall portion of the cuvette solution W1, the cuvette 3, the chamber solution W2, and the transparent portion 5b of the side wall portion of the chamber 5. And the light is collected by the immersion objective lens 11 via the immersion solution W3.

The fluorescence collected by the immersion objective lens 11 is reflected by the mirror 25 and imaged on the imaging surface of the camera 31 by the imaging lens 29. As a result, in the camera 31, a tomographic image orthogonal to the detection optical axis P of the sample S can be obtained. By driving the movable stage 7 by the control device 77 and moving the cuvette 3 in the X, Y, and Z directions in the chamber 5, the observation position of the sample S is changed and a tomographic image of each observation position is acquired. can do.

By matching the focal position of the cylindrical lens 21 with the detection optical axis P of the immersion objective lens 11 and aligning the focal plane of the immersion objective lens 11 with the incident plane of the laser beam, the focus of the immersion objective lens 11 The fluorescence generated in a wide range along the surface is focused at once by the immersion objective lens 11 and photographed by the camera 31, and a clear fluorescent image of the observation region in the sample S can be obtained. Further, since the laser beam is not emitted to the plane other than the imaging plane of the camera 31, fading of fluorescence can be suppressed and a good three-dimensional stereoscopic image can be obtained.

In this case, according to the microscope 71 according to the present embodiment, the control device 77 drives the movable stage 7 to rotate the cuvette 3 around a predetermined rotation axis orthogonal to the illumination optical axis Q and the detection optical axis P. By reversing the orientation of the sample S with respect to the immersion objective lens 11 and bringing the portion of the sample S far from the immersion objective lens 11 closer to the immersion objective lens 11, a good image can be obtained over substantially the entire area of the sample S. Can be obtained.

Further, even if the cubet 3 is moved in the chamber 5 by the movable stage 7 to change the observation position of the sample S, the amount of the immersion solution W3 arranged in the gap between the immersion objective lens 11 and the chamber 5 is (). Since it does not change (it is maintained as it is by surface tension even if the focus is finely adjusted), it is not necessary to prepare a large amount of immersion solution W3 or replenish it frequently, and the liquid of immersion solution W3. It can also prevent cutting.

Further, when the observation position of the sample S is changed, the focal position of the immersion objective lens 11 may shift depending on the refractive index distribution in the sample S, or the refractive index of the cubicle solution W1 and the refractive index of the chamber solution W2. Even if the focal position of the immersion objective lens 11 is deviated due to a slight difference from the above, the focus is simply adjusted by the aiming unit 12 in the direction along the detection optical axis P of the immersion objective lens 11. The displacement of the position can be eliminated.

In the present embodiment, the light sheet microscope has been described as an example, but it may be applied to a light field microscope. In this case, as in the second embodiment, the illumination optical system 9 further includes a cylindrical lens 43 having a negative power (see FIG. 4), and a cylindrical lens 43 is inserted between the cylindrical lens 21 and the chamber 5. It may be arranged so that a laser beam having a thickness in the direction along the photographing optical axis of the camera 31 is incident on the sample S. Further, the imaging optical system 13 has a microlens array 47 composed of a plurality of microlenses 48 for projecting an image on the imaging surface of the camera 31, and an imaging lens 29A for forming an image on the microlens array 47 (see also FIG. 4). ) And should be provided. By doing so, it is possible to acquire a plurality of image information having different parallax at one time.

[Sixth Embodiment]
Next, the microscope according to the sixth embodiment of the present invention will be described.
As shown in FIG. 12, the microscope 91 according to the present embodiment is different from the first to fifth embodiments in that it constitutes a light emitting microscope.
Hereinafter, the parts having the same configuration as the microscopes 1, 41, 51, 61, 71 according to the first to fifth embodiments are designated by the same reference numerals and the description thereof will be omitted.

The microscope 91 is a direction in which the cuvette 3, the chamber 5, the movable stage 7, the dry objective lens (objective lens) 93 that collects the fluorescence emitted from the sample S, and the dry objective lens 93 are along the detection optical axis P. It is provided with an aiming unit 12 that can be moved to the surface, an imaging optical system 13 that acquires an image of a sample S based on fluorescence focused by a dry objective lens 93, and a control device 77 that controls a movable stage 7 and the like. ..

In the present embodiment, the cuvette 3 has a transparent portion (light transmitting portion) 3b capable of transmitting fluorescence at the bottom.
The chamber 5 has a transparent portion (light transmitting portion) 5b capable of transmitting fluorescence at the bottom thereof.

The dry objective lens 93 is provided outside the chamber 5 in the vicinity of the transparent portion 5b at the bottom, and is arranged vertically upward facing the transparent portion 5b. Further, the dry objective lens 93 is arranged at a distance from the transparent portion 5b at the bottom of the chamber 5 without interposing the immersion solution W3.
The imaging optical system 13 includes an imaging lens 29 for forming an image of fluorescence focused by the dry objective lens 93, and a camera 31.

The operation of the microscope 91 configured in this way will be described.
In order to observe the sample S with the microscope 91 according to the present embodiment, first, the movable stage 7 supports the cuvette 3 containing the sample S and the cuvette solution W1 and is immersed in the chamber solution W2 in the chamber 5. Move to the desired observation position.

Among the fluorescence emitted by the sample S, the fluorescence radiated in the direction along the detection optical axis P includes the transparent portion 3b at the bottom of the cuvette solution W1, the cuvette 3, the chamber solution W2, and the transparent portion 5b at the bottom of the chamber 5. It is focused by the dry objective lens 93 and imaged on the imaging surface of the camera 31 by the imaging lens 29. As a result, in the camera 31, a tomographic image orthogonal to the detection optical axis P of the sample S can be obtained. By driving the movable stage 7 by the control device 77 and moving the cuvette 3 in the X, Y, and Z directions in the chamber 5, the observation position of the sample S is changed and a tomographic image of each observation position is acquired. can do.

In this case, according to the microscope 91 according to the present embodiment, when the cuvette 3 is moved in the chamber 5 to change the observation position of the sample S, the dry objective lens 93 is changed according to the refractive index distribution in the sample S. Even if the focal position of the dry objective lens 93 shifts due to a slight difference between the refractive index of the cuvette solution W1 and the refractive index of the chamber solution W2, the aiming unit 12 causes the dry objective to shift. The deviation of the focal position can be eliminated only by finely adjusting the lens 93 in the direction along the detection optical axis P.

Therefore, even if there is a slight difference in the refractive index distribution in the sample S, the refractive index of the cuvette solution W1 and the refractive index of the chamber solution W2, the movable stage 7 is driven to image in the Z direction at equal intervals. When acquiring a stack image, it is possible to obtain images at equal intervals by finely adjusting the focus, and it is possible to construct a distortion-free three-dimensional image. In addition, the configuration can be as simple and inexpensive as the light source and the illumination optical system are not required.

Although the embodiments of the present invention have been described in detail with reference to the drawings, the specific configuration is not limited to this embodiment, and design changes and the like within a range not deviating from the gist of the present invention are also included. For example, the present invention is not limited to the one applied to each of the above embodiments and modifications, and may be applied to an embodiment in which these embodiments and modifications are appropriately combined, and is not particularly limited. .. For example, in the above embodiment, the example in which the laser light is incident on the sample S from one direction or two directions is shown, but for example, the laser light may be incident on the sample S from three or more directions.

Further, in each of the above embodiments, the cuvette solution W1, the chamber solution W2, the immersion solution W3, the transparent portion 3b of the cuvette 3 and the transparent portion 5b of the chamber 5 all have substantially the same refractive index. If the thickness of the transparent portion 3b of 3 and the thickness of the transparent portion 5b of the chamber 5 are constant, the transparent portion 3b of the cuvette 3 through which excitation light and fluorescence are transmitted even if the cuvette 3 is moved in the chamber 5. Since the refractive index of the transparent portion 5b of the chamber 5 does not change, at least the cuvette solution W1 and the chamber solution W2 may have substantially the same refractive index as each other.

1,41,51,61,71,91 Microscope 3 Cuvette (specimen container)
3a containment unit (specimen storage unit)
3b, 5b transparent part (excited light transmitting part, light transmitting part)
5 chamber (medium container)
5a Opening 7 Movable stage 9 Illumination optical system 11 Immersion objective lens (objective lens)
12 Aiming unit 31 Camera (imaging unit)
21 Cylindrical lens (lens)
21A, 21B Cylindrical lens (lens)
24A, 24B Reflective surface (reflection mirror)
47 Microlens Array 93 Dry Objective Lens (Objective Lens)
P Detection optical axis Q Illumination optical axis S Sample (sample)
W1 cuvette solution (first immersion medium)
W2 chamber solution (second immersion medium)
W3 immersion solution (third immersion medium)

Claims (20)

A medium container for storing a second immersion medium having a refractive index equivalent to that of the first immersion medium, in which a sample container for accommodating the first immersion medium is immersed together with the sample.
An objective lens that is arranged outside the medium container and collects the light emitted from the sample.
An imaging unit that captures the light focused by the objective lens, and
An aiming unit that moves the objective lens in the direction along the detection optical axis,
A movable stage that movably supports the specimen container in the medium container at least in a direction along the detection optical axis is provided.
The sample container and the medium container each have a light transmitting portion capable of transmitting the light from the sample.
A microscope in which the objective lens is arranged so as to face the light transmitting portion of the specimen container via the light transmitting portion of the medium container. The microscope according to claim 1, wherein the objective lens is arranged at a distance from the light transmitting portion of the medium container. The first or second aspect of the present invention, wherein the objective lens is an immersion objective lens, and the immersion objective lens is arranged between the immersion objective lens and the light transmitting portion of the medium container via a third immersion medium. microscope. The immersion objective lens is arranged so that the detection optical axis is directed in a direction intersecting the vertical direction.
The microscope according to claim 3, wherein the third immersion medium is held by surface tension between the immersion objective lens and the light transmitting portion of the medium container. The specimen container and the medium container have the light transmitting portion on the side wall portion.
The microscope according to claim 4, wherein the immersion objective lens is arranged with the detection optical axis directed in a direction substantially orthogonal to the vertical direction. The microscope according to any one of claims 3 to 5, wherein the third immersion medium has a refractive index equivalent to that of the second immersion medium. The microscope according to any one of claims 1 to 6, wherein the light transmitting portion of the sample container has a refractive index equivalent to that of the second immersion medium. The microscope according to any one of claims 1 to 7, wherein the light transmitting portion of the medium container has a refractive index equivalent to that of the second immersion medium. The microscope according to any one of claims 1 to 8, wherein the movable stage movably supports the specimen container in a direction intersecting the detection optical axis. The microscope according to any one of claims 1 to 9, wherein the imaging unit captures the light emitted by the specimen by itself. An illumination optical system for irradiating the specimen with excitation light from a direction intersecting the detection optical axis is provided.
The microscope according to any one of claims 1 to 9, wherein the sample container and the medium container each have an excitation light transmitting portion that transmits the excitation light from the illumination optical system toward the sample. The medium container has the excitation light transmitting portion at the bottom.
The illumination optical system includes a reflection mirror arranged in the medium container, and the excitation light is incident on the medium container from the excitation light transmitting portion at the bottom and reflected by the reflection mirror toward the sample. The microscope according to claim 11. The medium container has the excitation light transmitting portion on the side wall portion.
The microscope according to claim 11, wherein the illumination optical system causes the excitation light to enter the medium container from the excitation light transmitting portion of the side wall portion. The microscope according to any one of claims 11 to 13, wherein the illumination optical system includes a lens having a positive power arranged in the medium container. The microscope according to claim 14, wherein the lens having a positive power is a cylindrical lens having a positive power in one direction intersecting the illumination optical axis of the illumination optical system. A microlens array formed by two-dimensionally arranging a plurality of microlenses in a direction intersecting the photographing optical axis of the imaging unit is provided.
The microscope according to any one of claims 11 to 13, wherein the illumination optical system causes the excitation light having a substantially parallel luminous flux to enter the specimen. The microscope according to any one of claims 11 to 16, wherein the movable stage movably supports a plurality of the specimen containers in the medium container around an axis parallel to the detection optical axis. The specimen container has a plurality of specimen accommodating portions arranged in one direction intersecting the illumination optical axis of the illumination optical system.
The microscope according to any one of claims 11 to 16, wherein the movable stage switchably supports the sample accommodating portion arranged on the detection optical axis. The microscope according to any one of claims 11 to 18, wherein the movable stage rotatably supports the specimen container in the medium container around the detection optical axis. The microscope according to claim 11, wherein the illumination optical system causes the planar excitation light to enter the specimen along a plane intersecting the detection optical axis of the objective lens.

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