JP2018032009A - microscope - Google Patents

Abstract

An object of the present invention is to reduce the amount of an immersion medium and the replenishment frequency of the immersion medium, prevent the liquid from running out of the immersion medium, and realize highly reliable observation with a simple and inexpensive configuration. A chamber (5) for storing a solution (W2) having a refractive index equivalent to that of the solution (W1) in which a cuvette (3) containing a solution (W1) is immersed together with a sample (S); An immersion objective lens 11 for condensing light, a camera 31 for photographing the light condensed by the immersion objective lens 11, and an aiming unit 12 for moving the immersion objective lens 11 in a direction along its detection optical axis P. and a movable stage 7 that supports the cuvette 3 in the chamber 5 so as to be movable in at least the direction along the detection optical axis P. The cuvette 3 and the chamber 5 are provided with transparent portions 3b and A microscope 1 is provided which has a transparent portion 5b and an immersion objective 11 is arranged opposite the transparent portion 3b of the cuvette 3 via the transparent portion 5b of the chamber 5. [Selection drawing] Fig. 1

Description

  The present invention relates to a microscope.

  Conventionally, there has been known a microscope in which excitation light is incident on a specimen from a direction intersecting the detection optical axis of the detection optical system and a three-dimensional stereoscopic image of the specimen is acquired based on fluorescence from the specimen detected by the detection optical system. (For example, see Patent Document 1 and Patent Document 2). In the microscopes described in Patent Document 1 and Patent Document 2, since excitation light is not irradiated other than on the imaging plane, it is possible to obtain a favorable three-dimensional stereoscopic image while suppressing fading of fluorescence.

  In recent years, this technology has been used not only for the purpose of obtaining stereoscopic images of organisms such as zebrafish with fluorescent proteins labeled as target molecules, but also for spheroids and organoids (artificial organs or parts thereof). It is also attracting attention for the purpose of application to so-called drug discovery screening, in which three-dimensional stereoscopic images of three-dimensional cultured cells are obtained and drug efficacy is evaluated by using image analysis technology, and applied to a wide range of applications. It is expected that. In addition, this observation technique requires a finer and higher-resolution observation due to a strong need from researchers who want to observe at a resolution that can recognize individual cells.

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

  On the other hand, in the microscope described in Patent Document 2, a sample is stored in a cuvette filled with an immersion medium such as a clearing solution, and the cuvette is further stored in a chamber filled with an immersion medium. It is placed on the XYZ stage. Further, the objective lens for observation is immersed in an immersion medium in the chamber through a sealing member whose tip is prevented from leaking. According to the configuration of the microscope disclosed in Patent Document 2, the immersion medium does not decrease even if the XYZ stage is moved. Therefore, the above-described problem of the microscope described in Patent Document 1 can be solved.

Japanese Patent No. 444432 International Publication No. 2015/184124

  However, in the microscope described in Patent Document 2 in which the objective lens cannot be moved, when the focus position of the objective lens shifts according to the refractive index distribution in the sample with the change of the observation position, the excitation light is emitted by the scanner. Therefore, 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-described circumstances, and has a simple and inexpensive configuration, which reduces the amount of the immersion medium and its replenishment frequency, and prevents the immersion medium from running out of liquid. It aims at providing the microscope which can implement | achieve 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 a sample container containing the first immersion medium is immersed together with the specimen. An objective lens that is arranged outside the medium container and collects light emitted from the sample, an imaging unit that photographs the light collected by the objective lens, and a direction along the detection optical axis of the objective lens And a movable stage that movably supports the sample container in the medium container so as to be movable in a direction along at least the detection optical axis, and the sample container and the medium container are separated from the sample. Each of the microscopes has a light transmitting part capable of transmitting light, and the objective lens is disposed to face the light transmitting part of the sample container via the light transmitting part of the medium container.

  According to this aspect, the specimen is accommodated in the specimen container together with the first immersion medium, and the whole specimen container is immersed in the second immersion medium in the medium container. Then, light emitted from the specimen passes through the first immersion medium, the light transmission part of the specimen container, the second immersion medium having the same refractive index as the first immersion medium, and the light transmission part of the medium container. The light is collected by the objective lens and photographed by the imaging unit. Therefore, the tomographic image of the specimen that intersects the detection optical axis of the objective lens can be acquired by moving the specimen container in the medium container in the direction along the detection optical axis of the objective lens by the movable stage.

  In this case, even if the observation position is changed by moving the sample container within the medium container by the movable stage, the relative position between the objective lens and the medium container does not change. 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 transmission part of the medium container, and to replenish the immersion medium frequently, and the immersion medium does not run out.

In addition, when the observation position of the specimen is changed, even if the focus position of the objective lens is shifted according to the refractive index distribution in the specimen, the position of the objective lens in the direction along the detection optical axis is set by the aiming unit. By fine adjustment, it is possible to eliminate the shift of the focal position.
Therefore, it is possible to configure a microscope that realizes highly reliable observation by preventing the liquid from running out of the immersion medium 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 with a space between the objective lens and the light transmission part of the medium container.
With this configuration, the objective lens can be switched to one having a different magnification.

In the above aspect, the objective lens may be an immersion objective lens, and the immersion objective lens may be disposed between the light transmission part 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, it is possible to increase the numerical aperture of the immersion objective lens and obtain higher resolution. In addition, since the amount of the third immersion medium does not change due to the movement of the sample container, it is not necessary to prepare a large amount of the third immersion medium or to replenish the third immersion medium frequently, and the third immersion medium runs out of liquid. There is nothing.

In the above aspect, the immersion objective lens is disposed with the detection optical axis directed in a direction intersecting a vertical direction, and the third immersion medium is the light transmission of the immersion objective lens and the medium container. It is good also as being hold | maintained by surface tension between parts.
With this configuration, there is no need for a mechanism for holding the third immersion medium between the tip of the immersion objective lens and the light transmission portion of the medium container, and a simple configuration can be achieved.

In the above aspect, the specimen container and the medium container have the light transmission part on a side wall part, and the immersion objective lens is arranged with the detection optical axis directed in a direction substantially perpendicular to the vertical direction. It is good.
With this configuration, the immersion objective lens can be disposed on the side of the medium container in the vicinity of the light transmission 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 specimen is changed, the focal position of the immersion objective lens is displaced according to the refractive index distribution in the specimen, and the detection optical axis of the immersion objective lens is caused by the aiming unit. Even if the fine adjustment of the focus is performed in the direction along the axis, the occurrence of spherical aberration can be suppressed.

In the above aspect, the light transmission part of the sample 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 transmission part of the sample container varies due to manufacturing errors.

In the above aspect, the light transmission part 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 transmission part of the medium container varies due to manufacturing errors.

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

In the above aspect, the imaging unit may take an image of the light emitted by the specimen.
By configuring in this way, a light emitting microscope can be configured, and a simple and inexpensive configuration can be achieved as long as a light source and an illumination optical system are unnecessary.

In the above aspect, an illumination optical system that irradiates the specimen with excitation light from a direction intersecting the detection optical axis is provided, and the specimen container and the medium container provide the specimen with the excitation light from the illumination optical system. It is good also as having each the excitation light transmission part permeate | 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 through each excitation light transmitting unit. Can be irradiated.

  In the above aspect, the medium container has the excitation light transmitting portion at a bottom, the illumination optical system includes a reflection mirror disposed in the medium container, and the excitation light is transmitted to the excitation light at the bottom. It is good also as making it inject into the said medium container from a permeation | transmission part, and reflecting toward the said sample with the said reflective mirror.

  With this configuration, the illumination optical system excluding the reflection mirror can be arranged below the medium container. As a result, mechanical interference between the movable stage and the sample container and the illumination optical system can be avoided, and excitation light is applied to the sample not only from one direction but also from a plurality of directions intersecting the detection optical axis of the objective lens. An incident illumination optical system can be easily configured.

In the above aspect, the medium container has the excitation light transmission part on a side wall part, and the illumination optical system causes the excitation light to enter the medium container from the excitation light transmission part of the side wall part. It is good.
With this configuration, the illumination optical system can be arranged on the side of the medium container. Thereby, it can be configured by adding a medium container, a movable stage and an illumination optical system to a normal 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 a positive power arranged in the medium container.
In order to increase the resolution by reducing the thickness of the sheet illumination, it is necessary to set the lens emission NA large. With this configuration, the distance from the lens to the sample can be shortened compared to the case where the lens is disposed outside the medium container, and the emission NA of the lens can be set large. Thereby, the resolution can be improved with a simple configuration in which a lens having a positive power is simply disposed in the medium container.

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

  In the above aspect, a microlens array in which a plurality of microlenses are two-dimensionally arranged in a direction intersecting the imaging optical axis of the imaging unit is provided, and the illumination optical system emits the excitation light of a substantially parallel light beam. It is good also as making it enter into the said sample.

  By configuring in this way, the focal plane of the objective lens coincides with the incident range of the excitation light, so that light generated in a wide range along the focal plane can be condensed at once by the objective lens. A plurality of pieces of image information with different parallaxes can be acquired at once by capturing an image projected by the microlens array by the imaging unit.

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

In the above aspect, the sample container has a plurality of sample storage portions arranged in one direction intersecting the illumination optical axis of the illumination optical system, and the movable stage is disposed on the detection optical axis. It is good also as supporting a sample storage part so that change is possible.
With this configuration, it is possible to observe a plurality of specimens in order simply by switching the specimen storage unit disposed on the detection optical axis of the objective lens by the movable stage.

In the above aspect, the movable stage may support the sample container so as to be rotatable around the detection optical axis in the medium container.
With this configuration, the excitation light can be incident from the different directions to the same observation position in the sample only by rotating the sample container around the detection optical axis by the movable stage. Thereby, the incident depth of the excitation light from each direction on the specimen can be reduced to suppress the influence of scattering on the specimen, and a clear image can be acquired.

In the above aspect, the illumination optical system may cause the planar excitation light to be incident on the sample along a plane intersecting the detection optical axis of the objective lens.
By configuring in this way, by making the focal plane of the objective lens coincide 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 obtaining a higher resolution image can be configured.

  According to the microscope of the present invention, it is possible to realize a highly reliable observation by preventing the immersion medium from running out while reducing the amount of the immersion medium and its replenishment frequency with a simple and inexpensive configuration. There is an effect that can be done.

1 is a schematic configuration diagram showing a microscope according to a first embodiment of the present invention. It is the top view which looked at the cuvette and prism of FIG. 1 in the direction in alignment with a 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 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 the schematic block diagram which looked at the microscope which concerns on 5th Embodiment of this invention in the perpendicular direction. It is the schematic block diagram which looked at the microscope of FIG. 10 in the direction in alignment with the detection optical axis of an immersion objective lens. It is a schematic block diagram which shows the microscope which concerns on 6th Embodiment of this invention.

[First Embodiment]
A microscope according to a 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 this embodiment includes a cuvette (specimen container) 3 that accommodates a sample (specimen) S, a chamber (medium container) 5 that can accommodate the cuvette 3, and a cuvette 3. A movable stage 7 to be supported, an illumination optical system 9 for irradiating the sample S with laser light (excitation light), an immersion objective lens (objective lens) 11 for collecting the fluorescence emitted from the sample S, and an immersion objective lens 11 is provided with an aiming unit 12 that can move 11 in a direction along the detection optical axis P, and an imaging optical system 13 that acquires an image of the sample S based on the fluorescence condensed by the immersion objective lens 11.

  As shown in FIG. 2, the cuvette 3 has an array structure in which three accommodating portions (specimen accommodating portions) 3 a that accommodate the sample S are arranged in one direction. Each container 3a is filled with a cuvette solution (first immersion medium) W1 such as a clearing solution, and a 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, light transmitting portion) 3b that can transmit 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 part (excitation light transmitting part, light transmitting part) 5b that can transmit laser light and fluorescence at the bottom. The transparent part 5b is formed in a wide range of the bottom part including the illumination optical axis Q of the illumination optical system 9 and the detection optical axis P of the immersion objective lens 11. The chamber 5 stores a chamber solution (second immersion medium) W2 having a refractive index substantially equal to that of the cuvette solution W1, and the cuvette 3 is immersed in the chamber solution W2.

  The movable stage 7 includes an arm portion 7a that holds the cuvette 3, and a support portion 7b that supports 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 positioned on the illumination optical axis Q. Thus, the cuvette 3 is supported.

  The movable stage 7 moves the cuvette 3 held in a vertical direction (hereinafter referred to as Z direction) and a two-dimensional direction (hereinafter referred to as X and Y directions) intersecting the vertical direction and orthogonal to each other. It can be made to. Thereby, the movable stage 7 can change the accommodation part 3a of the cuvette 3 arrange | positioned on the detection optical axis P, or can change the observation position of the sample S within the same accommodation part 3a. Yes.

  The immersion objective lens 11 is configured by combining a 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 disposed vertically upward to face the transparent portion 5b. An immersion solution (third immersion medium) W3 such as pure water is injected into the gap between the top surface 11a of the most advanced lens of the immersion objective lens 11 and the transparent portion 5b at the bottom of the chamber 5, and the immersion solution W3 is held in the gap by surface tension.

  The aiming unit 12 moves the immersion objective lens 11 within a range where the surface tension of the immersion solution W3 works in the gap between the top surface 11a of the most advanced 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 a laser light source 15 that generates laser light, an optical fiber 17 that guides the laser light emitted from the laser light source 15, and a laser light guided by the optical fiber 17 into a parallel light beam. Convex lenses 19A and 19B, cylindrical lenses (lenses) 21A and 21B for condensing the laser beams converted into parallel beams by the convex lenses 19A and 19B, and laser beams collected by the cylindrical lenses 21A and 21B toward the sample S And prism coatings 23A, 23B having mirror-coated reflective surfaces (reflecting mirrors) 24A, 24B.

  The optical fiber 17 has two tip portions 18A and 18B branched from a midway position in the longitudinal direction. The 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 disposed vertically upward to face the transparent portion 5b at the bottom of the chamber 5. Yes.

  The laser light emitted from one end portion 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 end portion 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 19 </ b> A and 19 </ b> B and the cylindrical lenses 21 </ b> A and 21 </ b> B are arranged outside the chamber 5 with a space in the direction intersecting the detection optical axis P, and the prisms 23 </ b> A and 23 </ b> B are detected light inside the chamber 5. It arrange | positions at intervals in the direction which cross | intersects this on the axis | shaft P, and is being fixed to the inner bottom part.

  The cylindrical lenses 21A and 21B have power in one direction orthogonal to the illumination optical axis Q. The cylindrical lenses 21 </ b> A and 21 </ b> B collect laser light having a substantially parallel light flux in a planar shape having a predetermined width dimension that is the same as the light beam diameter dimension, and focus on the substantially detection optical axis P of the immersion objective lens 11. Can be tied.

  The prisms 23A and 23B reflect the laser beams condensed in a planar shape by the cylindrical lenses 21A and 21B by the reflecting surfaces 24A and 24B, and cuvette 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 3 b on the side wall portion 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 adjusted in advance so that the focal positions of the respective laser beams coincide with each other.

  The imaging optical system 13 includes a mirror 25 that reflects fluorescence collected 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. An imaging lens 29 for imaging the image formed by the imaging lens 29, 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 equal to each other. The refractive index is

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

  Subsequently, 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 emitted from the two tip portions 18A and 18B. The laser light is converted into a parallel light beam by the convex lenses 19A and 19B, collected in a planar shape by the cylindrical lenses 21A and 21B, and incident on the transparent portion 5b at the bottom of the chamber 5.

  Laser light that has entered the chamber 5 from the bottom transparent portion 5b is incident on the prisms 23A and 23B and reflected by the reflecting surfaces 24A and 24B. The laser light 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.

  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, so that the laser light irradiated by the illumination optical system 9 is incident on the sample S without being refracted. Can be made.

  When the planar laser light is incident on the sample S, the fluorescent substance in the sample S is excited along the incident plane of the laser light to generate fluorescence. Among the fluorescence generated in the sample S, the fluorescence emitted in the direction along the detection optical axis P includes 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. It is condensed by the immersion objective lens 11 through the immersion solution W3.

  Also in this case, 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 immersion solution W3 have substantially the same refractive index, so that the fluorescence from the sample S Can be condensed by the immersion objective lens 11 without being refracted.

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

  By making a laser beam having a planar shape extending along an incident plane extending in a direction perpendicular to the detection optical axis P of the immersion objective lens 11 incident on the sample S, the focal positions of the cylindrical lenses 21A and 21B and the detection of the immersion objective lens 11 are detected. The optical axis P and the focal plane of the immersion objective lens 11 are made coincident with the incident plane of the laser light, so that the fluorescence generated in a wide range along the focal plane of the immersion objective lens 11 is generated. 11 can be condensed at a time and photographed by the camera 31. Thereby, a clear fluorescent image of the observation region in the sample S can be acquired. In addition, since laser light is not irradiated except for the imaging plane of the camera 31, it is possible to obtain a good three-dimensional stereoscopic image while suppressing the fading of fluorescence.

  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 disposed in the gap between the immersion objective lens 11 and the chamber 5 Therefore, it is not necessary to prepare a large amount of the immersion solution W3 or replenish it with a high frequency, 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 focus position of the immersion objective lens 11 is shifted according to the refractive index distribution in the sample S, the aiming unit 12 detects the immersion objective lens 11. By finely adjusting the position in the direction along the optical axis P, it is possible to eliminate the shift of the focal position. Thereby, the desired observation position in the sample S can be observed with high accuracy.

  Note that the immersion solution W3 has a refractive index substantially equal to that of the chamber solution W2, so that when the focus is finely adjusted in the direction along the detection optical axis P of the immersion objective lens 11 by the aiming unit 12, the spherical surface is obtained. Occurrence of aberration can be suppressed. Further, since the transparent part 3b of the cuvette 3 and the transparent part 5b of the chamber 5 have a refractive index substantially equal to that of the chamber solution W2, the thickness of the transparent part 3b of the cuvette 3 and the thickness of the transparent part 5b of the chamber 5 are caused by manufacturing errors. Even when there is a variation in thickness, the occurrence of spherical aberration can be suppressed.

  As described above, according to the microscope 1 according to this embodiment, even when the focus position of the immersion objective lens 11 is shifted when the observation position of the sample S is changed, the immersion objective lens 11 is caused by the aiming unit 12. The focus position shift can be eliminated simply by fine-tuning in the direction along the detection optical axis P. Therefore, unlike the conventional microscope, it is not necessary to use a complicated and expensive adjustment mechanism for correcting the shift of the focal position of the immersion objective lens by the scanner, and the amount of the immersion solution W3 can be obtained with a simple and inexpensive configuration. Further, it is possible to prevent the immersion solution W3 from running out while reducing the replenishment frequency, and to realize highly reliable observation.

  Further, the illumination optical system 9 allows laser light to enter the chamber 5 from the transparent portion 5 b at the bottom of the chamber 5, so that the illumination optical system 9 excluding the prisms 23 </ b> A and 23 </ b> B can be disposed below the chamber 5. . Thereby, 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. The illumination optical system 9 to be made can be easily configured.

  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. Instead of this, single illumination that allows the laser light to be incident on the sample S only from one direction is used. There may be. In many cases, the sample S absorbs and scatters light. Therefore, illumination with a plurality of illumination light beams is advantageous in illumination uniformity.

  Further, the movable stage 7 may support the cuvette 3 so as to be rotatable around the detection optical axis P in the chamber 5. In this way, laser light 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. Thereby, the incident depth of the laser light from each direction to the sample S can be reduced to suppress the influence of scattering in the sample S, and a clear fluorescent image can be acquired. It is more effective in the case of single illumination.

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

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

[Second Embodiment]
Next, a 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 negative power and shutters 45A and 45B, and by switching between the shutters 45A and 45B, The difference from the first embodiment is that the function as a light field microscope and the function as a light sheet microscope can be switched.
In the following, parts having the same configuration as those of the microscope 1 according to the first embodiment are denoted by the same reference numerals and description thereof is omitted.

  The cylindrical lens 43 having negative power is provided, for example, on the optical path of laser light emitted from the tip end portion 18A of the optical fiber 17, and is attached to the emission end of the prism 23A. With this cylindrical lens 43, 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 can be configured.

  The shutters 45A and 45B are detachably provided on both illumination optical axes Q of the illumination optical system 9, and are disposed 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 including a plurality of microlenses 48 disposed in front of the camera 31. Each microlens 48 is two-dimensionally arranged in a direction intersecting the imaging optical axis of the camera 31.

  The imaging optical system 13 includes an imaging lens 29A that forms an image on the microlens array 47, an imaging lens 29B that forms an image on the imaging surface of the camera 31, and the imaging lens 29A and the imaging lens 29B. An imaging lens turret 49 is provided.

  The microlens array 47 projects an image on the imaging surface of the camera 31. Thereby, the camera 31 can acquire a plurality of pieces of image information having different parallax at a time.

  The imaging lens turret 49 is provided so as to be rotatable about a rotation axis 49a, and the imaging lens 29A and the imaging lens 29B can be alternatively arranged on the optical path of fluorescence.

  The laser light source 15 of the illumination optical system 9, the optical fiber 17, the convex lens 19A, the cylindrical lens 21A, the prism 23A and the cylindrical lens 43, 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 camera 31 functions as a light sheet microscope.

The operation of the microscope 41 configured in this way will be described.
When the sample S is observed with the microscope 41 according to the present embodiment, the function as the light field microscope and the function as the 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 light emitted from the distal end portion 18A of the optical fiber 17, and the shutter is placed on the optical path of the laser light emitted from the distal end portion 18B. 45B is inserted. 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 distal end portion 18A of the optical fiber 17 passes through the convex lens 19A and the cylindrical lens 21A having a positive power, is reflected by the reflecting surface 24A of the prism 23A, and then has a 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 is incident on the sample S. By making the focal plane of the immersion objective lens 11 coincide with the incident range of the laser light, the fluorescence generated in a wide range along the focal plane can be condensed at once by the immersion objective lens 11.

  Fluorescence from the sample S collected 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 imaged by the camera 31 by each microlens 48. Projected onto a surface. Thereby, a plurality of pieces of image information having different parallaxes can be acquired at a time, and three-dimensional image data can be constructed from one image.

  On the other hand, when observing the sample S as a light sheet microscope, the shutter 45A is inserted on the optical path of the laser light emitted from the distal end portion 18A of the optical fiber 17, and the optical path of the laser light emitted from the distal end portion 18B is observed. 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, when observing as a light sheet microscope, since it is the same as that of a 1st embodiment, explanation is 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 position of the immersion objective lens 11 in the direction along the detection optical axis P is finely adjusted by the aiming unit 12. Thus, the shift of the focal position can be eliminated, and the desired observation position in the sample S can be observed with high accuracy.

The present embodiment can be modified as follows.
As a first modification, a variable stop may be provided on the optical path of the laser light emitted from the distal end portion 18 </ b> A of the optical fiber 17. By changing the thickness of the illumination light beam of the laser light by the variable aperture, it is possible to avoid irradiating unnecessary laser light to the depth obtained by the function as the light field microscope.

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

[Third Embodiment]
Next, a 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 of the chamber 5 and makes the laser light enter the sample S.
In the following, parts having the same configuration as those of the microscope 1 according to the first embodiment are denoted by the same reference numerals and description thereof is omitted.

The microscope 51 according to this embodiment includes an inverted microscope configuration unit 53, a light sheet illumination module 55, a laser light source 15, and an optical fiber 17.
The inverted microscope configuration unit 53 includes the 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 unit 12, a convex lens 19A, and a cylindrical lens 21A.
The cuvette 3 and the chamber 5 both 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 an additional (add-on) configuration can be achieved simply by placing the light sheet illumination module 55 on the inverted microscope configuration unit 53 which is a normal inverted microscope.

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

  When the sample S is observed with the microscope 51 configured as described above, the laser light emitted from the laser light source 15 is guided by the optical fiber 17 and enters 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 beam by the convex lens 19A, passes through the transparent portion 5b on the side wall portion of the chamber 5, and is collected by the cylindrical lens 21A. The laser beam condensed by the cylindrical lens 21 </ b> A passes through the transparent portion 3 b on the side wall portion of the cuvette 3 and enters the sample S.

  Even when the sample S is observed with the microscope 51 according to the present embodiment, even if the observation position of the sample S is changed in the chamber 5 by moving the movable stage 7, the aiming unit 12 detects the immersion objective lens 11. By finely adjusting the position in the direction along the optical axis P, it is possible to eliminate the shift of the focal position. Therefore, a 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 disposed 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 added to an inverted microscope constituting unit 53 that is a normal inverted microscope provided with the immersion objective lens 11, the aiming unit 12, and the camera 31. It can be configured by simply adding the light sheet illumination module 55 provided.
Although the light sheet microscope is used in the present embodiment, an illumination module for a light field microscope may be formed.

[Fourth Embodiment]
Next, a 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 moves the cuvette 3 around an axis parallel to the detection optical axis P. It is different from the first embodiment in that it is rotatably supported.
In the following, parts having the same configuration as those of the microscope 1 according to the first embodiment are denoted by the same reference numerals and description thereof is omitted.

  For example, as shown in FIG. 8, the cuvette 3 is a microplate in which a plurality of storage portions 3a in which the sample S is stored 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 the transparent portion 3 b over the entire area in the circumferential direction.

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

The operation of the microscope 61 configured as described above 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 any one of the accommodating portions 3a is disposed on the detection optical axis P. .

  In this state, the laser light emitted from the laser light source 15 is guided by the optical fiber 17 to be converted into a parallel light beam by the convex lens 19A, and then condensed in a planar shape by the cylindrical lens 21A to be transparent on the side wall of the chamber 5. It penetrates the part 5b. The laser light incident on the chamber 5 is incident on the sample S through the transparent portion 3 b on the side wall of the cuvette 3. Thereby, the fluorescence image of the sample S of the accommodating part 3a arrange | positioned on the detection optical axis P is acquirable.

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

  As described above, according to the microscope 61 according to the present embodiment, the sample S placed on the detection optical axis P can be obtained simply by moving the cuvette 3 around the axis parallel to the detection optical axis P by the movable stage 7. Can be switched. Accordingly, the laser light is sequentially incident on the sample S of each storage unit 3 a by the illumination optical system 9, and the fluorescence from the sample S of each storage unit 3 a is sequentially collected by the immersion objective lens 11 and continuously collected by the camera 31. Can be taken. Thereby, a large amount of images of the sample S can be acquired efficiently and at high speed.

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

[Fifth Embodiment]
Next, a microscope according to a fifth embodiment of the 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 of the chamber 5 with a single illumination and makes the laser light incident on the sample S, so that the liquid is immersed. The objective lens 11 collects the fluorescence transmitted from the sample S through the side wall of the chamber 5, and the movable stage 7 can move the cuvette 3 in the X, Y, and Z directions in the chamber 5, respectively. It differs from the first to fourth embodiments in that it is rotatably supported around a predetermined rotation axis orthogonal to the optical axis Q and the detection optical axis P.
In the following, portions having the same configuration as those of the microscopes 1, 41, 51, 61 according to the first to fourth embodiments are denoted by the same reference numerals and description thereof is 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. Aiming unit 12 for moving immersion objective lens 11 supported by revolver 73 in the direction along detection optical axis P, imaging optical system 13, water replenisher 75 for replenishing immersion solution W3, and movable stage 7 And a control device 77 for controlling the control and the like. In FIG. 11, reference numeral 79 denotes a drain tank.

In the present embodiment, the cuvette 3 is a single 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 portions, light transmitting portions) 3b on all side wall portions in the circumferential direction.
The chamber 5 has a transparent part (excitation light transmission part, light transmission part) 5b on two side wall parts 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 laser light guided by the optical fiber 17 into a parallel light beam, a cylindrical lens 21 having the same configuration as the cylindrical lenses 21 </ b> A and 21 </ b> B, And a variable aperture 81.

  The variable diaphragm 81 is disposed between the cylindrical lens 21 and the transparent portion 5 b on the side wall portion of the chamber 5. By changing the beam diameter of the laser beam by the variable aperture 81, the thickness of the laser beam condensed in a planar shape by the cylindrical lens 21 can be changed. This change is performed according to the immersion objective lens 11 inserted in the optical path.

  The distal end portion 18 of the optical fiber 17, the convex lens 19, the cylindrical lens 21, and the variable diaphragm 81 are arranged to face the transparent portion 5 b on one side wall portion of the chamber 5, and emit laser light emitted from the laser light source 15. The light is incident on the sample S through the transparent part 5 b on one side wall of the chamber 5 and the transparent part 3 b on any side wall of the cuvette 3.

  As shown in FIG. 10, the immersion objective lens 11 is disposed outside the chamber 5 so as to face the transparent portion 5 b of the other side wall portion with the detection optical axis P orthogonal to the illumination optical axis Q. . An immersion solution W3 such as pure water is injected into a gap between the top surface 11a of the most advanced 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. Is held in the gap.

  The revolver 73 can be alternatively disposed on the fluorescent light 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 75 a at the tip, and when the immersion objective lens 11 is switched, the top surface 11 a of the most advanced lens of the immersion objective lens 11 and the transparent part 5 b on the side wall of the chamber 5. The immersion solution W3 can be replenished from the nozzle 75a into the gap.

  The imaging optical system 13 includes a mirror 25, an imaging lens 29 that forms an image of the 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, and 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 beam diameter of the laser beam by the variable aperture 81, switches the immersion objective lens 11 by the revolver 73, and the immersion objective lens 11 by the aiming unit 12. The fine adjustment of the position in the direction along the detection optical axis P and the replenishment of the immersion solution W3 by the water replenisher 75 are controlled.

The operation of the microscope 71 configured as described above will be described.
In order to observe the sample S with the microscope 71 according to this 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. The laser light source 15 generates laser light.

  The laser light emitted from the laser light source 15 is guided by the optical fiber 17, converted into a parallel light beam by the convex lens 19, condensed in a planar shape by the cylindrical lens 21, passes through the variable aperture 81, and The light passes through the transparent portion 5 b of the side wall and enters the chamber 5.

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

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

  The fluorescence condensed 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. Thereby, in the camera 31, a tomographic image orthogonal to the detection optical axis P of the sample S is obtained. The movable stage 7 is driven by the control device 77, and the cuvette 3 is moved in the X, Y, and Z directions in the chamber 5, thereby changing the observation position of the sample S and acquiring a tomographic image at each observation position. can do.

  The focal position of the immersion objective lens 11 is made to coincide with the focal position of the cylindrical lens 21 and the detection optical axis P of the immersion objective lens 11, and the focal plane of the immersion objective lens 11 is made to coincide with the incident plane of the laser light. Fluorescence generated in a wide range along the surface is condensed at once by the immersion objective lens 11 and photographed by the camera 31, and a clear fluorescence image of the observation region in the sample S can be acquired. In addition, since laser light is not irradiated except for the imaging plane of the camera 31, it is possible to obtain a good three-dimensional stereoscopic image while suppressing the fading of fluorescence.

  In this case, according to the microscope 71 according to the present embodiment, the movable stage 7 is driven by the control device 77 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 direction 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 close to the immersion objective lens 11, a good image can be obtained over substantially the entire area of the sample S. Can be acquired.

  Even when the cuvette 3 is moved in the chamber 5 by the movable stage 7 and the observation position of the sample S is changed, the amount of the immersion solution W3 disposed in the gap between the immersion objective lens 11 and the chamber 5 is ( Even if there is a fine adjustment of the focal point, it does not change (it is maintained as it is due to the surface tension), so it is not necessary to prepare a large amount of the immersion solution W3 or replenish it with a high frequency. Cutting can also be prevented.

  Further, when the observation position of the sample S is changed, the focal position of the immersion objective lens 11 is shifted according to the refractive index distribution in the sample S, or the refractive index of the cuvette solution W1 and the refractive index of the chamber solution W2. Even if the focus position of the immersion objective lens 11 is shifted due to a slight difference from the above, the sighting unit 12 makes the focus only by finely adjusting the position of the immersion objective lens 11 in the direction along the detection optical axis P. The position shift can be eliminated.

  In the present embodiment, the light sheet microscope has been described as an example, but the present invention 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 (see FIG. 4) having negative power, and the cylindrical lens 43 is interposed between the cylindrical lens 21 and the chamber 5. The laser beam having a thickness in the direction along the photographing optical axis of the camera 31 may be incident on the sample S. The imaging optical system 13 includes a microlens array 47 including a plurality of microlenses 48 that project an image onto the imaging surface of the camera 31, and an imaging lens 29A that forms an image on the microlens array 47 (see also FIG. 4). ). In this way, a plurality of pieces of image information having different parallaxes can be acquired at a time.

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

  The microscope 91 includes a cuvette 3, a chamber 5, a movable stage 7, a dry objective lens (objective lens) 93 that collects fluorescence emitted from the sample S, and a direction along the detection optical axis P of the dry objective lens 93. A sighting unit 12 that can be moved to the image sensor, an imaging optical system 13 that acquires an image of the sample S based on the fluorescence condensed by the dry objective lens 93, and a control device 77 that controls the movable stage 7 and the like. .

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

The dry objective lens 93 is provided outside the chamber 5 in the vicinity of the transparent portion 5b at the bottom, and is disposed vertically upward to face the transparent portion 5b. Further, the dry objective lens 93 is arranged with a space between the transparent portion 5b at the bottom of the chamber 5 without the immersion solution W3 interposed therebetween.
The imaging optical system 13 includes an imaging lens 29 that forms an image of the fluorescence condensed by the dry objective lens 93 and a camera 31.

The operation of the microscope 91 configured as described above 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 from the sample S, the fluorescence emitted in the direction along the detection optical axis P passes through the cuvette solution W1, the transparent portion 3b at the bottom of the cuvette 3, the chamber solution W2 and the transparent portion 5b at the bottom of the chamber 5. Then, the light is condensed by the dry objective lens 93 and imaged on the imaging surface of the camera 31 by the imaging lens 29. Thereby, in the camera 31, a tomographic image orthogonal to the detection optical axis P of the sample S is obtained. The movable stage 7 is driven by the control device 77, and the cuvette 3 is moved in the X, Y, and Z directions in the chamber 5, thereby changing the observation position of the sample S and acquiring a tomographic image at each observation position. 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 and the observation position of the sample S is changed, the dry objective lens 93 according to the refractive index distribution in the sample S. Even if the focus position of the dry objective lens 93 shifts due to a deviation in the focal position of the lens or 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 move. Only by finely adjusting the lens 93 in the direction along the detection optical axis P, the shift of the focal position can be eliminated.

  Therefore, even if there is a slight difference between the refractive index distribution in the sample S and the refractive index of the cuvette solution W1 and the refractive index of the chamber solution W2, the movable stage 7 is driven and images are taken in the Z direction at equal intervals. When acquiring the stack image, it is possible to obtain images at equal intervals by performing fine adjustment of a slight focus, and to construct a three-dimensional image without distortion. Further, the configuration can be made simple and inexpensive as much as the light source and the illumination optical system are unnecessary.

  As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the specific structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included. For example, the present invention is not limited to those applied to the above-described embodiments and modifications, but may be applied to embodiments in which these embodiments and modifications are appropriately combined, and is not particularly limited. . For example, in the above-described embodiment, an example in which laser light is incident on the sample S from one direction or two directions has been described. However, for example, laser light may be incident on the sample S from three or more directions.

  In 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. 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 is used. Since the refractive index of the transparent portion 5b of the chamber 5 does not change, it is sufficient that at least the cuvette solution W1 and the chamber solution W2 have substantially the same refractive index.

1, 41, 51, 61, 71, 91 Microscope 3 Cuvette (specimen container)
3a container (specimen container)
3b, 5b Transparent part (excitation light transmission part, light transmission part)
5 Chamber (medium container)
5a Aperture 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
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 containing the first immersion medium is immersed together with the specimen;
An objective lens that is arranged outside the medium container and collects light emitted from the specimen;
An imaging unit that captures the light collected by the objective lens;
An aiming unit for moving the objective lens in a direction along the detection optical axis;
A movable stage that supports the sample container in the medium container so as to be movable at least in the direction along the detection optical axis;
The sample container and the medium container each have a light transmission part capable of transmitting the light from the sample,
A microscope in which the objective lens is arranged to face the light transmission part of the sample container via the light transmission part of the medium container.   The microscope according to claim 1, wherein the objective lens is disposed with a space between the objective lens and the light transmission portion of the medium container.   The objective lens according to claim 1 or 2, wherein the objective lens is an immersion objective lens, and the immersion objective lens is disposed between the light transmission portion of the medium container and a third immersion medium. microscope. The immersion objective lens is disposed with the detection optical axis directed in a direction intersecting a 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 transmission portion of the medium container. The sample container and the medium container have the light transmission part on a side wall part,
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 transmission part of the specimen 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 transmission part 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 supports the sample container so as to be movable in a direction intersecting the detection optical axis.   The microscope according to any one of claims 1 to 9, wherein the imaging unit photographs the light that the specimen self-emits. An illumination optical system for irradiating the sample with excitation light from a direction intersecting the detection optical axis;
The microscope according to any one of claims 1 to 9, wherein each of the sample container and the medium container has an excitation light transmission unit that transmits the excitation light from the illumination optical system toward the sample. The medium container has the excitation light transmission part at the bottom,
The illumination optical system includes a reflection mirror disposed in the medium container, and the excitation light is incident on the medium container from the excitation light transmission part at the bottom and is reflected toward the sample by the reflection mirror. The microscope according to claim 11. The medium container has the excitation light transmission part on a side wall part,
The microscope according to claim 11, wherein the illumination optical system causes the excitation light to enter the medium container from the excitation light transmission 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 disposed in the medium container.   The microscope according to claim 14, wherein the lens having the positive power is a cylindrical lens having a positive power in one direction intersecting an 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 imaging optical axis of the imaging unit;
The microscope according to any one of claims 11 to 13, wherein the illumination optical system causes the excitation light of a substantially parallel light beam to enter the sample.   The microscope according to any one of claims 11 to 16, wherein the movable stage supports a plurality of the sample containers so as to be movable around an axis parallel to the detection optical axis in the medium container. The sample container has a plurality of sample storage 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 supports the sample storage unit disposed on the detection optical axis in a switchable manner.   The microscope according to any one of claims 11 to 18, wherein the movable stage supports the sample container so as to be rotatable around the detection optical axis in the medium container.   The microscope according to claim 11, wherein the illumination optical system causes the excitation light having a planar shape to enter the specimen along a plane intersecting the detection optical axis of the objective lens.

Images