Specimen fixation device and microscope device containing it

Abstract

To provide a novel sample fixation device, and a microscope device equipped with the same.SOLUTION: A sample fixation device of the present invention is designed to be attached to a microscope, and comprises a polyhedron or a sphere for having a sample fixed therein by filling an inside of the polyhedron or sphere with a transparent fixing substance.SELECTED DRAWING: Figure 5

Description

Technical Field

[0001] The present invention relates to a specimen fixing device and a microscope device including the same.

Background Art

[0002] In microscopic observation, a system that enables 3D imaging is being developed. For example, a system has been developed in which a transparent specimen is fixed in a transparent material and rotated while being photographed from the side with a microscope, and a system that can rotate in six axes and can perform 3D photography including non-transparent specimens (see Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, heretofore, there has been no microscope stage that enables free photography of a specimen from all directions.

[0005] Therefore, an object of the present invention is to provide a novel specimen fixing device and a microscope device including the same.

Means for Solving the Problems

[0006] One embodiment of the present invention is a specimen fixing device for mounting on a microscope, comprising a polyhedron or sphere for fixing a specimen inside by filling it with a transparent fixing material. The outer wall of the polyhedron or sphere may include acrylic or glass. The polyhedron may have a frame along each edge. The polyhedron may have parallel faces. All faces of the polyhedron may have the same shape. The polyhedron may be a regular polyhedron. The polygons formed by the frame may have parallel faces. The fixing material may include a gel, resin, or paraffin. The fixing material may be a histochemical mounting agent. The transmittance of the outer wall and / or the fixing material to light from a light source used for observing the specimen may be 80-100% or 95-100%.

[0007] Another embodiment of the present invention is a microscope stage or microscope apparatus having any of the specimen fixing devices described above. The microscope apparatus may include a stereomicroscope, a bright-field microscope, a fluorescence microscope, or a laser microscope. The microscope apparatus may also include a transmitted illumination device, a coaxial reflected illumination device, or a side illumination device. [Effects of the Invention]

[0008] The present invention makes it possible to provide a novel specimen fixation device and a microscope device including the same. [Brief explanation of the drawing]

[0009] [Figure 1] This is a schematic diagram of a microscope stage according to an embodiment of the present invention. [Figure 2] This figure shows the process of fabricating a specimen fixation device in one embodiment of the present invention. [Figure 3] In one embodiment of the present invention, the completed specimen fixation device is shown from various angles. A is a photograph of the entire specimen fixation device, and B is a magnified photograph of the specimen. [Figure 4] This is a photograph of a 3D stage manufactured in one embodiment of the present invention. [Figure 5] This is a photograph of a 3D stage equipped with a specimen fixing device containing a specimen, in one embodiment of the present invention. [Modes for carrying out the invention]

[0010] The object, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description herein, and will be easily reproducible to those skilled in the art from the description herein. The embodiments of the invention and specific examples described below are examples of preferred embodiments of the invention and are provided for illustrative or explanatory purposes only, and do not limit the invention thereto. It will be apparent to those skilled in the art that various modifications and modifications can be made based on the description herein, within the intent and scope of the invention disclosed herein.

[0011] ==Specimen fixation device== The specimen fixation device disclosed herein is a device for mounting on a microscope and includes a polyhedron or sphere for fixing a specimen inside by filling it with a fixative material.

[0012] The specimens used here are not particularly limited, but when observing a specimen fixed in a specimen fixing device with transmitted light, it is preferable that the transmittance of the specimen to the light source used for observation is high. The transmittance of the specimen to visible light depends on the size of the specimen / polyhedron and the intensity of the light used, but for example, it is preferably 10-100%, more preferably 20-100%, preferably 40-100%, and even more preferably 80-100%. Also, the thickness of the specimen in the optical axis direction is not particularly limited, but it is preferable that it be thin, preferably 1 cm or less, more preferably 5 mm or less, and even more preferably 1 mm or less. When observing a specimen fixed in a specimen fixing device with coaxial incident light or side-illuminated light, neither the light transmittance of the specimen nor the thickness of the specimen is particularly limited.

[0013] The polyhedron or sphere is filled with a fixed material. The polyhedron or sphere has an outer wall to hold the fixed material. The outer wall is preferably highly transparent, but the transmittance to visible light depends on the size of the specimen / polyhedron and the intensity of the light used. For example, the transmittance of the outer wall to the light from the light source used to observe the specimen is preferably 80-100%, more preferably 90-100%, and even more preferably 95-100%. The outer wall may contain, or be made of, acrylic or glass. In terms of shape, it may be plate-shaped or lens-shaped.

[0014] A polyhedron may have frames along its edges. The material of the frames is not particularly limited, but may be made of acrylic, glass, plastic, etc. Not all edges need to have frames; as long as the shape of the polyhedron is maintained, parts other than the edges may be made of the same material as the frames.

[0015] The fixing material used to secure the specimen inside the polyhedron or sphere only needs to be transparent. The transmittance of the fixing material to visible light depends on the size of the specimen and polyhedron and the intensity of the light used, but for example, the transmittance to the light source used for observing the specimen is preferably 80-100%, more preferably 90-100%, and even more preferably 95-100%. A histochemical encapsulant can be used as the fixing material. Examples of encapsulants include MGK-S (Matsunami Glass) and Biolite (Oken Shoji). The fixing material may also include gels such as agarose, gelatin, and Pluronic PF-127, resins, paraffin, etc.

[0016] Examples of the polyhedron include, for example, a rhombic dodecahedron, a rhombic triacontahedron, a truncated icosahedron, a hexagonal octahedron, a trigonal icosahedron, a pentagonal icosahedron, a kite-shaped hexacontahedron, a pentagonal hexacontahedron, a hexagonal icosahedron, etc. The polyhedron preferably has parallel opposing faces. Examples of such polyhedra include a rhombic dodecahedron, a truncated icosahedron, a trigonal icosahedron, a kite-shaped hexacontahedron, a pentagonal hexacontahedron, a hexagonal icosahedron, etc. In addition, in the polyhedron, it is preferable that all faces have the same shape, and particularly preferably, the polyhedron is a regular polyhedron.

[0017] The radius of the sphere or the maximum length from the center of gravity of the polyhedron to the outer wall is not particularly limited. For example, it may be 1 mm or more, more preferably 1.5 mm or more, even more preferably 2 mm or more, may be 100 mm or less, more preferably 75 mm or less, and even more preferably 50 mm or less. Note that a plurality of specimen fixing devices having different maximum lengths may be prepared, and the optimum size of the polyhedron may be selected according to the size of the specimen. Also, when it is desired to photograph the entire specimen, the radius of the sphere or the maximum length from the center of gravity of the polyhedron to the outer wall is related to the maximum length of the specimen. For example, when the maximum length of the specimen is 1 mm, the radius of the sphere or the maximum length from the center of gravity of the polyhedron to the outer wall is preferably 2 mm or more, when the maximum length of the specimen is 5 mm, it is preferably 10 mm or more, when the maximum length of the specimen is 8 mm, it is preferably 16 mm or more, and when the maximum length of the specimen is 10 mm, it is preferably 20 mm or more.

[0018] ==Method for manufacturing a specimen fixing device== An example of a method for manufacturing a specimen fixing device having a polyhedron with a frame or a sphere is given below. However, the method for manufacturing the specimen fixing device is not limited to these, and those skilled in the art can easily fabricate the specimen fixing device by known methods.

[0019] First, design a frame in the shape of a polyhedron using a computer. Fabricate the designed polyhedron frame with a 3D printer. Cut an acrylic plate or a glass plate to a size that fits the frame and fit it into the frame. Leave one side unfitted with the acrylic plate or the glass plate, pour melted gel up to about half of the volume of the polyhedron, and solidify the gel in that state. Place a specimen for observation in the solidified gel state, add more melted gel, and solidify the gel. When fitting the acrylic plate or the glass plate to the last side, the specimen fixing device is completed. The position of the specimen within the specimen fixing device can be finely adjusted, for example, by centrifugal force, addition of gel, use of small tools, magnets, etc. In particular, it is preferable to use a gel that is not cross-linked (e.g., edible gelatin, etc.) because the traces generated in the gel will not remain due to fine adjustment.

[0020] Note that for the gelation method of the gel, an appropriate one may be selected from temperature changes such as heating and cooling, light irradiation such as light, visible light, and UV, addition of chemical substances such as a gelling agent, and physical stimuli such as pressure, vibration, and shear force, depending on the type of the gel.

[0021] In the case of a sphere, manufacture a glass sphere with a hole in part, and fix the specimen inside with gel in the same manner as in the case of the polyhedron. Then, when closing the hole, the specimen fixing device is completed.

[0022] Note that the polyhedron does not have to be a perfect polyhedron in the mathematical sense, and the sphere does not have to be a perfect sphere, and manufacturing distortions and necessary concavities and convexities for manufacturing are allowed.

[0023] ==Microscope device equipped with a specimen fixing device== By using the specimen fixing device disclosed in this specification for microscopic observation, not only movement in the x-axis, y-axis, and z-axis directions but also rotation along three rotation axes of roll, pitch, and yaw becomes possible. For example, a microscope stage for mounting this specimen fixing device is shown in FIG. 1.

[0024] The microscope stage shown in Figure 1A has a 3D stage 10 and can be fitted with a specimen fixing device 11. A specimen 12 is placed in the specimen fixing device 11 before it is fitted. This 3D stage is movable in the x, y, and z directions, with each stage being movable. Furthermore, this 3D stage is a gimbal and is rotatable around a first rotation axis 17 (roll axis), a second rotation axis 19 (pitch axis), or a third rotation axis 18 (yaw axis).

[0025] The microscope stage shown in Figure 1B is a gimbal comprising a circular outer ring 20, two first rotation axes 27 for fixing a specimen fixing device 21 containing a specimen 22, two second rotation axes 29 for fixing the specimen fixing device 21, and a base (not shown) on which the outer ring 20 is mounted and which can move the outer ring 20 in the x, y, and z directions. The first rotation axes 27 are roll axes, and the second rotation axes 29 are pitch axes, and by extending and retracting, they can support the specimen fixing device 21. For example, the first rotation axes 27 and the second rotation axes 29 can be manufactured by combining rotary actuators with telescopic arms. The specific operation method is described below. When the first rotation axis 27 supports the specimen fixing device 21, the second rotation axis 29 moves away from the specimen fixing device 21. In this state, when the first rotation axis 27 rotates, the specimen fixing device 21 rotates around the roll axis. Next, with the second rotation axis 29 supporting the specimen fixing device 21 and the first rotation axis 27 moving away from the specimen fixing device 21, the specimen fixing device 21 rotates around the pitch axis as the second rotation axis 29 rotates. Finally, with either or both of the first rotation axis 27 and the second rotation axis 29 supporting the specimen fixing device 21, the specimen fixing device 21 rotates around the yaw axis as the outer ring 20 rotates. Meanwhile, by moving the base, the specimen fixing device 21 can move in the x, y, and z axes.

[0026] By combining these movements and rotations, the specimen can be positioned and oriented in any direction within the range of the stage's movement. These stages can be movably and rotatably attached to the stage portion of a conventional microscope to manufacture a microscope apparatus. Furthermore, the movement and rotation of the microscope stage can be computer-controlled.

[0027] Here, the microscope is not particularly limited and may be, for example, a stereomicroscope, bright-field microscope, fluorescence microscope, or laser microscope. This specimen fixation device is compatible with either observation method.

[0028] Furthermore, this microscope apparatus includes an illumination device, which may be, for example, a transmitted illumination device, a coaxial reflected illumination device, or a side illumination device. The illumination device may include a mirror for adjusting the direction of light. When using a transmitted illumination device, the specimen is observed using transmitted light. When using a coaxial reflected illumination device or a side illumination device, the specimen is observed using reflected light or side illumination, respectively. This specimen fixing device is compatible with both observation methods. In addition, this microscope apparatus may include a lens for correcting light distortion. This lens is particularly effective when the specimen fixing device is spherical. Furthermore, this microscope apparatus may include an imaging device such as a CCD camera.

[0029] ==Instructions for using a microscope equipped with a specimen fixation device== First, the specimen fixation device containing the specimen is attached to the microscope stage. Next, the microscope stage is attached to the microscope body.

[0030] When using transmitted light, the 3D stage is moved so that the transparent part of the outer wall of the specimen fixing device lies on the straight line connecting the optical axis and the objective lens for observation. It is preferable that the outer wall from which light enters and the opposite outer wall from which light exits are parallel and perpendicular to the optical axis.

[0031] When using incident or side-illuminated light, the 3D stage is moved so that light shines on the specimen through the transparent part of the outer wall, and the light reflected from the specimen on the outer wall enters the objective lens through the transparent part. [Examples]

[0032] (1) Manufacturing of specimen fixation devices A truncated icosahedron-shaped frame with a 1mm recess was fabricated using Fusion 360 software (Figure 2A), and the frame was printed using a high-resolution 3D printer, AGILISTA-3100 (Figure 2B). The frame was coated with a matte black spray (Figure 2C). Pentagons and hexagons were cut from a 1mm thick transparent acrylic sheet using a laser cutter, and then fitted and glued into the frame of the model using Aron Alpha Tough Power (Konishi Co., Ltd.) (Figure 2D). The polygons that serve as the attachment points for the stage axes were designed to be filled with 3D printed material (Figure 3D; for gimbal). In addition, one of the smallest polygons, the pentagon, was left unfitted with an acrylic sheet to serve as an entrance for inserting the transparent gel and specimen (Figure 3D; opening). The size of this truncated icosahedron is 40mm in diameter.

[0033] (2) Preparation of specimens Pururun Agar (Matsuki Agar Industry Co., Ltd.) was dissolved in water at a concentration of approximately 4% (w / v) and microwaved until transparent. As a biological specimen, a black-headed orb-weaver spider, approximately 5 mm in total length (excluding legs), was frozen and then gelled in a small amount of dissolved agar.

[0034] Agar, partially dissolved, was placed in the specimen fixation device to form a gel. The black-headed orb-weaver spider was placed in the gel within the device, and the specimen fixation device was filled with dissolved agar. It was left at room temperature until the agar had completely gelled. The specimens were arranged so that they would fit within the prisms connecting opposite polygons, as shown in Figure 3.

[0035] (3) Manufacturing of 3D stages Based on the 3D stage in Figure 1, the connecting parts for the pitch axis and roll axis of the 3D stage were designed using the Pro ENGINEER software and fabricated using AGILISTA. In this embodiment, only two axes, the pitch axis and the roll axis, were provided. Other components used included stainless steel hex socket head bolts and nuts and bearings, an aluminum shaft holder and frame (Figure 4). Finally, the specimen fixing device containing the specimen was fixed to the pitch axis with double-sided tape (Figure 5). [Explanation of symbols]

[0036] 10 3D Stages 11 Specimen fixation device 12 specimens 17. First axis of rotation 18. Third axis of rotation 19. Second axis of rotation 20 Outer ring 21 Specimen fixation device 22 specimens 27 First axis of rotation 29 Second axis of rotation

Claims

1. A specimen fixing device for attachment to a microscope, The specimen fixation device includes a sphere for fixing the specimen inside by filling it with a transparent fixing material, A circular outer ring, Two first rotation axes and two second rotation axes for fixing the specimen fixing device, A base on which the outer ring is mounted and which can move the outer ring in three different directions, Equipped with, When the first rotation axis supports the specimen fixing device, the second rotation axis moves away from the specimen fixing device. When the second rotation axis supports the specimen fixing device, the first rotation axis moves away from the specimen fixing device. Microscope stage.

2. The microscope stage according to claim 1, wherein the outer wall of the sphere includes acrylic or glass.

3. The microscope stage according to claim 1, wherein the fixing substance comprises a gel, resin, or paraffin.

4. The microscope stage according to claim 1, wherein the fixation material is a mounting agent for histochemistry.

5. The microscope stage according to claim 2, wherein the transmittance of the outer wall and / or the fixing material to light from a light source used for observing the specimen is 80 to 100%.

6. The microscope stage according to claim 2, wherein the transmittance of the outer wall and / or the fixing material to light from a light source used for observing the specimen is 95 to 100%.

7. A microscope apparatus having a microscope stage as described in claim 1.

8. The microscope apparatus according to claim 7, comprising a stereomicroscope, a bright-field microscope, a fluorescence microscope, or a laser microscope.

9. The microscope apparatus according to claim 7 or 8, comprising a transmitted illumination device, a coaxial reflected illumination device, or a side illumination device.

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