Sample fixation device and microscope device including the same

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 apparatus including the same. [Background technology]

[0002] Systems that enable 3D imaging in microscopic observation are being developed. For example, a system has been developed in which a transparent specimen is fixed in a transparent material and rotated while photographing it from the side using a microscope, and a system that can rotate on six axes and photograph 3D images of even non-transparent specimens has been developed (see Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication WO2010 / 014244 Summary of the Invention [Problem to be solved by the invention]

[0004] However, until now, there has been no microscope stage that allows specimens to be photographed freely from all directions.

[0005] SUMMARY OF THE PRESENT EMBODIMENTS Accordingly, an object of the present invention is to provide a novel specimen fixing device and a microscope apparatus including the same. [Means for solving the problem]

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

[0007] Another embodiment of the present invention is a microscope stage or a microscope apparatus having any one of the above-mentioned specimen fixing devices. The microscope apparatus may have a stereomicroscope, a bright-field microscope, a fluorescence microscope, or a laser microscope. The microscope apparatus may have a transmitted illumination device, a coaxial epi-illumination device, or a lateral illumination device. Effect of the Invention

[0008] According to the present invention, it is possible to provide a novel specimen fixing device and a microscope apparatus including the same. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram of a microscope stage according to an embodiment of the present invention. [Diagram 2] 1A to 1C are diagrams illustrating a process for producing a specimen fixation device in one embodiment of the present invention. [Diagram 3] 1 shows photographs of a completed specimen fixation device from various angles in one embodiment of the present invention. A is a photograph of the entire specimen fixation device, and B is a photograph of an enlarged specimen. [Figure 4] 1 is a photograph of a 3D stage manufactured in one embodiment according to the present invention. [Diagram 5] 1 is a photograph of a 3D stage equipped with a specimen fixation device containing a specimen in one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The objectives, features, advantages, and ideas of the present invention will be clear to those skilled in the art from the description of this specification, and those skilled in the art can easily reproduce the present invention from the description of this specification. The embodiments of the invention and specific examples described below show preferred embodiments of the present invention, and are shown for illustrative or explanatory purposes, and do not limit the present invention thereto. It is clear to those skilled in the art that various changes and modifications can be made based on the description of this specification within the intent and scope of the present invention disclosed in this specification.

[0011] ==Specimen fixation device== The specimen fixation device disclosed herein is a device for mounting to a microscope, which includes a polyhedron or sphere for fixing a specimen therein by filling it with a fixative substance.

[0012] The specimen used here is not particularly limited, but when observing a specimen fixed in a specimen fixing device with transmitted light, it is preferable that the specimen has a high transmittance with respect to the light from the light source used for observation. The transmittance of the specimen with respect to visible light depends on the size of the specimen, the polyhedron, and the intensity of the light used, but is preferably 10 to 100%, more preferably 20 to 100%, more preferably 40 to 100%, and even more preferably 80 to 100%. In addition, the thickness of the specimen in the optical axis direction is not particularly limited, but is preferably 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 lateral light, neither the light transmittance nor the thickness of the specimen is particularly limited.

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

[0014] The polyhedron may have a frame along the sides. The material of the frame is not particularly limited, and may be, for example, acrylic, glass, plastic, etc. It is not necessary for all sides to have a frame, and as long as the shape of the polyhedron is maintained, there may be a portion other than the sides that is made of the same material as the frame.

[0015] The fixative for fixing the specimen inside the polyhedron or sphere may be transparent. The transmittance of the fixative to visible light depends on the size of the specimen, the polyhedron, and the intensity of the light used. For example, the transmittance to the light of the light source used for observing the specimen is preferably 80 to 100%, more preferably 90 to 100%, and even more preferably 95% to 100%. As the fixative, a histochemical sealant can be used. Examples of the sealant include MGK-S (Matsunami Glass), Biolite (Oken Shoji), and the like. The fixative may also include gels such as agarose, gelatin, and Pluronic PF-127, resin, and paraffin.

[0016] Examples of polyhedrons include an even-angled icosahedron, a rhombic triocahedron, a truncated icosahedron, a hexagonal octahedron, a triangular icosahedron, a pentagonal icosahedron, a kite-shaped hexahedron, a pentagonal hexahedron, and a hexagonal icosahedron. It is preferable that the polyhedron has parallel opposing faces, and examples of such polyhedrons include an even-angled icosahedron, a truncated icosahedron, a triangular icosahedron, a kite-shaped hexahedron, a pentagonal hexahedron, and a hexagonal icosahedron. It is also preferable that all of the faces of the polyhedron have the same shape, and it is particularly preferable that 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, but may be, for example, 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. A plurality of specimen fixing devices having different maximum lengths may be prepared, and the optimal size of the polyhedron may be selected according to the size of the specimen. In addition, 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, and for example, 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 1 mm, preferably 10 mm or more when the maximum length of the specimen is 5 mm, preferably 16 mm or more when the maximum length of the specimen is 8 mm, and preferably 20 mm or more when the maximum length of the specimen is 10 mm.

[0018] ==Manufacturing method of specimen fixation device== Below, we will give an example of a method for manufacturing a specimen fixation device having a polyhedron or a sphere with a frame, but the method for manufacturing the specimen fixation device is not limited to this, and a person skilled in the art can easily prepare a specimen fixation device using known methods.

[0019] First, a polyhedron-shaped frame is designed using a computer. The designed polyhedron frame is then printed using a 3D printer. An acrylic or glass plate is cut to a size that fits into the frame, and then fitted into the frame. On one side, the acrylic or glass plate is not fitted, and dissolved gel is poured into the polyhedron up to about half its volume, and the gel is allowed to harden in this state. A specimen to be observed is placed on the solidified gel, and dissolved gel is added to harden the gel. An acrylic or glass plate is fitted onto the last side, completing the specimen fixation device. The position of the specimen in the specimen fixation device can be fine-tuned, for example, by centrifugal force, adding gel, using small tools, or using magnets. In particular, it is preferable to use a non-crosslinked gel (e.g., edible gel) since this does not leave any traces on the gel caused by fine adjustments.

[0020] Depending on the type of gel, an appropriate method for gelation can be selected from among temperature changes such as heating and cooling, light irradiation such as light, visible light and UV light, addition of chemical substances such as gelling agents, and physical stimuli such as pressure, vibration and shear force.

[0021] In the case of spheres, a spherical glass with a hole in it is manufactured, and the specimen is fixed inside with gel in the same way as in the case of polyhedrons.Then, the hole is sealed and the specimen fixing device is completed.

[0022] It should be noted that the polyhedron does not have to be a perfect polyhedron in the mathematical sense, the sphere does not have to be a perfect sphere, and distortions and irregularities necessary for manufacturing are allowed.

[0023] ==Microscope equipment with specimen fixation device== By using the specimen fixation device disclosed in this specification for microscopic observation, it is possible to perform not only x-axis, y-axis, and z-axis movements, but also rotation along three rotation axes: roll, pitch, and yaw. For example, a microscope stage for mounting this specimen fixation device is shown in Figure 1.

[0024] The microscope stage shown in FIG. 1A has a 3D stage 10, and a specimen fixing device 11 can be attached to it. A specimen 12 is placed in the specimen fixing device 11 before attachment. This 3D stage can move along the x-axis, y-axis, and z-axis. This 3D stage is a gimbal, and can rotate around a roll axis, which is a first rotation axis 17, a pitch axis, which is a second rotation axis 19, or a yaw axis, which is a third rotation axis 18.

[0025] The microscope stage shown in FIG. 1B is a gimbal, and includes a circular outer ring 20, two first rotation shafts 27 for fixing the specimen fixation device 21 including the specimen 22, two second rotation shafts 29 for fixing the specimen fixation 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-axis, y-axis, and z-axis directions. The first rotation shaft 27 is a roll axis, and the second rotation shaft 29 is a pitch axis, and the specimen fixation device 21 can be supported by expanding and contracting. For example, the first rotation shaft 27 and the second rotation shaft 29 can be manufactured by combining a rotary actuator with a telescopic arm. A specific operation method will be described below. When the first rotation shaft 27 supports the specimen fixation device 21, the second rotation shaft 29 moves away from the specimen fixation device 21. When the first rotation shaft 27 rotates in this state, the specimen fixation device 21 rotates around the roll axis. Next, when the second rotation shaft 29 supports the specimen fixation device 21 and the first rotation shaft 27 moves away from the specimen fixation device 21, the specimen fixation device 21 rotates around the pitch axis. Finally, when the outer ring 20 rotates with either or both of the first rotation shaft 27 and the second rotation shaft 29 supporting the specimen fixation device 21, the specimen fixation device 21 rotates around the yaw axis. Meanwhile, by moving the base, the specimen fixation device 21 can be moved in the directions of the x-axis, y-axis, and z-axis.

[0026] By combining these movements and rotations, the specimen can be oriented in any position and in any direction within the range of the stage movement. By movably and rotatably mounting these stages to the stage of a conventional microscope, a microscope device can be manufactured. 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, any of a stereomicroscope, a bright-field microscope, a fluorescent microscope, and a laser microscope. This specimen fixing device is compatible with any of the observation methods.

[0028] Furthermore, the microscope apparatus includes an illumination device, but may also include, for example, a transmitted illumination device, a coaxial incident illumination device, or a side illumination device. The illumination device may include a mirror for adjusting the direction of light. When a transmitted illumination device is used, the specimen is observed by transmitted light. When a coaxial incident illumination device or a side illumination device is used, the specimen is observed by incident light or side light, respectively. This specimen fixing device is compatible with either observation method. The microscope apparatus may also include a lens for correcting optical distortion. This lens functions effectively, particularly when the specimen fixing device is spherical. Furthermore, the microscope apparatus may also include an imaging device such as a CCD camera.

[0029] ==How to use a microscope equipped with a specimen fixation device== First, the specimen fixing device containing the specimen is attached to the microscope stage, and then 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 fixation device is on the straight line connecting the optical axis and the objective lens. It is preferable that the outer wall through which the light enters and the opposing outer wall through which the light exits are parallel and perpendicular to the optical axis.

[0031] When using incident or lateral light, the 3D stage is moved so that the light illuminates 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 frame with a 1 mm recess was created using the software Fusion 360 (Figure 2A), and the frame was printed using a high-resolution 3D printer AGILISTA-3100 (Figure 2B). The frame was coated with a black matte spray (Figure 2C). Pentagons and hexagons were cut out of a 1 mm thick transparent acrylic plate using a laser cutter, and then fitted into the frame of the model using Aron Alpha Tough Power (Konishi Co., Ltd.) and glued (Figure 2D). The polygons that serve as the adhesion points for the stage axis were designed to be filled with 3D printer material (Figure 3D; for gimbal). In addition, the acrylic plate was not fitted into one of the pentagons, which is the smallest polygon, as an entrance for inserting the transparent gel and specimen (Figure 3D; opening). The size of this truncated icosahedron is 40 mm in diameter.

[0033] (2) Preparation of specimens Pururun agar (Matsugi Kanten Sangyo Co., Ltd.) was added to water at a concentration of about 4% (w / v) and dissolved in a microwave oven until it became transparent. As a biological specimen, a spider of the Japanese genus Araneus with a total length of about 5 mm (excluding legs) was frozen and placed in a small amount of dissolved agar to gel it.

[0034] The specimen fixation device was filled with about half of the dissolved agar, which was then allowed to gel. The spider, along with the gel, was placed on the gel inside the device, and the specimen fixation device was filled with the dissolved agar. It was left at room temperature until the agar gelled completely. The specimen was placed so that it would fit within the rectangular prism that connected both opposing 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 software Pro ENGINEER and modeled using AGILISTA. In this example, only two axes, the pitch axis and the roll axis, were provided. Other components used included stainless steel hexagon socket bolts, nuts, and bearings, as well as aluminum shaft holders and frames (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 Stage 11 Specimen fixation device 12 specimens 17 First Rotation Axis 18 Third Rotation Axis 19 Second Rotation Axis 20 Outer ring 21 Specimen fixation device 22 specimens 27 First Rotation Axis 29 Second Rotation Axis

Claims

1. A specimen fixing device for mounting on a microscope, A specimen fixation device comprising a polyhedron or sphere for fixing a specimen therein by filling it with a transparent fixation substance.

2. The specimen fixation device according to claim 1 , wherein the outer wall of the polyhedron or sphere comprises acrylic or glass.

3. 3. The specimen fixing device according to claim 1, wherein the polyhedron has a frame along each side.

4. The specimen fixation device according to claim 1 , wherein the polyhedron has parallel opposing faces.

5. 2. The specimen fixing device according to claim 1, wherein all faces of the polyhedron have the same shape.

6. The specimen fixation device according to claim 1 , wherein the polyhedron is a regular polyhedron.

7. 7. The specimen fixation device according to claim 6, wherein the polygon formed by the frame has parallel opposing faces.

8. The specimen fixation device of claim 1 , wherein the fixation substance comprises a gel, a resin, or paraffin.

9. 2. The specimen fixation device of claim 1, wherein the fixative is a histochemical mounting medium.

10. 2. The specimen fixing device according to claim 1, wherein the transmittance of the outer wall and / or the fixing substance to light from a light source used for observing the specimen is 80 to 100%.

11. 2. The specimen fixing device according to claim 1, wherein the transmittance of said outer wall and / or said fixing substance to light from a light source used for observing said specimen is 95 to 100%.

12. A microscope stage having the specimen fixing device according to claim 1.

13. A microscope apparatus comprising the microscope stage according to claim 12.

14. The microscope apparatus according to claim 13, comprising a stereo microscope, a bright field microscope, a fluorescence microscope, or a laser microscope.

15. 15. The microscope apparatus according to claim 13, further comprising a transmitted illumination device, a coaxial epi-illumination device, or a lateral illumination device.

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