JP6887611B2 - Blade holder and sample holder - Google Patents

Description

The present invention relates to a blade holder and a sample holder capable of accurately trimming a very small area.

When observing a specific part of a sample such as a living body with a TEM (Transmission Electron Microscope), the operator first trims the solid sample embedded with an embedding agent such as Epon resin, and the observation target is Shape the included protrusions. Hereinafter, the protrusion is referred to as a sample block.
In trimming, an operator may use a blade (razor blade, glass knife, etc.) to cut a part of a solid sample into a right-angled column such as a trapezoidal column.

Specifically, the operator sets the solid sample under a stereomicroscope and looks at the surface of the solid sample through the stereomicroscope, or directly looks at the surface of the solid sample set under the stereomicroscope from diagonally above with the naked eye. While using the blade, cut two parallel lines, which are the upper and lower bottoms of the trapezoid, and two non-parallel lines, which are the legs of the trapezoid, in the vertical direction with respect to the surface of the solid sample. Next, a trapezoidal columnar sample block is produced by cutting in the horizontal direction with respect to the side surface of the solid sample and cutting off unnecessary parts.
Next, the operator attaches the solid sample with the sample block or the sample block itself to the ultramicrotome, and slices the sample block to a thickness of 50 nm to 100 nm to form a thin section for high-quality transmission electron microscope observation. To make.

When it is intended to continuously obtain thin sections, a plurality of prepared thin sections may be connected to the water surface in a ribbon shape and arranged. Here, it is known that in order to ensure the linearity of continuous thin sections, it is preferable that each thin section has two extremely strict parallel lines (see Non-Patent Document 1).
Therefore, for example, if the sample block is accurately cut out into a trapezoidal columnar or rectangular parallelepiped shape, the shape of each thin section becomes a trapezoidal or rectangular shape, and it is preferable because it has two accurate parallel lines. It is particularly preferable to cut out in a columnar shape because the shape of each thin section becomes an isosceles trapezoid and the orientation (top and bottom) of the thin section can be easily confirmed.

In recent years, a method of continuously taking cross-sectional images using SEM (Scanning Electron Microscopes) and reconstructing the taken continuous images in three dimensions has been in the limelight in the field of biological tissue structure analysis. ing.
As a method for acquiring a continuous cross-sectional image by SEM, for example, a block surface observation method using a focused ion beam (FIB-SEM: Focused Ion Beam Scanning Electron Microscopes) and a cutting block surface observation method using a microtome incorporated in a mirror body (SBF). -SEM: Serial Block-Face SEM), continuous section SEM method (Array tomography) in which continuous ultrathin sections are placed on a hard substrate such as glass and the images taken by SEM are reconstructed in three dimensions are known. ..
In either method, if the sample contains a large area other than a specific part, rough cutting before imaging takes time, and the efficiency of continuous cross-sectional image acquisition and / or 3D reconstruction is reduced. There is. Especially in the case of FIB-SEM, the consumption of expensive gallium ion guns increases due to rough cutting of unnecessary areas other than specific parts, and the cost increase due to this also becomes a big problem. In addition, since the number of images taken by the three-dimensional reconstruction method is usually 1,000 to 10,000, there is a problem that the amount of data becomes enormous unless the area unnecessary for observation is removed as much as possible. Further, especially in the case of Array tomography, if the parallelism of the two lines of each thin section is low, there is a problem that the efficiency is lowered because it is necessary to correct the positional deviation at the time of three-dimensional reconstruction. Therefore, accurate trimming within the smallest possible range is required.

Harris et al., The Journal of Neuroscience, November 22, 2006, 26 (47): 12101-12103

However, the above-mentioned conventional technique has the following problems.
That is, when the operator looks at the surface of the solid sample using a stereomicroscope, it is difficult to confirm the position of a specific part or the position of the blade because the magnification is low, and in the case of epi-illumination, the internal structure of the solid sample is examined. There is a problem that it cannot be seen at all.
Further, with the naked eye of the operator, the objective lens becomes an obstacle and the surface of the solid sample is viewed from diagonally above, which causes a problem that it is difficult to make two cutting lines accurately in parallel.
Due to these problems, it is difficult for even a skilled worker to reduce the distance between the two notch lines, and the distance between the two cut lines can be reduced to about 0.5 mm at most. If the distance between the two parallel cut lines is large, the area of the thin section becomes large, so that the number of thin sections that can be held in the grid / mesh for TEM is limited, and there is also a problem that the observation efficiency is lowered.
Further, as described above, the method of reconstructing a continuous cross-section SEM image in three dimensions requires accurate trimming within a very small range as much as possible, so that there is a problem that it is difficult to deal with it by the prior art.

An object of the present invention is to provide a blade holder and a sample holder capable of accurately trimming a very small range in consideration of the above problems.

The blade holder of the present invention includes a holder main body portion mounted on an objective lens of an optical microscope and a blade for cutting a solid sample while being held by the holder main body portion, and the holder main body portion is the objective. The blade has a through hole through which the optical axis of the lens passes, and the blade is held so that its cut surface is parallel to the optical axis and intersects at least a part of the inner peripheral surface of the through hole. It is those that, and at least a part is positioned on the optical axis.
Further, the lens barrel of the objective lens is inserted into the through hole, and the side surface of the lens barrel is pressed by the inner peripheral surface of the through hole by the action of a force for narrowing the inner diameter of the through hole, and the holder. The main body is mounted on the objective lens.
Further, the cutting edge of the blade is housed inside the through hole.
Further, the holder main body is provided with a holder main body attached to the objective lens of the optical microscope and a blade for cutting a solid sample while being held by the holder main body, and the holder main body has an optical axis of the objective lens. The blade has a through hole through which the blade is held so that its cut surface is orthogonal to the optical axis and intersects at least a part of the inner peripheral surface of the through hole. To do.
It is also characterized by being provided with a lighting device for illuminating the solid sample.

The sample holder of the present invention is a sample holder for fixing the solid sample on the stage of the optical microscope when trimming the solid sample with the blade holder, and the plate held on the stage and the plate. A sample fixing means for fixing the solid sample to the plate so that the solid sample faces the blade is provided , the plate is a square in plan view, and the sample fixing means is at the center of the plate and the solid sample. The solid sample is also rotated by 90 ° or 180 ° as the plate is rotated by 90 ° or 180 ° .

Further, the sample fixing means includes a sample holding portion that extends in the vertical direction with respect to the plate and holds the solid sample inside, and the sample holding portion has the lowermost end of the solid sample as the stage. The solid sample is held so as to be lower than the surface of the sample.

The blade holder of the present invention is used by being attached to the objective lens of an optical microscope. The optical axis of the objective lens passes through the through hole formed in the holder body, and the blade is held inside the cylinder including the inner peripheral surface of the through hole. When held in, the blade does not interfere with the observation of the solid sample surface when the operator looks into the microscope eyepiece. Further, since the blade is defocused by a certain amount, when the sample surface is in focus, the operator can determine that the constant amount of distance is maintained between the blade and the sample. it can.
In addition, by fixing the solid sample to the stage of the microscope, the operator can operate the coarse movement handle and the fine movement handle of the microscope to move the solid sample relative to the blade in the desired amount vertically and horizontally. It can be moved in a direction. As a result, the operator can accurately move the blade to the very vicinity of the specific part to be observed in the solid sample and make the first cut line. Specifically, from the state where the sample surface is in focus, the solid sample and the objective lens are brought close to each other in the vertical direction by using the coarse or fine movement mechanism of the microscope, and the blades are brought into contact with each other. The amount of movement during this period coincides with the amount of defocus of the blade. Further, if the solid sample and the objective lens are brought closer to each other beyond the contact point, a notch line having a depth equal to the brought-in amount can be made. Here, when the blade is held by the holder body so as to pass through the center of the through hole, the blade is located on the optical axis of the objective lens, so that the cut surface also passes through the center of the field of view of the microscope. Become.
In addition, by moving the solid sample accurately in parallel with the stage in the horizontal plane by operating the XY fine movement knob of the microscope stage, the second cut line can be accurately translated with the first line at an extremely narrow interval of 0.1 mm or less. Can be done. At this time, by making the surface of the sample including the first cut line in focus before moving in the horizontal plane, the operator does not have to pay special attention to the above-mentioned between the sample surface and the blade. Since the clearance equal to the defocus amount is secured, there is no concern that the blade will inadvertently collide with the sample surface.
As described above, according to the blade holder of the present invention, accurate trimming within a very small range is possible, so that the observation efficiency can be improved and the cost related to the observation can be suppressed. In particular, by applying it to trimming in the case of FIB-SEM, it is possible to significantly reduce the time and cost for rough cutting with a gallium ion gun in the FIB-SEM chamber. In addition, the amount of digital data itself for reconstructing a three-dimensional image by applying it to the trimming in the previous stage of creating a continuous section for Array tomography and shaping the sample block so that it is small and has high parallelism. Not only is the number of sections reduced, but the linearity of the ribbon of continuous sections is increased, and the calculation time for alignment correction in each thin section is shortened. Further, according to the blade holder of the present invention, the observation of the sample and the operation of moving the blade are facilitated, so that the risk of destroying the valuable sample due to carelessness of the operator can be reduced.

In the present specification, the “through hole” refers to a hole formed in the holder main body and through which the optical axis of the objective lens passes. Therefore, for example, when a groove extending in the vertical direction is formed on the surface of two parts and the surfaces forming the groove of each part are made to face each other, a hole is formed by the two grooves. Is also included in the "through hole". Further, even if a part of the hole is closed with a translucent member such as glass, it is included in the "through hole" as long as the optical axis passes (light passes through).
Further, in the present specification, the “cut surface” refers to a surface formed by cutting the solid sample when the blade is brought into contact with the solid sample and relatively moved.

If the solid sample is embedded with a translucent embedding agent such as Epon resin, the blade holder of the present invention can be applied to a transmission microscope.
Further, if at least a part of the blade is positioned on the optical axis, accurate positioning of the blade in the plane direction of the microscope stage becomes easy.
Further, if the blade holder is attached to the objective lens by inserting the lens barrel of the objective lens into the through hole, the number of parts of the blade holder can be reduced.

Further, if the cutting edge of the blade is housed inside the through hole, the cutting edge is not exposed to the outside from the through hole, so that it is possible to prevent an operator from unexpectedly contacting the blade and getting injured.
The holder main body may hold the blade in the horizontal direction, that is, the blade may be held inside the through hole so that the cut surface of the blade is orthogonal to the optical axis of the objective lens. In this case, it is possible to accurately make a cut line in the horizontal direction with respect to the solid sample.
Further, a lighting device for illuminating the solid sample may be attached to the blade holder. In this case, the surface of the solid sample is illuminated by the light of the lighting device, which has the effect of making it easier to see.

The sample holder for fixing the solid sample on the stage of the optical microscope is required to have a mechanism capable of reliably fixing the solid sample at a position where it is easy to see during trimming and so that the solid sample does not move.
The sample holder of the present invention includes a plate to be fixed on the stage and a sample fixing means, and the sample fixing means can hold a solid sample in a state of facing the blade.
Also, by rotating the plate accurately at 90 ° or 180 °, the solid sample held integrally with the plate can also be rotated accurately at 90 ° or 180 °. Here, if the plate is a square in a plan view, the center of the plate does not move by rotating 90 ° or 180 °.
Further, if the mechanism is such that the lowermost end of the solid sample can be held at a position lower than the surface of the stage by the sample holding portion, the position of the uppermost end of the solid sample is also lowered, so that the working distance of the objective lens can be easily secured.

The blade holder of the first embodiment is viewed from diagonally upper right (a) and diagonally lower right (b). Front view (a), plan view (b), bottom view (c), left side view (d) and right side view (e) of the blade holder Front view (a) showing the state where the blade holder is attached to the objective lens and front view (b) showing the state where the notch line is inserted. Perspective view of optical microscope A modified example of the blade holder seen from diagonally above right (a) and diagonally below right (b) Front view showing the blade holder of the second embodiment (a) A view of the blade holder of the third embodiment as viewed from diagonally lower right. Perspective views (a) to (c) showing the sample holder and the solid sample 100 of the first embodiment. Top view (a) and front view (b) of the sample holder A plan view (a) showing a state in which the sample holder is set on the stage using Kremmel and a plan view (b) showing a state in which the plate is rotated by 90 °. A plan view (a) showing a state in which the sample holder is set on the stage using an L-shaped plate and a plan view (b) showing a state in which the plate is rotated by 90 °. The plan view (a) of the sample holder of the second embodiment, the plan view (b) showing the state of holding the solid sample, and the cross-sectional view taken along the line A-A'(c). Top view of solid sample showing trimming method (a)-(f) Photographs (a) to (f) showing the trimming method in the examples Photographs (a) to (f) showing the trimming method in the examples Micrograph of sample block

[First Embodiment of Blade Holder]
A first embodiment of the blade holder of the present invention will be described.
As shown in FIGS. 1 and 2, the blade holder 1 includes a holder main body 10 and a blade 20, and is used for trimming a solid sample 100 (see FIGS. 3, 8, etc.). The solid sample 100 of the present embodiment is embedded in Epon resin and has translucency.
As shown in FIG. 3, the blade holder 1 is attached to the objective lens 203 of the transmission type optical microscope 200. Since the structure of the optical microscope 200 is well known, detailed description thereof will be omitted, but as an example, as shown in FIG. 4, the eyepiece 201, the revolver 202, the objective lens 203, the stage 204, the condenser 205, the coarse movement handle 206, and the fine movement handle It is roughly composed of 207, light source 208, XY fine movement knob 209, and the like. The optical microscope 200 to which the blade holder 1 is attached may be not only an upright microscope but also an inverted microscope.

The holder main body 10 is a rectangular parallelepiped member, and includes a through hole 11 extending in the vertical direction. The material of the holder main body 10 is not particularly limited, and for example, metals such as aluminum, stainless steel, and iron, and plastic can be used. Further, the shape of the holder main body 10 is not limited to a rectangular parallelepiped, and may be a cube, a cylinder, or the like.
The through hole 11 is provided to allow the holder main body 10 to pass through the optical axis of the objective lens 203 while being mounted on the objective lens 203. The light emitted from the light source 208 and traveling in the vertical direction passes through the solid sample 100, the through hole 11, and the objective lens 203, and further passes through other lens groups depending on the configuration of the optical microscope 200, and finally reaches the eyepiece lens 201. To reach.

The holder body 10 has a first slit 12 extending from the upper part of the right side surface into the horizontal plane to reach the through hole 11, and a second slit 13 extending from the upper part of the right side surface into the vertical plane to reach the through hole 11. Is formed. The upper part of the holder body 10 is divided into two pieces, a front piece 14 and a rear piece 15, by the first slit 12 and the second slit 13, and one end of the front piece 14 and the rear piece 15 is the holder body part 10. It is in the state of a cantilever supported by the left part of.
Further, a screw hole 16 is formed which penetrates from the front surface to the rear surface of the front piece 14 and further extends from the front surface of the rear piece 15 to the inside thereof, and a screw 17 (for example, a set screw) is screwed into the screw hole 16. There is.
When the screw 17 is rotated in the tightening direction, the front piece 14 and the rear piece 15 are relatively close to each other, which narrows the distance between the second slit 13 and thus the inner diameter of the through hole 11. It can be made smaller. When the operator inserts the lens barrel of the objective lens 203 from above the through hole 11 and rotates it in the direction of tightening the screw 17, the inner diameter of the through hole 11 becomes smaller and the side surface of the lens barrel becomes the inner circumference of the through hole 11. Since it is pressed by the surface, the holder body 10 can be attached to the objective lens 203. That is, by tightening the screw 17, a force that tries to narrow the inner diameter of the through hole 11 acts, and the side surface of the lens barrel is pressed by the inner peripheral surface of the through hole 11, so that the holder body is attached to the objective lens. it can. When removing the holder body 10 from the objective lens 203, the screw 17 may be rotated in the loosening direction to increase the inner diameter of the through hole 11.

In the present embodiment, the holder main body 10 is a rectangular parallelepiped, and the holder main body 10 is attached to the objective lens 203 by tightening with screws 17, but the present invention is not limited to this, for example, around the holder main body 10. A well-known fastener such as a band or a clamp may be attached and tightened with these to narrow the distance between the second slits 13 and attach the holder main body 10 to the objective lens 203. Alternatively, a ring-shaped elastic member may be attached to the upper part of the inner peripheral surface of the through hole 11 without providing the first slit 12 and the second slit 13. In this case, the elastic member is inserted into the through hole 11 while being expanded outward in the radial direction by the lens barrel of the objective lens 203, so that the holder main body 10 is attached to the objective lens 203 by the contraction force of the elastic member. ..

The blade 20 is a member for cutting the solid sample 100 while being held by the holder main body 10. The type of the blade 20 is not particularly limited as long as a linear notch can be made in the solid sample 100 such as a razor blade, a glass knife, and a wire. In this embodiment, a razor blade is used.
The blade 20 is held inside the through hole 11 so that its cut surface is parallel to the optical axis and intersects at least a part of the inner peripheral surface of the through hole 11.
Specifically, a third slit 21 is formed in the lower part of the holder main body 10 extending in the vertical direction from the right side surface thereof to the left side surface through the center of the through hole 11. The blade 20 is inserted into the third slit 21 so that the cutting edge 22 is not exposed downward from the through hole 11 with the cutting edge 22 facing downward. That is, the cutting edge 22 of the blade 20 is housed inside the through hole 11.

Further, screw holes 23 extending from the front surface of the holder main body 10 to the third slit 21 are formed at two positions on the left and right, and screws 24 (for example, set screws) are screwed into the screw holes 23.
When the left and right screws 24 are rotated in the tightening direction, the screws 24 move backward inside the screw holes 23, and the tips of the screws 24 come into contact with the side surfaces of the blade 20. By further tightening the screw 24 from the state where the screw 24 is in contact with the side surface of the blade 20, the blade 20 is pressed against the inner surface of the third slit 21 by the tip of the screw 24 and fixed.
Since the optical axis of the objective lens 203 extends in the vertical direction and the third slit 21 also extends in the vertical direction when the holder body 10 is attached to the objective lens 203, the cut surface of the blade 20 is parallel to the optical axis. It will be held inside the through hole 11 so as to form a. Further, since the third slit 21 is formed so as to pass through the center of the through hole 11, the blade 20 is held so as to pass through the center of the through hole 11. As a result, the blade 20 is positioned on the optical axis of the objective lens 203. In addition, in order to position the blade 20 more accurately on the optical axis of the objective lens 203, a special screw for finely moving the blade 20 so as to slightly bend it may be added.
As shown in FIG. 5, the holder main body 30 may be composed of two front and rear blocks 31 and 32. In this case, semi-cylindrical grooves 31a and 32a are formed in each block 31 and 32, and both blocks 31 and 32 are joined with screws 33 or the like with the blade 20 sandwiched between the blocks 31 and 32. Will be done.

[Second Embodiment of Blade Holder]
Next, a second embodiment of the blade holder of the present invention will be described, but the same reference numerals will be given to the parts having the same configuration as the first blade holder 1 and the description thereof will be omitted.
As shown in FIG. 6, the blade holder 2 of the present embodiment is characterized in that it includes a lighting device 40 for illuminating the solid sample 100. Specifically, an LED penlight is used as the lighting device 40, and the penlight 40 is inserted and fixed in a hole 41 diagonally provided in the holder main body 10. Since the surface of the solid sample 100 is brightly illuminated by the light of the penlight 40, the workability of trimming is improved.
As the lighting device 40, a well-known lighting means such as an optical fiber that allows light from a light source (not shown) to pass through from one end to the inside and irradiates from the other end can be used. Further, the lighting device 40 may be attached to the outside of the holder main body 10 instead of being inserted into the hole of the holder main body 10.
If the lighting device 40 is provided, the surface of the solid sample 100 is illuminated by the light of the lighting device 40, so that the sample surface 100 can be easily observed.

[Third Embodiment of the blade holder]
Next, the third embodiment of the blade holder of the present invention will be described, but the same reference numerals are given to the parts having the same configuration as the blade holders 1 and 2 of the first and second embodiments. And the explanation is omitted.
As shown in FIG. 7, the present embodiment is characterized in that the blade 20 is held inside the through hole 11 so that the cut surface of the blade 20 is orthogonal to the optical axis of the objective lens 203.
Specifically, a fourth slit 50 extending in the horizontal direction is formed in the lower part of the holder main body 10. The blade 20 is fixed to the fourth slit 50 by a well-known fixing means with its cutting edge 22 located inside the through hole 11.
Since the optical axis of the objective lens 203 extends in the vertical direction and the fourth slit 50 extends in the horizontal direction when the holder body 10 is attached to the objective lens 203, the cut surface of the blade 20 is orthogonal to the optical axis. It will be held inside the through hole 11 so as to form a structure and intersect with at least a part of the inner peripheral surface of the through hole 11.
The blade holder 3 of the present embodiment is useful for making a horizontal cut in the solid sample 100 at the time of trimming.
The lighting device 40 shown in the second embodiment can also be attached to the blade holder 3 of the present embodiment.

[First Embodiment of Sample Holder]
Next, the first embodiment of the sample holder 60 of the present invention will be described.
As shown in FIGS. 8 and 9, the sample holder 60 is used to fix the solid sample 100 on the stage 204 of the optical microscope 200 when trimming the solid sample 100 with the blade holders 1 to 3.
The sample holder 60 includes a plate 61 and a sample fixing means 62.
The plate 61 is a member held on the stage 204 and has an opening 61a in the center thereof. The plate 61 of the present embodiment has a square view in a plan view. The material of the plate 61 is not particularly limited, and for example, a metal such as aluminum, stainless steel, or iron, or plastic can be used. The plate 61 is held by the Clemmel 210 attached to the stage 204 so as not to be misaligned.

The sample fixing means 62 is used to fix the solid sample 100 to the plate 61 so that the upper surface of the solid sample 100 faces the blade 20.
Specifically, the sample fixing means 62 includes a cylindrical sample holding portion 62a and a ring 62b that surround the opening 61a of the plate 61 and extend downward. The sample holding portion 62a is turret-processed. That is, a thread is cut on the outer peripheral surface of the sample holding portion 62a, and the sample holding portion 62a is further divided by a plurality of slits extending in the vertical direction. With the solid sample 100 inserted inside the sample holding portion 62a, the diameter of the sample holding portion 62a is reduced by turning the ring 62b screwed into the thread, and the lowermost end of the solid sample 100 is closer to the surface of the stage 204 than the surface of the stage 204. Hold it in a low position. As a result, the position of the upper surface of the solid sample 100 is lowered, and a sufficient working distance of the objective lens 203 can be secured. The plate 61 can also be used upside down, in which case the position of the upper surface of the solid sample 100 can be further lowered.
By making the plate 61 square, the solid sample 100 is rotated exactly 90 ° or 180 ° by rotating the plate 61 90 ° or 180 ° in a horizontal plane and holding it with the Clemmel 210 as shown in FIG. be able to. Since the sample is fixed to the center of the plate 61, the observation site of the sample does not deviate from the field of view of the microscope even when rotated by 90 ° or 180 °, which is convenient for trimming work.
As shown in FIG. 11, an L-shaped plate 63 may be attached to the stage 204 instead of the Clemmel 210. In this case, the plate 61 can be held by abutting the two sides of the plate 61 against the right-angled bent portion of the L-shaped plate 63 so that the plate 61 is not misaligned, and the plate 61 is further rotated by 90 ° or 180 ° to be held. it can.

[Second Embodiment of Sample Holder 60]
Next, the second embodiment of the sample holder of the present invention will be described, but the parts having the same configuration as the sample holder 60 of the first embodiment will be described with the same reference numerals. Omit.
As shown in FIG. 12, the sample holder 70 of the present embodiment is characterized in that the plate is divided into a trapezoidal plate 71 and a rectangular plate 72. The trapezoidal plate 71 is fixed to the stage 204 by bolts 73 instead of the Clemmel 210. Recesses 71a and 72a are formed in the trapezoidal plate 71 and the rectangular plate 72, and the solid sample 100 is moved to the trapezoidal plate 71 side by tightening the bolt 74 with the solid sample 100 sandwiched between the recesses. It is a mechanism to hold.

Next, the trimming method of the present invention will be described.
The trimming method of the present invention uses an optical microscope 200 provided with any of the blade holders 1 to 3 and sample holders 60 and 70. In the following description, it is assumed that the optical microscope 200 includes a blade holder 1 and a sample holder 60.
First, the operator attaches the blade holder 1 to an appropriate objective lens 203 and causes the sample holder 60 to hold the solid sample 100.
Next, the operator operates the coarse movement handle 206, the fine movement handle 207, the XY fine movement knob 209, etc. while looking into the eyepiece 201 of the optical microscope 200 to move the solid sample 100 in the direction along the optical axis with respect to the blade 20. By moving the lens 203 in a direction orthogonal to the optical axis, the focus of the objective lens 203 is adjusted to a specific part of the solid sample 100, and at the same time, the blade 20 is also positioned (FIG. 13 (a)). At this time, since the blade 20 is defocused by a certain amount on the side close to the objective lens 203, the blade 20 is located very close to a specific portion of the solid sample 100.

Then, the operator moves the solid sample 100 in the vertical direction to make the first linear notch C1 in the solid sample 100 as shown in FIG. 13 (b).
Next, the operator moves the solid sample 100, positions the blade 20 at a position parallel to the first notch as shown in FIG. 13 (c), and the solid sample as shown in FIG. 13 (d). Make a second straight cut C2 in 100. At this time, the plate 61 may be rotated by 180 °. In addition, an eyepiece micrometer (reticle) may be used to appropriately control or observe the distance between the two straight lines C1 and C2.
Then, the operator moves the solid sample 100 and makes two notches C3 and C4 that intersect the two parallel notches as shown in FIG. 13 (e). As a result, a trapezoidal columnar or rectangular parallelepiped notch is made in the solid sample 100. At this time, the plate 61 may be rotated by 90 °.
Finally, the operator makes a horizontal cut in the solid sample 100 and cuts off an unnecessary portion to prepare a trapezoidal columnar or rectangular parallelepiped sample block B as shown in FIG. 13 (f). The horizontal cutting may be made by the operator holding the blade 20 by hand and moving it in the horizontal direction, or by using the blade holder 3 shown in the third embodiment. ..

Using the blade holder 1 and the sample holder 60 of the present invention, a part of the brain tissue was used as a solid sample 100 for trimming.
As shown in FIG. 14 (a), a thin slice of brain tissue having a thickness of 50 μm was fixed with an adhesive on a table made of epoxy resin. Brain tissue contains large and small blood vessels.
As shown in FIG. 14 (b), five parallel cut lines were made on the surface of the solid sample 100 using the blade holder 1. The distance between the second and third cut lines is about 80 μm, and a small blood vessel to be observed is included between them.
Next, the operator holds the blade 20 with his left and right hands and moves the blade 20 horizontally from the side surface of the solid sample 100 (Fig. 14 (c)), and first removes unnecessary parts up to the first cut line (Fig. 14 (c)). Figure 14 (d)).
Next, the blade 20 was moved horizontally to the second cut line (Fig. 14 (e)), and the unnecessary part up to the second cut line was deleted (Fig. 14 (f)).

The operator then rotated the solid sample 100 180 ° in a horizontal plane (Fig. 15 (a)). Then, the blade 20 was moved horizontally from the opposite side surface of the solid sample 100 to the fifth cut line (Fig. 15 (b)), and the unnecessary part up to the fifth cut line was deleted (Fig. 15 (c)). )).
In the same procedure, unnecessary parts up to the 4th and 3rd cut lines were deleted (Figs. 15 (d) and 15 (e)). The reference numeral H in FIG. 15 (e) is a hair (diameter 0.1 mm or less) placed for comparison in size.
Next, the operator rotated the solid sample 100 90 ° in the horizontal plane, and deleted the area between the second and third cut lines that was far from the observation target, and the trimming was completed (Fig. 15). (f)).
The distance between the second and third cut lines was about 0.1 mm.
FIG. 16 is an electron microscope image of a sample block obtained by trimming in the same procedure as in this example. The width of the two parallel lines is about 30 μm.

The present invention has a blade holder and a sample holder capable of accurately trimming a very small area, and has industrial applicability.

B sample block
C1 to C4 cut line
H code
1 blade holder
2 blade holder
3 blade holder
10 Holder body
11 Through hole
12 1st slit
13 2nd slit
14 Front piece
15 Rear piece
16 screw holes
17 screws
20 blades
21 3rd slit
22 Cutting edge
23 screw holes
24 screws
30 Holder body
31 blocks
31a groove
32 blocks
32a groove
33 screws
40 Lighting equipment
41 holes
50 4th slit
60 Sample holder
61 plate
61a opening
62 Sample fixing means
62a Sample holder
62b ring
63 L-shaped plate
70 Sample holder
71 Trapezoidal plate
71a recess
72 rectangular plate
72a recess
73 bolts
74 bolts
100 solid sample
200 light microscope
201 Eyepiece
202 Revolver
203 Objective lens
204 stage
205 condenser
206 Coarse handle
207 Fine movement handle
208 light source
209 XY fine movement knob
210 Clemmel

Claims (7)

The holder body attached to the objective lens of the optical microscope and
It is equipped with a blade that cuts a solid sample while being held by the holder body.
The holder main body has a through hole through which the optical axis of the objective lens passes.
The blade is held so that its cut surface is parallel to the optical axis and intersects at least a part of the inner peripheral surface of the through hole , and at least a part thereof is on the optical axis. A blade holder characterized by being located in. The lens barrel of the objective lens is inserted into the through hole, and the side surface of the lens barrel is pressed by the inner peripheral surface of the through hole by the action of a force for narrowing the inner diameter of the through hole, and the holder main body portion. The blade holder according to claim 1 , wherein the blade holder is mounted on the objective lens. The blade holder according to claim 1 or 2 , wherein the cutting edge of the blade is housed inside the through hole. The holder body attached to the objective lens of the optical microscope and
It is equipped with a blade that cuts a solid sample while being held by the holder body.
The holder main body has a through hole through which the optical axis of the objective lens passes.
The blade holder is characterized in that the cut surface is held so as to be orthogonal to the optical axis and so as to intersect at least a part of the inner peripheral surface of the through hole. The blade holder according to any one of claims 1 to 4 , further comprising a lighting device for illuminating the solid sample. A sample holder for fixing the solid sample on the stage of the optical microscope when trimming the solid sample with the blade holder according to any one of claims 1 to 5.
The plate held on the stage and
A sample fixing means for fixing the solid sample to the plate so that the solid sample faces the blade is provided .
The plate is a square in plan view
The sample fixing means fixes the solid sample at the center of the plate.
A sample holder characterized in that the solid sample also rotates 90 ° or 180 ° as the plate rotates 90 ° or 180 °. The sample fixing means includes a sample holding portion that extends vertically with respect to the plate and holds the solid sample inside.
The sample holder according to claim 6 , wherein the sample holding portion holds the solid sample so that the lowermost end of the solid sample is at a position lower than the surface of the stage.

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