JP2008101964A - Ultra microtome - Google Patents

Abstract

To provide an ultramicrotome for producing a well-continuous slice sample without stopping the movement of an arm.
An ultramicrotome for producing a thin piece sample by cutting a cross section of a sample block 10 using a diamond knife 4d.
An ultramicrotome comprising means 37 for removing water adhering to the cross section of the sample block on a track 51 drawn by the cross section of the sample block 10 during the cutting of the cross section.
[Selection] Figure 6

Description

  The present invention relates to an ultramicrotome for making a flake sample from cross-section cutting of a solid sample.

Conventionally, an ultramicrotome has been used as a cross-section of a resin-embedded sample block, which is a pretreatment operation of a sample for observation with an optical microscope, an electron microscope, or the like, or as a thin piece preparation device (see, for example, Non-Patent Document 1). ). Resin embedding is an operation performed to prevent breakage and deformation due to the impact of cutting when the sample is soft or very thin. The embedded resin is shaved until the sample surface embedded therein is exposed, and finally finished with a glass knife or a diamond knife so as to be smooth until the shavings become inconspicuous. The ultramicrotome is equipped with an arm that moves up and down, a knife base that allows a small amount of movement, and a stereomicroscope. When cutting, the sample block is fixed to the sample holder, the sample holder is inserted and fixed to the arm, the knife base is brought close to the vicinity of the sample, and the arm is moved up and down to cut. At this time, cutting is performed repeatedly, and these can also be performed automatically. When the sample cross section is repeatedly cut, it is possible to make a sliced piece by moving a small amount of the knife base for each cutting. In many cases, a wet cutting method using water is used as a method for collecting a thin piece sample formed by cutting a cross section with a diamond knife. The wet cutting method is limited to the case of using a sample that is insoluble and capable of cutting at room temperature. It is a method of floating on the water surface on a knife boat. At this time, since the cross-section cutting is continuously performed, the thin sample is connected every time the thin sample is formed by cutting, becomes a group of thin pieces, and floats on the water surface. And it can use as a thin piece sample for electron microscopes by extract | collecting the thin piece group with a mesh. Especially, in order to observe with a transmission electron microscope (TEM), since it is necessary to permeate | transmit an electron beam, it is necessary to form the very thin thin piece sample about 10-100 nm in thickness.
Kentaro Asakura and Yasuhisa Hirohata, “Ultra-microtome technique Q & A for electron microscope researchers”, 1st edition, Agne Jofusha, September 30, 1999, p. 3-5, p. 18-20, p. 24-27, p. 33-34, p. 44-45, p. 47-48, p. 51, p. 57-59, p. 61, p. 63, p. 66, p. 75-77

  However, in the conventional wet cutting method in which water is poured into a knife boat, the water in the knife boat may adhere to the cutting surface of the sample block depending on the sample cross-sectional shape, water surface height, and knife angle. In particular, in organic samples, there is work of electron beam dyeing that performs chemical treatment to give contrast to transmission images due to differences in components. After this treatment, fine irregularities, stain residue etc. Since it is on the block surface, the hydrophilicity increases, and the water on the knife boat easily adheres to the sample block surface even with slight contact. Further, once water has adhered, the hydrophilicity of the sample cross section is increased even with a small amount of water, and water is deposited on the sample surface every time the diamond knife contacts the sample surface. When water adheres to the sample surface, the cutting force decreases due to the contact state between the knife and the flakes and the change in tension. In addition, the generated flakes are wound on the back of the knife blade by the tension of water, and are dragged from the water surface on the knife boat side, and do not float on the water surface. Therefore, when water adheres to the sample surface, it is necessary to stop the movement of the arm and wipe the sample cutting surface with a filter paper or the like. There was a problem.

  Then, this invention makes it a subject to provide the ultra microtome for producing the thin piece sample favorably continuous, without stopping the motion of an arm.

In order to achieve the above object in the present invention, first, in the invention of claim 1, an ultramicrotome for producing a thin piece sample by cutting a cross section of a sample block using a diamond knife,
An ultramicrotome comprising means for removing water adhering to the cross section of the sample block on a track drawn by the cross section of the sample block at the time of cutting the cross section.

  The invention according to claim 2 is characterized in that the means removes water adhering to the cross section of the sample block by bringing a water absorbent into contact with the cross section of the sample block. The ultra microtome.

  According to the first aspect of the present invention, when the sample is cut in the ultramicrotome, water adhering to the cross section of the sample block can be continuously removed from the diamond knife water surface, and an arm is used to wipe off the water. As a result, the cutting efficiency is not interrupted, and the working efficiency can be dramatically improved.

  According to the second aspect of the present invention, water adhering to the cross section of the sample block can be efficiently removed without causing vibration of the diamond knife water surface.

  As described above, the present invention has an effect that it is possible to provide an ultramicrotome for producing a favorable continuous thin piece sample without stopping the movement of the arm.

  Hereinafter, the best embodiment of the present invention will be described with reference to FIGS.

  First, the ultra microtome according to the present embodiment will be described. As shown in FIG. 1, the ultramicrotome includes a sample holder 1 for holding a sample block to be cut in a cross section, a segment arc 2 for adjusting the angle of the holder, an arm 3 that swings up and down, a pedestal 6 for moving a knife 4 forward, an entity It consists of a microscope 7 and the like. The sample block is fixed by the sample holder 1, the sample holder 1 is fixed to the segment arc 2, the knife 4 is fixed to the knife holder 8, and the pedestal 6 of the knife 4 moves back and forth to cut the sample block. In addition, when wet cutting is performed using a diamond knife as the knife 4, water is injected onto the knife boat, and there is also a water level adjuster 5 that adjusts the water level after injection. The state of cutting is confirmed by the stereomicroscope 7 and the mirror surface, which is a pretreatment for thinning, is also confirmed by the stereomicroscope 7.

In general, as shown in FIG. 2, the sample block 10 is an embedded resin sample block in which a sample is embedded in an embedded resin, and originally has a rectangular parallelepiped shape. Sharpen and trim the tip. However, when the sample has sufficient strength for cutting the cross section, the sample block may be used as it is without being embedded. When performing cross-section cutting, it is desirable that the resin be present in at least three directions around the sample. Further, it is desirable that the remaining amount of the resin is small along the sample, and the cross-section that becomes a flake is 0.2 to 0.4 mm square. It is desirable to have a size of about. Further, in order to reduce the impact of cutting and obtain a flatter flake, it is desirable that not only the cross section but also the resin portion on the side surface of the cross section be a smooth mirror finish.

  As shown in FIG. 3, the diamond knife 4 d is a type that can be wet-cut with water, and has a structure in which a diamond cutting edge 21 is provided at the end of the uppermost knife boat 23. . When installing on the pedestal 6, in order to create a clearance angle, it is often inclined about 4 to 6 degrees. The angle at which the knife edge is tilted is a unique value for each knife. When cutting a thin piece, a little more water is injected onto the knife boat 23 so that the water is covered up to the blade edge 21, and the water level is adjusted by the water level adjuster 5 attached to the ultramicrotome. If the amount of water is too large, water will overflow from the blade edge 21 or it will be easy to hit the sample. Therefore, it is desirable that the water surface be level so that it does not rise due to surface tension. When the water surface is horizontal, when the water surface is observed with the stereomicroscope 7, the surface is often reflected white. When cutting with an ultramicrotome, it is desirable for sampling with a mesh that the thin specimens 22 are successively connected starting from the cutting edge 21 and are continuous in a strip shape on the water surface.

  The ultramicrotome according to the present embodiment is provided with a water wiping jig 41 as shown in FIG. 4 as means for removing water adhering to the cross section of the sample block. The water wiping jig 41 is a jig for wiping water. As shown in FIG. 4, the water wiping jig 41 has a support 34, a screw rod 39 so as to go straight to the support 34, and a screw 36 in the horizontal direction. Adjust the position. The water-absorbing material 37 for wiping off water is sandwiched and fixed by clips 35. If the sample holder 1 side is the front direction, as shown in FIG. 5, the water wiping jig 41 is installed on the left side to which the diamond knife 4d is fixed. The space on the right side of the knife pedestal 6 included in the ultramicrotome is narrow and is not suitable for installing the water wiping jig 41. The diamond knife 4d in FIG. 5 is in a state viewed from the back side where the blade edge 21 is not present. The bottom 38 of the water wiping jig 41 is a magnet and is fixed on the metallic base 6 of the knife. As the water-absorbing material 37 for wiping off water, a filter paper cut to about 1 to 2 cm square, a non-woven fabric such as Kimwipe (manufactured by Crecia), Bencott (manufactured by Asahi Kasei Co., Ltd.), etc. cut to a 5 cm square 2-3 times. It is desirable that it has been raised to a certain extent. If the filter paper is coated for liquid separation, moisture may not be absorbed instantly, so qualitative or quantitative filter paper is preferable. In the case where the sample is soft and the exposed surface of the filter paper may be damaged, a soft material such as Kimwipe but having high water absorption is desirable.

  The order of setting the water wiping jig 41 is such that the knife base 6 is moved so that the cutting edge 21 of the diamond knife 4d is in contact with the sample block, and the diamond knife 4d or It is desirable to set the position of the water wiping jig 41 without changing the angle of the sample holder 1 or the like. FIG. 6 shows the trajectory 51 drawn by the cross section of the sample block 10 when the arm 3 of the ultramicrotome moves, but the cross section of the sample block 10 draws a slightly arc-shaped trajectory until the arm 3 makes one round. When the arm 3 is swung down, it moves in an arc shape on the side close to the diamond knife 4d. When the arm 3 reaches the lower part, it is drawn slightly to the arm 3 side to avoid contact with the diamond knife 4d and rises in an arc shape from the lower part to the upper part. The wiping position of the cross section of the sample block 10 is preferably a position where the cross section of the sample block 10 is separated from the cutting edge 21 of the diamond knife 4d in a state where the movement of the arm 3 is swung down from the upper part to the lower part. If the water is wiped when the arm 3 is swung up from the lower part to the upper part, there is a risk that the cross section of the sample block 10 is pressed against the water wiping jig when the arm 3 is swung down. Further, as another means for removing water adhering to the cross section of the sample block 10, a means for removing water by applying wind to the cross section can be used. However, when water is to be removed by air drying, it is generated. The wind to be generated may cause vibration of the water surface and the like, and means for removing water with a water-absorbing material is preferable.

Next, a method for forming a thin piece sample will be described using an ultramicrotome and a water wiping jig installed on the ultramicrotome. First, the sample block is trimmed with a glass knife or the like until the sample surface is exposed to a mirror surface state. After trimming, replace the glass knife with a diamond knife, and adjust the angle with a segment arc or knife holder while confirming with a stereomicroscope so that the angle of the specimen surface and the knife edge is parallel. When wet cutting is performed, water is poured into a knife boat on a diamond knife. After adjusting the water level, the distance between the sample surface and the knife is shortened, but if the light in the gap between the sample surface and the knife is reduced from gold to red, the arm is set to the automatic cutting mode and the time is shortened. desirable. When automatic cutting is performed with a cutting thickness setting of about 0.1 μm in a state where the light between the sample surface and the knife changes from gold to red, a thin sample is often generated several times. The water wiping jig is installed when the slice sample starts to be generated. When the trajectory is raised to the top, the automatic cutting mode is canceled, and the hand is moved up and down from the top to the knife blade. When the position of the water wiping jig is confirmed, the position of the water-absorbing material can be finely adjusted without damaging the knife, which is desirable.

  In the present embodiment, a sheet or film sample can be used as the observation sample. Examples of the sheet or film sample include a laminate film for packaging materials, a barrier film, and an optical film for display. The sheet or film-like sample has a multilayer structure, and it is necessary to observe the cross section in detail for observing these layer structures. These observation samples are made of a thermosetting resin or an ultraviolet curable resin. Embedded and becomes a sample block. When the observation sample has sufficient strength for cutting the cross section, the sample block may be used as it is without being embedded.

  A sample block obtained by embedding a film sample in which a SiOx film of about 50 nm is laminated on a PET base film with an epoxy resin is sectioned and trimmed with a glass knife so that the sample surface is exposed. The mirror surface was made smooth. Thereafter, gas phase dyeing was performed with ruthenium tetroxide crystals, and the sample block after dyeing was thinned. After water was put into the knife boat of the diamond knife, the vertical and horizontal angles of the cutting surface of the sample and the cutting edge of the diamond knife were adjusted. A water-absorbing material for wiping off water was sandwiched between clips, and as this water-absorbing material, 5 types C-type filter paper cut into about 1.5 cm square was used. A water wiping jig was placed so that the surface of the filter paper fixed with a clip would come to the place where it seems to be on the run-out path of the sample holder, and the arm was moved with the manual dial on the side of the ultramicrotome body. The jig support was rotated little by little from the knife side to the sample holder side, and when the arm was swung from the upper part to the lower part, it was fixed with a slight touch without causing the arm speed to stall. After that, when automatic cutting is performed and thin piece cutting is performed at a film thickness of 0.1 μm, the thin piece sample floats on the water surface in sequence, but before the sample is cut with a knife, water drops do not form on the sample surface. It was. Therefore, it became possible to move the arm continuously, and to obtain a thin sample efficiently.

The figure which shows the structural example of an ultramicrotome. The figure which shows a mode that the sample surface is exposed by the embedding resin sample block which embedded the sample in embedding resin. The figure which shows a mode that the sample sliced on the water in the knife boat of a diamond knife floats. The figure which shows the structural example of the jig for water wiping off. The figure which shows the positional relationship with the diamond knife at the time of installing the jig for water wiping off. The figure which shows the positional relationship of the track | orbit which the cross section of a sample block draws, and the absorber for water wiping off.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Sample holder 2 ... Segment arc 3 ... Arm 4 ... Knife 4d ... Diamond knife 5 ... Water level adjuster 6 ... Base 7 ... Stereomicroscope 8 ... Knife holder 10 ... Sample block 11 ... Resin 12 ... Sample 21 ... Cutting edge 22 ... Thin piece Sample 23 ... Knife boat 34 ... Post 35 ... Clip 36 ... Screw 37 ... Absorbent 38 for wiping water ... Bottom 39 ... Screw rod 41 ... Jig 51 for wiping water ... Orbit

Claims (2)

An ultra microtome for making a thin sample by cutting a cross section of a sample block using a diamond knife,
An ultramicrotome comprising means for removing water adhering to the cross section of the sample block on a track drawn by the cross section of the sample block at the time of cutting the cross section.   2. The ultramicrotome according to claim 1, wherein the means removes water adhering to the cross section of the sample block by bringing a water absorbent into contact with the cross section of the sample block.