JP5626615B1 - Loading device for ultra-thin sections of electron microscope specimens - Google Patents

Abstract

Even if a groove is provided in an annular body and water droplets are absorbed by the absorbent paper from the gap between the grid and the annular body and the gap between the annular body and the grid becomes narrow, the water droplet flows without stagnation in the groove and is absorbed by the absorbent paper. Further, the area of the annular body is reduced by penetrating the long hole in the annular body, the grid and the annular body are quickly dried, and the support membrane is not broken. Furthermore, the present invention provides a loading tool for an ultrathin section of an electron microscope observation sample in which movement of an ultrathin section by extrusion of a water surface caused by landing of an annular body is extremely reduced. An annular body (2c) is formed by passing through a circular hole (1) in the center of a disk with an extremely thin flat shape, and a long hole (3) is formed at the periphery of the annular body (2c) in the same direction as the periphery. ), A concave groove that crosses from the inner edge to the outer edge on the lower side of the central band (4), with a central gap (4) provided as a central gap (4) between the long hole (3) and the long hole (3). (5) is provided, a thin shaft (6) is provided on the side opposite to the groove (5), and a grip handle (7) is provided on the thin shaft (6). [Selection] Figure 11

Description

  The present invention relates to a loading tool for preparing an electron microscope observation sample by an ultrathin section method and placing an ultrathin section floating on the water surface of a knife boat on a grid of an electron microscope observation sample stage.

Conventionally, an ultrathin section produced by an ultrathin section method is used to observe a medical / biological organism with an electron microscope. In the ultrathin section method, a biological tissue is cut into 1 mm ³ , and an ultrathin section cut into a thickness of about 0.1 μm or less is electronically stained and observed. Briefly describing the series of procedures for ultra-thin sections, fixation (glutar, osmic acid) ⇒ water washing (buffer) ⇒ dehydration (alcohol) ⇒ resin penetration (epoxy resin, other resins) ⇒ resin embedding (epoxy resin) , Other resins) ⇒ Ultra-thin slice (use ultra-microtome, ultra-thin cut on the surface of knife boat) ⇒ Collect (put ultra-thin slice on a grid with a support membrane or put on a grid without a support membrane) ) ⇒Electronic staining (penetrating heavy metals) ⇒Electron microscope observation (using transmission electron microscope) ⇒Recording images (film / TV) The only part of the ultrathin section placed on the grid can be observed by the transmission electron microscope. The small membrane that falls from the grid hole and the ultrathin section that easily breaks are covered with a support membrane. Put on the grid. Ultra-thin sections that are not torn without a support membrane and do not fall out of the grid holes can be placed directly on the grid. Images with better resolution can be observed without the support film. The shape of the grid holes and the size of the holes are commercially available. There are the following methods for loading an ultrathin section on a grid.

  The water surface of the knife boat is made concave so that water does not adhere to the ultra-thin cut surface of the sample during the pressing method or ultra-thin cutting, and the ultra-thin cut is made below the blade edge. To load by pressing, add water to the knife boat and raise the water surface to make it convex. The grid is previously subjected to a hydrophilic treatment by sticking an adhesive treatment or a supporting film. Grab the edge of the grid with tweezers and bend the grip so that the grid is horizontal. A grid is pressed horizontally against an ultrathin section floating under a stereomicroscope (see Non-Patent Document 1).

  The edge of the grid that has been pulled up, pre-adhered to the grid, or hydrophilically treated with a support film is grasped with tweezers, and the grid is placed vertically and placed near the ultrathin section floating under the stereomicroscope. Then, the ultrathin slice is pulled and attached to the grid surface using a tool such as eyelashes (see Non-Patent Document 1).

The method of the loop method 1 will be described with reference to FIGS.
The loading tool (A) has a thickness of 20 to 50 μm and a hole with a diameter of 2 mm (1 mm) in the range of a circular metal plate having the same outer diameter as that of the grid (10) and 3 mm, which can be observed with an electron microscope. ) Through which the annular body (2a) is formed, the narrow shaft (6) is provided on one side of the annular body (2a), and the grip (7) is attached to the thin shaft (6). Is used. As an example, if an appropriately shaped hole is provided on the peripheral surface of the annular body (2a), drying after water droplet absorption can be accelerated. When the angle between the annular body (2a) and the thin shaft is bent at 30 degrees, it is difficult to adjust the angle of each person's preference. To use this loader, use an ordinary ultrathin section method to cut an ultrathin slice that floats on the water surface of a knife boat under a stereomicroscope and hold the handle of the loader in your hand. Gently lower the ultrathin section so that the ultrathin section fits in the hole, and when the lower surface of the annular body has landed, gently lift the loader as it is, and the ultrathin section will It floats on the water surface inside, and water drops form on the entire lower surface of the annular body. Next, when water drops on the lower surface of the annular body are brought into contact with the upper surface of the grid prepared in advance, the grid adheres concentrically to the water drops on the annular body (2a). Next, the absorbent paper is pressed against the side surface of the annular body from any side of the annular body to absorb water droplets from the gap between the annular body and the grid. The ultrathin section floating on the water surface of the inner diameter of the annular body is loaded on the grid when there are no more water droplets. Since the grid drops when the joint surface with the annular body is dried, the grid is moved to a predetermined position (see Patent Document 1).

The method of the loop method 2 will be described with reference to FIGS.
The loop method 2 loading tool (B) is an ultra-thin 50 μm thick hole with an inner diameter of 2 mm in the center of a 3 mm flat metal disk having the same outer diameter as the grid (10) and can be observed with an electron microscope. (1) A thin shaft (6) is formed on one side of the annular body (2b) which forms an annular body (2b) penetrating the inner edge of the annular body (2b) from the inner edge to the outer edge. And use a loader with a handle on the thin shaft (6). Since the angle between the annular body (2b) and the thin shaft is bent at 30 degrees, the angle is adjusted to the desired angle for use. To use this loader, use an ordinary ultrathin section method to cut an ultrathin slice that floats on the water surface of a knife boat under a stereomicroscope and hold the handle of the loader in your hand. Gently lower the top of the ultrathin section so that the ultrathin section fits in the hole. When the water droplets on the lower surface of the annular body are brought into contact with the upper surface of the grid that has been subjected to a sticking treatment or a support film in advance and subjected to a hydrophilic treatment, the water droplets on the annular body are gridded. Adheres concentrically. Next, as shown in FIG. 5, the absorbent paper is pressed against the side surface of the annular body from an arbitrary side of the annular body to absorb water droplets from the gap between the annular body and the grid. The ultrathin section floating on the water surface of the inner diameter of the annular body is loaded on the grid when there are no more water droplets. Since the grid falls when the joint surface with the annular body is dried, the grid is moved to a predetermined position. The loop method 2 is a loading tool made of an annular body that does not damage the support membrane (see Patent Document 2).

Utility Model Registration No. 1973277 Japanese Patent No. 3433292

"Electron Microscopic Guidebook" 47-48 pages August 10, 2004 First Edition Editor Japan Society of Microscopy

  Regarding the loading tool A of the loop method 1, the utility model registration No. 1937277 of the patent document 1 and the patent No. 3433292 of the patent document 2 regarding the loading tool B of the loop method 2 have the following drawbacks.

  In the process of absorbing water droplets from the gap between the grid and the annular body to the absorbent paper, the gap between the annular body and the grid becomes narrower as the process progresses, resulting in a bad absorption efficiency and a long absorption time. In addition, when the pressing position is shifted or separated during absorption of water droplets on the absorbent paper, the gap between the grid and the annular body is closed and dried, and is not absorbed even when pressed again. In order to make it absorb again, it is made to press and absorb to the location where priming water was dripped on the absorbent paper.

  The conventional loading tool puts an ultra-thin section floating on the water surface of the knife boat into the inner diameter of the annular body, and causes the annular body to land horizontally, but the annular body cannot land accurately and horizontally. That is, since the annular body lands in a slightly inclined state, some part around the annular body lands first. The water surface is pushed out in the direction of the annular body on the opposite side with the annular body that has landed first, and the floating ultrathin slice is moved as indicated by the arrow in FIG. However, considering the fact that the moving direction is uncertain, the grid surface has a wide margin and is used effectively in order to reduce the number of ultrathin sections and allow the inner diameter of the annular body to be loaded. Not. The present invention has been made to eliminate the above drawbacks.

7 to 11,
The loading tool B of the present invention forms an annular body (2) through a circular hole (1) in the center of a disc made of an extremely thin flat, and the circumferential edge of the annular body (2) is in the same direction as the circumferential edge. A plurality of the long holes (3) are penetrated, and one part of the gap between the long hole (3) and the long hole (3) is made wider than the other gaps to be provided as a flat central band (4). A concave groove (5) crossing from the inner edge to the outer edge is provided on the lower side of the central band (4), a thin shaft (6) is provided on the opposite side of the groove (5), and the grip is extended to the thin shaft (6). A handle (7) is provided.
The present invention is an ultrathin section loading tool for an electron microscope observation sample having the above-described configuration.

  Due to the provision of a concave groove that crosses from the inner edge to the outer edge on the lower side of the annular body, the water droplet remains in the annular body even if the space between the annular body and the grid is closed. The water channel is secured, and it flows without any delay and is absorbed by the absorbent paper in a short time. Even if the absorbent paper is separated from the annular body during the absorption, if it is pressed again, it is absorbed by the absorbent paper without delay. Since the groove is located between the water surface and the grid and the water surface of the annular body does not open to the groove until the water droplets are completely absorbed, the ultrathin slice remains in the annular body. The movement of the ultrathin section to the groove side on the water surface surrounded by the annular body is very slow. The reason for this is that the surface of the water surrounded by the annular body is mixed with an invisible hydrophobic substance and an ultrathin slice that were generated during ultrathin cutting. In the case of an open shape, when water droplets are absorbed by the water-absorbing paper, a flow occurs on the water surface, and the hydrophobic substance flows into the groove and moves the ultrathin slice to the groove side.

  The loader of the present invention is provided with a plurality of through holes in the periphery of the annular body in the same direction as the periphery, so that the area of the annular body is reduced. Since the extrusion of the water surface is minimal and the movement of the ultra-thin slice is extremely small, it is possible to place a large amount of the ultra-thin slice on the inner diameter of the annular body of the loading tool and land the annular body with peace of mind. Moreover, since the area of an annular body is small, drying is also quick. Furthermore, the grid support membrane does not break.

  Plan view of the prior art   Side view of conventional technology   An enlarged plan perspective view of the main part of the prior art   An enlarged plan perspective view of the main part of the prior art   Enlarged perspective view showing the state of use of a conventional loading tool   It is an expanded sectional view which shows the use condition of the loading tool of a prior art, and shows the movement condition of the ultra-thin slice floating on a water drop   Plan view of the present invention   Side view of the present invention   Main part enlarged plan view of the present invention   Main part enlarged plan perspective view of the present invention   Enlarged back perspective view of essential parts of the present invention   The expanded perspective view which shows the use condition of this invention   The expanded perspective view which shows the use condition of this invention   The enlarged side view which shows the use condition of this invention   The enlarged side view which shows the use condition of this invention   The enlarged side view which absorbs the water droplet between a grid and an annular body to absorption paper by reversing up and down of the present invention   The enlarged sectional view which absorbs the water droplet between a grid and an annular body in absorption paper by reversing up and down of the present invention   Sectional drawing which finished absorbing water droplets between the grid and the annular body of the present invention on absorbent paper   Plan view in which ultra-thin sections are loaded on the grid of the present invention

Hereinafter, embodiments for carrying out the present invention will be described with reference to FIGS.
An annular body (2) having a circular hole (1) penetrated in the center of a flat metal disc having a thickness of 50 μm to 100 μm is provided, and the outer diameter of the annular body (2) is the same as that of the grid (10), The inner diameter is a circular hole (1) that can be observed by an electron microscope.

  Unlike the conventional loading tool (A) and the conventional loading tool (B), the loading tool (C) of the present invention is provided with a plurality of elongated holes (3) penetrating the periphery of the annular body (2c) in the same direction as the periphery. , One part of the gap between the long hole (3) and the long hole (3) is widened to form a flat central band (4).

  A concave groove (5) is provided on the lower side of the central band (4) to cross from the inner edge to the outer edge of the annular body (2c). The concave groove (5) has a width of 0.4 mm to 0.9 mm and a depth of about half the thickness of the annular body (2c).

  A thin shaft (6) is provided at a position facing the central band (4) of the annular body (2c), and a grip handle (7) is provided in the extending direction of the thin shaft (6). The present invention has the above structure.

The use of this loading tool will be described with reference to FIGS.
In order to use this, an ultrathin section that has been ultrathinned by a conventional ultrathin section method and floated on the water surface of a knife boat is subjected to a groove (5) of the loading tool (C) of the present invention under a stereomicroscope. Hold the annular body (2c) horizontally above the ultrathin section (9) floating on the water surface (8) of the knife boat with the bottom facing down. Then, the ultrathin section (9) is accommodated in the inner diameter of the annular body (2c), and is landed. When the loading tool (C) is pulled up vertically, the water droplet (11) adheres to the lower part of the annular body (2c). The ultrathin slice (9) floats on the water surface (8) having the inner diameter of the annular body (2c). Next, the water droplet (11) at the lower part of the annular body (2c) is gently brought into contact with the grid (10) that has been subjected to hydrophilic treatment in advance by sticking an adhesive treatment or a support film (12). The grid (10) adheres concentrically with the annular body (2c) through the water droplet (11). Then, in order not to drop the grid (10) when dried, the top and bottom are reversed and the absorbent paper (13) is pressed against the mouth portion of the groove (5) on the side surface of the annular body (2c). The water droplet (11) between the annular body (2c) and the grid (10) is absorbed by the absorbent paper (13) through the groove (5), but is floated surrounded by the inner diameter of the annular body (2c). The ultrathin section (9) does not move. When the water droplet (11) is completely absorbed by the absorbent paper (13), the ultrathin slice (9) is loaded on the grid (10).

DESCRIPTION OF SYMBOLS 1 hole 2 annular body 3 long hole 4 center belt 5 groove 6 thin shaft 7 grip handle 8 water surface 9 ultrathin section 10 grid 11 water droplet 12 support film 13 absorbent paper A conventional loading tool B conventional loading tool C loading of the present invention Ingredients

Claims (1)

An annular body (2) is formed through a circular hole (1) in the center of an ultra-thin flat disk, and a plurality of elongated holes (3) are penetrated in the same direction as the peripheral edge of the annular body (2). In addition, a gap between the long hole (3) and the long hole (3) is provided as a flat central band (4), and a concave groove crossing from the inner edge to the outer edge on the lower side of the central band (4) ( 5) A tool for loading an ultra-thin section of an electron microscope observation sample, characterized in that a fine shaft (6) is provided at a position opposite to the groove (5) across the hole (1) .

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