JPH10206301A - Method for making biological specimen for observation by electron microscope, and biological specimen card - Google Patents

Abstract

PROBLEM TO BE SOLVED: To appropriately cut a biological specimen part by preparing a sliced piece, holding the piece in the embedding process with a synthetic resin sheet to be polymerization hardened, and providing the adhesion sealing with a pouch film to simplify the embedding operation. SOLUTION: A biological specimen is sliced into a sliced piece 2 of 50-100μm in thickness, and then, fixed, dehydrated, and substituted. The substituted sliced piece 2 is immersed in the embedding resin solution 1 so as to be penetrated. The sliced piece 2 wet with the resin solution 2 is held with two synthetic resin films 4, and the resin solution 2 is polymerization hardened. The sandwiched piece is adhesion sealed in a card shape with a pouch film 5. A part to be observed is selected from the card by a projector, etc., punched and taken out, and the films 4, 5 are peeled from the sliced piece 2. The sliced piece 2 is adhered to a synthetic resin pedestal, and then, super-sliced and dyed, and examined with a transmission type microscope. Selection of the target part is facilitated, and the applied observation at the electron microscope level is facilitated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for preparing a biological specimen for observation with an electron microscope from a biological sample collected from a living body for conducting an electron microscopic search, and a biological specimen holding card.

[0002]

2. Description of the Related Art In order to determine whether the nature of a tumor removed by surgery, biopsy or the like is a malignant tumor (cancer, sarcoma) or a benign tumor, the tumor is referred to a pathological search.

As a pathological search method, (1) macroscopic search (2) optical microscopic search (= histological search) 50-1000 times (3) electron microscopic search (= cytological search) 1000 times -100,000 times.

[0004] Light microscopy (a method in which a tissue section embedded in paraffin is sliced into sections having a thickness of 2.5 µ to 5.0 µ and stained for examination) occupies the mainstream of pathological search. ing. In other words, the nature of the tumor (malignant or benign?) If it is malignant, is it cancer or sarcoma? Then, it is searched whether tumor cells have invaded the blood vessels or lymph vessels, etc. to determine the range of removal of organs at the time of surgery, and to determine the subsequent treatment policy (radiation therapy, chemotherapy, immunology Is determined. However, when it is difficult to determine the character of the tumor in the light microscopic search, the determination is left to (3) electron microscopic search as the next search. That is, a search at the cell level is performed. At this time, the nature of the tumor is clarified by observing the presence of special fibers and neuronal secretory granules that could not be confirmed by an optical microscope.

Next, a conventional method for preparing a specimen for observation with an electron microscope will be described.

[0006] The preparation of a specimen from a biopsy for observation with an electron microscope is as follows: (1) Tissue excision → (2) Fixation → (3) → Dehydration → (4)
Substitution → (5) → embedding → (6) ultra-slicing → (7) staining →
(8) This is performed according to the procedure of a speculum. (Refer to Fujita Planning Publishing Co., Ltd. “Detailed Explanation of Pathological Techniques”, pp. 2-47) (1) Excision of a tissue piece: The tissue to be extirpated is thin, small, and normally 1 mm in thickness under an electron microscope to improve the penetration of fixative.
To cut, to shredded in ^triangular 1mm.

(2) Fixation: In order to preserve tissues and cells in a state close to that of a living body, protein component molecules are stabilized and insolubilized to prevent migration and diffusion of substances inside and outside the cells and to improve chromaticity. The tissue is fixed with a fixing solution such as osmium.

(3) Dehydration: Since epoxy resin, polyester resin and the like are water-insoluble embedding agents, they are usually dehydrated using ethanol or acetone in order to remove water in tissues.

(4) Substitution: Since ethanol is hardly compatible with an epoxy resin, a polyester resin, and a methacryl resin as an embedding resin, it is substituted with a substitution agent such as propylene oxide which has an affinity for both a dehydrating agent and an embedding resin. .

(5) Embedding: An embedding resin solution (for example, EPON 812; Shell Chem) in which an embedding resin such as an epoxy resin is penetrated into a tissue and injected into a gelatin capsule or a polyethylene capsule (8 mm diameter).
ical CO. USA).

(6) Ultra-slicing: Slicing is performed to a thickness of 0.08 to 0.1 μm using a microtome for an electron microscope.

(7) Staining: Use of heavy metals such as uranium acetate and lead citrate to utilize transmitted electrons in an electron microscope.
Observe by staining the carbohydrates.

[0013] The diameter 8mm a biopsy sample shredded Usually 1 mm ³ square as shown in the above-described procedure contains the embedding resin solution
It is embedded in a gelatin capsule or polyethylene capsule.

As described above, a method for embedding a tissue piece in a gelatin capsule or a polyethylene capsule is described in Luft.
Since the embedding method using epoxy resin was established by (1961) et al., The method is still widely used in the electron microscope embedding method. However, the skin, myocardium,
In many tissues such as the renal cortex and gastrointestinal mucosa, it is necessary to pay attention to the directionality. Further, the 1 mm ³ squares limit the range of the amount of information.

That is, since the tissue piece is sliced into 1 mm × ³ mm pieces, it is difficult to know the condition of the surrounding area of the tissue, and it may not be essentially suitable for the area to be observed with an electron microscope. In addition, it is necessary to slice a 1 mm ^3- sided biopsy sample hardened in a gelatin capsule in various orientations.
It takes time and skill. In addition, since the embedding position of the biopsy sample in the gelatin capsule is different for each sample, it takes a lot of time and labor to ultra-slice and cut out the sample.

[0016]

SUMMARY OF THE INVENTION An object of the present invention is to overcome the above-mentioned problems, to simplify the embedding operation, and to cut out a portion of a biopsy sample to be appropriately observed with an electron microscope without skill. It is an object of the present invention to provide a biological specimen preparation method and a biological specimen holding card which can perform the above.

[0017]

According to the present invention, according to the present invention, a biological sample is sliced in a fixing solution to a thickness of 50 to 100 μm to form a sliced section, which is fixed as conventionally known. After dehydration, replacement, and infiltration into the embedding resin solution, the sliced section in which the embedding resin has penetrated is sandwiched between sandwiches with a transparent synthetic resin sheet, and the embedding resin is polymerized and cured. The problem is solved by adhesively sealing the structure with a pouch film. Furthermore, according to the present invention, a thin slice section cut out from a biological sample to a thickness of 50 to 100 μm is fixed, dehydrated, replaced, and after penetrating an embedding resin agent, sandwiched between sandwiches with a transparent synthetic resin sheet. We propose a biological specimen card for electron microscopy observation, which is obtained by bonding and sealing a polymerized and cured structure with a pouch film.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION

Excision and fixation of tissue pieces Organs obtained from humans and various animals, such as kidneys, are subjected to 50-100 μm with a microslicer in a fixative solution at the maximum cutting plane.
Cut into thin slices of m thickness.

The sliced slices are fixed in a fixing solution for an electron microscope, for example, a 2.5% glutaraldehyde / buffer solution in a known manner.

Dehydration The slices fixed as known are dehydrated with alcohol or acetone.

Replacement Known dehydrated slices are replaced, for example, with propylene oxide.

Embedding The sliced section, which has been replaced in a known manner, is converted into an embedding resin solution, for example, an epoxy resin solution, using EPON 812 (Shell).
Chemical CO. USA) or TAAB812
(TABB Chem. CO. USA) or Polybe
d812 (Polyscience CO. USA) or Quetol 812 (Nissin EM Japan) or EPOK
812 (Oken Shoji Japan) as DDSA as a curing agent
(Dodecenyl succinic anhyd
ride) or MNA (methyl nadic a)
immersion in a mixed solution obtained by mixing DMP-30 as an accelerator.

FIG. 1 schematically shows this process. In the figure, 1 is an embedding resin liquid, 2 is a replaced sliced section, and 3 is a beaker. At this stage, the embedding resin liquid is viscous and has fluidity. In this immersion state, the embedding resin is well penetrated into the sliced section 2.

In the conventional steps of fixing, dehydrating, replacing, and infiltrating the embedded resin, shaking is performed using a shaker.

Next, as shown in FIG. 2, the thin slice section wet with the embedding resin liquid removed from the beaker is sandwiched between two transparent synthetic resin films 4, for example, two 1.5 mm thick vinyl sheets.

As a synthetic resin sheet, ACLAR Em is used.
Beding Film (Allidesignal
Inc. USA) was preferred. This synthetic resin film is preferably transparent without water absorption, flame-retardant, and resistant to osmium tetroxide, ethanol, acetone propylene oxide and the like. In the sandwich structure shown in FIG. 2, the embedding resin is polymerized and cured in a thermostat.

The sandwich piece embedded and polymerized and cured is adhesively sealed with a pouch film (Meiko Shokai) 5 as shown in FIG. The pouch film is a film adhered on a photograph or the like to prevent forgery of a passport, and has a sealant layer on one surface of a biaxially oriented polyethylene terephthalate, a biaxially oriented polypropylene, or a biaxially oriented nylon sheet.

The adhesive sealing with the pouch film is performed in such a state that the side surface of the sandwich structure in a cured state is in contact with air and the transparent synthetic resin film 4 is easily peeled from the cured embedded resin. When the peeling of the transparent synthetic resin film from the embedding resin is performed while protecting the transparent synthetic resin film 4 from the embedding resin and preventing the transparent synthetic resin film 4 from peeling from the embedding resin while preventing the side surface of the sandwich structure from contacting the air. Second, it is for peeling off integrally with the pouch film.

FIG. 4 is a plan view of the structure sealed and sealed by the pouch film 5 as described above. A target site where the pouch film adhesive seal structure shown in FIG. 4A is desired to be observed with a projector or an optical microscope is selected, and the target site indicated by a solid line circle 6 is punched out with, for example, punch scissors and taken out. The diameter of the punched portion is, for example, 3 to 5 mm. The punched portion is shown in an enlarged manner in a sectional view.

The punched portion of the pouch film 5 and the transparent synthetic resin film adhered thereto are peeled off from the embedded resin thin slice.

The thin section thus embedded is cut into a synthetic resin pedestal having a diameter of, for example, 8 mm (preferably a pedestal made of the same resin as the embedding resin, such as an epoxy resin-based EPON).
For example, as shown in FIG. 5, a thickness of 0.08 to 0.08 to be suitable for observation with an electron microscope.
Ultra-slice to 0.1 μm.

The ultra-thin sliced sections are stained electronically (double staining of uranium acetate and citric acid) and then examined with a transmission electron microscope.

When the above-mentioned structure obtained by bonding the pouch film is selected to have a card size that can be projected by a 35 mm projector as shown in FIG. 6A, it is very difficult to select a site to be observed with an electron microscope. It will be easier.

As shown in FIG. 6B, for example, as a card size such as a telephone card, a marking such as a bar code is printed for classification and storage, and the date and time, patient name, disease name, organ name, and family name are displayed. Providing an area for recording names and the like greatly simplifies the handling process in searching for an electron microscope.

[0035]

According to the present invention, a tissue-embedded piece is formed on a card covered with a pouch film, so that it can be projected under an optical microscope and projected on a projector, and the target site can be selected in a conventional gelatin capsule or polyethylene capsule. This can be easily performed with a remarkable difference compared to the information amount obtained from 1 mm ³ corners by embedding.

Further, as a derivative effect, (1) the position of a lesion such as an infarct or bleeding site can be grasped and the site can be observed.

(2) A large number of targets such as amyloid can be selected and observed on a tissue piece.

(3) In a tumor, the tumor itself can be compared with a normal tissue site and a boundary site on the same plane.

Application observation at the level of an electron microscope can be easily performed.

[Brief description of the drawings]

FIG. 1 is a schematic side view showing a process of permeating an embedded resin into a thin slice.

FIG. 2 is a schematic cross-sectional view showing a process of polymerizing and curing thin slices impregnated with an embedded resin sandwiched between transparent synthetic resin sheets.

FIG. 3 is a schematic cross-sectional view of a process in which a pouch film is bonded and sealed to the sandwich composition that has been polymerized and cured in FIG. 2;

FIG. 4 is a schematic plan view showing a place where a part of a thin slice covered with the pouch film prepared in FIG. 3 is extracted by punching.

FIG. 5 is a schematic perspective view showing a state where thin slices extracted by punching are attached to a base.

FIG. 6 is a schematic plan view showing a biological specimen card according to the present invention in a card shape.

Claims (2)

[Claims] 1. A biological sample in a fixative solution of 50 to 100 μm
Creating a sliced section by slicing to a thickness of
Fixing, dehydrating, and substituting the sliced section; immersing the substituted sliced section in an embedding resin solution to allow the embedding resin to penetrate into the sliced section tissue; and Sandwiching the sliced slices in a sandwich between transparent synthetic resin sheets to polymerize and cure the embedding resin, and bonding and sealing a pouch film to the polymerized and cured embedded slices sandwiched between the sandwiches A method for preparing a biological specimen for electron microscopic observation, comprising the steps of: punching out an electron microscopic observation site of an embedded thin slice section covered with the pouch film by punching. 2. A thin slice cut out from a biological sample to a thickness of 50 to 100 μm is fixed, dehydrated, replaced, penetrated with an embedding resin agent, and then sandwiched between sandwiches with a transparent synthetic resin sheet. A biospecimen card for electron microscope observation, wherein the polymerized and cured structure is bonded and sealed with a pouch film.