JPH07270286A - Method for preparing organism sample for microscope for obtaining continuous sectional image - Google Patents

Abstract

PURPOSE:To provide a method for preparing an organism sample for microscope for obtaining continuous sectional images which can easily and properly form a number of observation sample sections for continuous sectional images and can indicate a fine and continuous structure of a large organism sample. CONSTITUTION:An organism sample or a sample extracted from an organism is embedded with resin, and a number of observation sample sections 7 being adjacent in the back and forth, up and down, and left and right directions are successively formed from an embedded body 6. The surface of a part where the observation sample sections are formed out of the embedded body 6 is mechanically polished into a mirror-surface every time one observation sample section 7 is formed and then the surface of the part where mirror-surface-shaped observation sample sections are formed is exposed to plasma beams for etching, thus forming the observation sample sections 7. Firstly, the resin-embedded organism samples are polished by a machine to prepare a mirror-shaped section. Then, the mirror-shaped section is etched by the plasma beam method for performing a relief of the section in the organism soft structure in the two-step system (polishing and etching).

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 sample for a microscope.

[0002]

2. Description of the Related Art One method for knowing the one-dimensional structure of a biological sample is to obtain a microscope image of a cross section of a biological sample for a microscope cut in an imaginary section perpendicular to a specific central axis. A large number of microscopic images are sequentially created in the central axis direction, and these are arranged in the central direction using a computer or the like to obtain a continuous cross-sectional image,
Information on the 1- to 3-dimensional structure of the living body may be obtained.

The conventional method for preparing a biological sample for a microscope to obtain information on the one-dimensional structure of a biological sample is as follows: 1) With the biological tissue as it is, or with a chemical or (usually) plasma method. Etching with an ion beam or the like, and observing the surface with an optical microscope or a scanning electron microscope. 2) Embedding biological tissue in resin etc. to make thin slices, then etching by chemicals, ion beam, (normal) plasma method, etc., and observing the surface by scanning and transmission electron microscope or optical microscope (Thomas RS , “Ultrastructural loc
alization of mineral matter in bacterial spores by
microincineration ", J. Cell Biol. 23 (1964) 113.).
(Erlandsen., Thomas A. and Wendelshafer G., “A s
imple technique for correlating SEM with TEM on bi
ological tissue originally embedded in epoxy resin
for TEM ", Scanning Electron Microscopy, III (1973)
349.) (Hiroshi Sasaki, Haruo Kishida, "SEM observation of resin-embedded thick sections by ion etching", Scanning electron microscopy,
18 (1989) 43.). 3) Method of embedding resin, etc., mechanically polishing, etching (usually) by the plasma method, and then taking a replica and observing with a scanning and transmission electron microscope (Faberge AC, “III.
Development of a replica process for the electron
microscopy of biological material ", Studies in Gen
etics, IV, Research Report, Univ. of Texas (1968) 2
1.). 4) A method of observing the surface of a living tissue as it is, or after freeze-drying to obtain a replica, or after cleaving a replica and observing the surface with an optical microscope or a scanning and transmission electron microscope (Tanaka K. and Lino A., “Frose
n resin cracking method for scanning electron micro
scopy and its application to cytology ", Proc 30th
Annual Meeting Electron Microscopy Society of Amer
ica, CJ Arceneaux (ed.). Claitor´s Publishing Di
v., USA., (1972) 408.). 5) After embedding the biological tissue in resin etc., make a block section mirror surface with a microtome (thin section cutting device),
(Normal) Etching by plasma method or chemicals, and then observing with scanning electron microscope (Kuzirian AM
and Leighton SB, “Oxygen plasma etching of entir
e block faces improves the resolution and usefulne
ss of serial scanning electron microscopic image
s ", ScanningElectorn Microspocy, IV (1983) 1877.)
There is.

[0004]

However, in the method 1), the internal structure is unknown, and in the method 2), a large sample cannot be obtained because a thin piece is prepared.
The method of 3) has heat damage to the specimen, the method of 4) cannot examine the internal structure, and the method of 5) is (usually) heat damage due to plasma use, chemical damage and deformation are large, There is a problem such as.

To summarize the above problems, the conventional method is
1) When observing with a transmission electron microscope from a thin piece or after taking a replica, there is a problem in maintaining image continuity, and a large cross section cannot be observed. 2) A biological sample as it is, or ( When observing the surface by (frozen) fracturing or the like, there is a problem in relation to the internal structure, and 3) (normal) plasma etching causes large thermal damage to the sample.

The present invention has been made in view of the above circumstances, and it is possible to easily and satisfactorily form a large number of observation sample cross sections for a continuous cross-sectional image, and a fine continuous structure of a large biological sample. It is an object of the present invention to provide a method for producing a microscope sample for obtaining a continuous cross-sectional image capable of showing

[0007]

In order to achieve this object, a method of preparing a biological sample for a microscope for obtaining continuous cross-sectional images of the vertical, horizontal, or front-back (depth direction) of the present invention is a biological sample or A method for preparing a sample for a microscope, in which a sample extracted from a living body is embedded in a resin, and a plurality of observation sample cross sections that are adjacent to each other in the front-back, top-bottom, or left-right directions are sequentially formed from the embedded body,
Whenever one observation sample cross section is formed, the surface of the embedding body's observation sample cross section forming area is first mechanically polished to a mirror surface, and then the surface of the mirror-like observation sample cross section forming area is irradiated with a plasma beam. It is characterized by a two-step method (polishing and etching) in which a cross section of an observation sample is formed by etching.

[0008]

[Function] A resin-embedded biological sample is mechanically polished to form a mirror-shaped cross section. Next, the mirror-shaped cross section is etched by the plasma beam method to emboss the cross section of the biological soft structure.

[0009]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The details of the present invention will be described below with reference to the drawings showing an embodiment. As shown in FIG. 1, the method for producing a biological sample for a microscope of the present invention includes a resin embedding step 1, a mechanical polishing step 2 and an etching step 3.

The resin embedding process is a process in which the following specimen moves to the embedding body 6. In the resin embedding step 1, the optic nerve, which is the target site 5 of the study of the subject 4 such as a rat, is subjected to Os staining, etc., and then embedded in a resin such as an epoxy-based, acrylic-based, styrene-based or polyester-based resin. To form the embedded body 6. This resin embedding step 1 itself has been conventionally performed and is well known. The part 5 to be studied is a living body or a sample removed from the living body.

Next, in the mechanical polishing step 2, the embedding body 6 mechanically polishes the cross section 7 of the observation sample into a mirror surface. The polishing depth is 0.1 μm to 10 μm. For mechanical polishing, a conventionally used polishing method for soft metals (copper, aluminum) or the like can be used.

Next, in the etching step 3, the embedding body 6 exposes the target site by irradiating the cross section 7 of the observation sample with a plasma beam and etching it. Etching depth is 0.0
It is 1 μm to 1 μm. The plasma beam method is a Kaufman type method of extracting ions from plasma as a beam and irradiating the observation sample cross section of the embedded body installed elsewhere, and as a device for performing such a plasma beam method, An ion shower device (trade name, manufactured by Elionix) can be used. In the plasma beam method, only thermal energy is generated when the ion particles collide with the sample, so that the influence of heat can be minimized.

The observation sample cross section 7 thus formed is photographed by a scanning electron microscope, and the image is processed by an electronic computer 8 and then stored.

In order to obtain a continuous cross-sectional image of the sample, it is necessary to form the next cross-section of the observed sample that is continuous in the depth direction. Therefore, the embedded body 6 after being photographed by a scanning electron microscope is mechanically polished again. Return to step 2 and perform automatic polishing (0.1 μm to 10 μm
Depth), the surface of the next sample to be observed is cut out, and it is further etched in etching step 3 to form a cross section of the next sample to be observed. This process is repeated until the required number of continuous sectional images is obtained.

The mechanical polishing step 2 is 1) surface ditching (grindin
g), 2) fine grinding, 3) poli
shing), 4) Final polishing. Table 1 shows each condition.

[0016]

[Table 1]

The etching process 3 includes the following three processes. A gas such as Ar is brought into a plasma state by the ECR method. A Kauffman-type plasma beam is produced by applying a voltage (-250 V pressure) to derive plasma ions. The sample is etched by neutralizing the plasma beam.

The conventional plasma method suffers considerable heat damage because the sample is placed in a furnace in which plasma is generated. However, the above method of the present invention can be minimized because it depends only on the thermal shock of the extracted plasma ions.

The conditions of the etching process are shown in Table 2.

[0020]

[Table 2]

Experimental Example A sample cross section of a rat optic nerve was obtained under the following conditions. (1) Resin embedding / thermosetting This is basically the same as the resin embedding preparation for a transmission electron microscope. -Embedding resin (of Table 3, ...) is mixed well with a stirrer (stirring bar), and after 15 minutes, DMP30 (of Table 3) is dripped and mixed well. -After injecting this into a polyethylene container having an inner diameter of 25 mmφ and a depth of 20 mmd, support the optic nerve with a thin wire or the like so that the X shape is vertical (vertical), and fix it to the bottom surface. This is to facilitate the “leveling” of the cross section of the nerve bundle when mechanically polishing the resin sample. Epon mixture (50%) + propylene oxide (5
Shake at 0% overnight. -Heat cure at 45 ° C for 12 hours and 60 ° C for 36 hours in a constant temperature bath, and remove from the container.

[0022]

[Table 3]

(2) Polishing This is basically the same as the polishing method for soft metals. The polishing length can be controlled to be 0.1 μm or less while it is installed in the sample rotating machine, but an error when re-polishing, that is, when the nerve sample is re-installed is unknown, and is preferably 1 μm or less. -The device (Struers, Denmark) is equipped with a sample rotating machine (semi-automatic) on a rotary polishing table.・ 45m between the center of rotation of the rotary polishing table and that of the sample rotator
The same rotation speed of 150 rp in the same direction
Turn around m. Therefore, at an arbitrary position between the sample surface attached to the sample rotating machine and the rotary polishing table, a frictional force of the same magnitude in the direction perpendicular to the sample surface is applied to the sample surface. Therefore, the force in all directions is uniformly applied during one rotation of the sample rotating machine, which results in ideal polishing. Insert the sample resin (1 piece) and the balancing resin (2 pieces, dummy) created by the above procedure into the sample embedding plate (part of the sample rotating machine, 160 mmφ diameter, maximum 6 holes). A pressure of 100 N is applied to these three resin surfaces,

[Equation 1] Then, the weight becomes 6.7 g weight / mm ² .・ Grinding: Because epoxy resin is soft,
While adding (cooling) water, polishing is started using 1000 (FEPA particle size) abrasive paper (water resistant, SiC) to expose the nerve cross section (see Table 4). -Fine grinding: 2400 (FEPA grain size) → 4000 Grit While adding water, the polishing paper is replaced and polished at 1 minute intervals. Polishing: Polishing cloth (wool) with ethanol lubrication / coolant (lubricant blue) is sprayed with diamond spray (DP, particle size 3 μm) for 1 minute. Similarly, the polishing cloth is replaced and polishing is performed again with DP spray having a particle size of 1 μm.・ Final polishing: Polishing cloth (velvet)
A colloidal suspension (water + silica, particle size 0.04 μm) is added to, and the nerve preparation is ultrasonically cleaned, and then subjected to finishing polishing for 1 minute.

[0024]

[Table 4]

(3) Kaufman type plasma beam etching Ar flow rate: 0.5 sccm, accelerating voltage: 0.75 k
V, ion flow rate: 0.1 mA, irradiation time: 5 minutes. The result is shown in FIG. FIG. 2A: Scanning electron microscope section embedded in resin and mechanically polished. The unevenness is emphasized in terms of signals. FIG. 2B: Scanning electron microscope cross section etched by the plasma beam method. One ring pattern is a myelin sheath surrounding a single nerve.

[0026]

According to the method for producing a biological sample for a microscope for obtaining a continuous cross-sectional image of the present invention, particularly in the etching step, it depends only on the impact of plasma ions extracted from the inside of the furnace where plasma is generated. Since etching is performed, a wide mirror-like good living body cross-sectional structure can be obtained with minimum heat damage. Furthermore, by combining the three techniques of the sample preparation according to the method of the present invention, the scanning electron microscope continuous cross-sectional image and the computer, the fine three-dimensional structure of the living body can be reconstructed as an image.

[Brief description of drawings]

FIG. 1 is an explanatory diagram showing steps included in a method for producing a biological sample for a microscope to obtain a continuous cross-sectional image of the present invention.

FIG. 2 is a micrograph of a cross section of an observed sample.

[Explanation of symbols]

 1 Resin Embedding Process 2 Mechanical Polishing Process 3 Etching Process 4 Specimen 5 Target Area of Research 6 Embedded Body 7 Cross-section of Specimen 8 Electronic Computer

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location G01N 1/32 B G01N 1/28 R

Claims (2)

[Claims] 1. A method for preparing a microscope sample in which a biological sample or a sample excised from a biological body is embedded in a resin, and a plurality of adjacent observation sample cross sections are sequentially formed from the embedded body, which is one observation method. Each time a sample cross section is formed, the surface of the observation sample cross section forming portion of the embedded body is mechanically polished to a mirror surface,
Next, a biological sample for a microscope for obtaining a continuous cross-sectional image, characterized in that the surface of the mirror-shaped observation sample cross-section forming portion is irradiated with a plasma beam and etched to form the observation sample cross-section. Method. 2. The method for producing a biological sample for a microscope according to claim 1, wherein the sample for a microscope is a sample for a scanning electron microscope.