JP2686398B2 - Method for preparing sample for transmission electron microscope observation - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[Field of Industrial Application] The present invention relates to tissue (animal and plant) of a living body
The present invention relates to a method for preparing a transmission electron microscope observing sample useful for observing the morphology, ultrafine structure, etc. of a polymer at an arbitrary site of a cell or a polymer material by a transmission electron microscope. More specifically, the type of material, creation of a particular independent of the presence or absence of functional groups, that Ru gives a clear contrast to any structure-polymer present in the sample, which can be observed by a transmission electron microscope sample Is the way.

[0002]

2. Description of the Related Art Conventionally, several methods described below are known for observing macromolecules and ultrafine structures in tissues and cells of living bodies and macromolecular materials with an electron microscope. There are also many. For example, ultrathin sectioning, scanning electron microscopy,
Freeze replica method, in such shadowing method, Ru impossible der that there is a limit to the resolution to fully reveal the hyperfine structure of the polymer morphology and organization.

In observation with a transmission electron microscope using an ultra-thin section method, which is frequently used in the field of biology, it is necessary to fix and stain the tissue in advance. It is often unobservable because the fine structure is not fixed and dyed sufficiently. On the other hand, there is a negative staining method as a method of knowing the morphology and ultrastructure of a polymer or virus, but this requires the use of a material in which an observation target is purified to be almost pure.

[0004]

Therefore, for this reason, a polymer or ultrafine structure that exists in a specific site in a tissue / cell or a mixture of a plurality of polymer substances, or is expected to exist is left as it is. Observing the condition is often not possible with previously known methods alone. Therefore, the inventor of the present invention does not depend on the type of substance or the presence or absence of a specific functional group, has a wide range of application, and has a macromolecule or an ultrafine structure of a tissue or cell of a living body or an arbitrary site in a polymer material as it is. As a result of conducting a study on a method of knowing in this state, the present invention has been achieved.

[0005]

[Means for Solving the Problems] That is,
It is characterized by fixing and embedding samples such as cells and polymer materials to make ultrathin sections, then completely or partially deembedding, and then negatively staining the entire material with a negative staining agent. And a method for preparing a sample for transmission electron microscope observation.

[0006] More specifically, after fixing a sample of a tissue, cell or polymer material of a living body and embedding it in an epoxy resin to prepare an ultrathin section, it is placed on a metal mesh coated with a carbon film,
The epoxy resin is completely or partially de-embedded with an alkaline alcohol solvent, and then the whole material is
After hydrophilic treatment, only the ultrathin section surface of the sample is negatively stained
Then , the black color of the background of the sample is increased to observe the morphology of the polymer and the ultrafine structure of the tissue at an arbitrary site.

The fixing and embedding of the sample referred to here is to fix the tissue and the like with a fixing agent and embed and embed this in an epoxy resin as in the case of conventional transmission electron microscope samples. is there. The ultra-thin section for an electron microscope is to slice this fixed and embedded sample with an ultramicrotome to make an ultra-thin section.

[0008] Datsutsutsumiuma is embedding agent operation for removing such a resin from the ultra-thin section, metal main strung mosquitoes Bon film
Ash, for example alkaline copper
The resin is melted out with a solvent and completely or partially removed.

The remaining sample on the membrane was then contacted with water.
Hydrophilic treatment with a negative stain on the sample surface ,
For example, phosphotungstic acid increases the black color of the background of the sample to raise the sample. At this time, both sides of the membrane
When the both surfaces are dyed, the resolution of the ultrafine structure decreases. Trial
The drop method is preferred for dyeing only one side of the material.

[0010]

[Action] by the fixed-embedding, the sample such as tissues and cells in vivo truth can be solid wear without purification. Usually, an ultrathin section is directly observed under an electron microscope as it is, but in that case, the resolution is insufficient.

Since only one side of the test piece which is completely or partially deembedded in the ultrathin section is negatively stained, the black color of the background is increased, and therefore, the in-vivo macromolecule is left in its natural state without purification. It enables observation of polymer morphology and ultrafine structure with excellent resolution.

[0012]

Embodiments will be described in detail below with reference to embodiments. Example 1 Kidney tissue was collected by renal biopsy of a patient with nephrotic syndrome,
After fixing glutaraldehyde and osmic acid by a conventional method, it was embedded in Epon 812 (epoxy resin, manufactured by TAAB, England). After the ultra-thin sections by which the ultramicrotome, placed on a copper mesh stretched carbon film, 3 hours immersed partially in the liquid diluted 10-fold by the liquid to use immediately before ethanol dissolved sodium hydroxide 4g in ethanol 100ml
It was Datsutsutsumiuma to. Then, the mesh was suspended in distilled water for 15 minutes with the surface on which the tissue was placed as a lower part to be hydrophilically treated, and then 1 drop of 1% phosphotungstic acid aqueous solution was dropped on the tissue surface of the mesh. After absorbing the aqueous solution, the renal tissue of the nephrotic syndrome patient was observed by a transmission electron microscope according to the method of the present invention. The result is shown in FIG.

FIG. 1 shows the ultrafine structure of the glomerular basement membrane of renal tissue. The fine fibers forming the mesh structure have a diameter of about 1.9 nm, the fine fibers disappear at the arrowheads, and the nephrosis has a diameter of about 50 nm. It had formed a tunnel. It was the first time in the world that such a nephrotic tunnel was proved, and it is a lesion that is thought to cause proteinuria in patients with nephrotic syndrome. The present invention enables such observation.
Was. Note that the magnification in FIG. 1 is 100,000 times.

On the other hand, when the above ultrathin section was observed under a microscope as it was, the resolution was insufficient as shown in FIG. 2, and the details of the network structure of the glomerular basement membrane (G) could not be read at all. 50,000 times magnification.

Example 2 A sample was prepared from a normal human kidney biopsy material by the same method as in Example 1 and the glomeruli were observed. As shown in FIG. 3, the hemoglobin molecule of capillary erythrocyte (R) was clear. Was observed. From this, the shape and sequence of the hemoglobin molecule could be identified. 50,000 times magnification. FIG. 4 is a photomicrograph of a conventional ultrathin section method. Erythrocyte cut hemoglobin molecules in the (R) is difficulty considered much larger molecule itself than the estimated value
Was . 50,000 times magnification.

Example 3 When a sample was prepared from a renal biopsy material of a patient with nephritis by the same method as in Example 1 and the interstitium of renal tissue was observed, it was as shown in FIG. 5, and the collagen fiber striped pattern was clear. Was observed.
This revealed the ultrastructure of collagen fibers. Magnification 100,000 times. Figure 6 is a photomicrograph of the conventional ultramicrotomy, or ultrastructure of the collagen fibers, such less clear
Was . Magnification 100,000 times.

[0017]

As described above, according to the present invention, a method for creating a transmission electron microscope observation samples of the present invention, is not to be dependent on the presence or absence of the type and specific functional groups of the substance, the application range is wide, The biological It has become possible to know the polymer and ultrastructure of any part of the tissue / cell or polymer material.

In particular, it can be used for observing and analyzing the morphology of macromolecules in the living body, which are difficult to take out and purify, as they are, and greatly contribute to the discovery of new facts. Is.

[Brief description of the drawings]

FIG. 1 is a transmission electron micrograph of the glomerular basement membrane of the renal tissue of a patient with nephrotic syndrome according to the method of the present invention (× 1
0,000 times).

FIG. 2 is an electron micrograph by a conventional ultrathin section method.
(× 50,000 times).

FIG. 3 is a transmission electron micrograph by a method of the present invention of a hemoglobin molecule in erythrocytes in capillaries of glomeruli of a normal human kidney biopsy material (× 50,000).

FIG. 4 is an electron micrograph by a conventional ultrathin section method.
(× 50,000 times).

FIG. 5 is a transmission electron micrograph (× 100,000) of interstitial collagen fibers of renal tissue from renal biopsy material of a patient with nephritis according to the method of the present invention.

FIG. 6 is an electron micrograph by a conventional ultrathin section method.
(× 100,000 times).

Continued front page (56) References Open 61-202050 (JP, U) Open 63-35940 (JP, U) Open 58-132870 (JP, U) Japanese Patent Publication 2-25447 (JP , B2) Japanese Patent Publication Sho 64-4148 (JP, B2) Terutaro Nakamura, Ichiro Nakata Responsible Editing “Experimental Physics Course 13 Preparation and Processing of Samples” First Edition (56-7-10) Kyoritsu Edition P. ． 711-713

Claims (1)

(57) [Claims] 1. An ultrathin section is prepared by fixing and embedding a sample of biological tissue, cells, polymer material, etc. in an epoxy resin, and then placing it on a metal mesh coated with a carbon film to make the epoxy resin alkaline. Complete or partial de-embedding with alcohol solvent and then hydrophilic treatment of the whole material
After that only the sample Ultrathin sections surface by negative staining, for transmission electron microscopy by increasing the black sample background, characterized in that to allow observation of the hyperfine structure of the polymer morphology and tissue How to make a sample.