JP2003004606A - Method for selecting thin slice suitable as tissue specimen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for selecting a thin slice suitable as a tissue specimen, adapted to a method for forming the tissue specimen certainly observed and capable of being quantitatively analyzed by an image analyzer. SOLUTION: The tissue specimen free from dyeing irregularity can be formed by measuring the exposure time of the thin slice before the tissue specimen is formed by the use of a biological microscope and the exposure meter of a photographic device to select the thin slice of a constant range of an exposure time as the tissue specimen. This tissue specimen can be certainly observed and quantitatively analyzed by the image analyzer to contribute to the enhancement of work efficiency.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for selecting a suitable thin section for producing a useful tissue specimen in a wide field of histology and pathological diagnosis.

[0002]

2. Description of the Related Art A tissue specimen is prepared by first slicing a tissue or the like into thin slices and staining the slices. Then, it is evaluated by observing the sample or performing a quantitative analysis by an image analyzer. At that time, in particular, the production of thin tissue slices of uniform thickness is an important point in the production of a tissue specimen. The adjustment of the thickness of the thin slice during the production of the tissue slice is controlled by the feed lever or the fine movement device of the microtome, and when multiple thin slices with the same thickness are produced, the scale of the feed lever or the fine movement device is set to It is done according to the thickness of.

[0003]

However, paraffin blocks, frozen blocks, resin blocks, etc. used for thin sectioning of tissue are subtly changed by cooling or excessive humidity, and the thin sectioning process is performed. Since it is also affected by the temperature and humidity at the time, there is always a slight difference in the thickness of the section. Therefore, it is very difficult to produce thin slices of uniform thickness when thinning,
Such delicate adjustment of the thickness cannot be controlled by the above-mentioned device attached to the microtome, and can only be adjusted by the operator's intuition. Further, it is impossible to visually measure a subtle difference in the thickness of the sliced slices, and it is more difficult to directly measure each sliced slice. On the other hand, when a tissue sample is prepared using thin slices having irregular thicknesses, the difference in thickness appears as the difference in density such as staining of the sample. Therefore, the difference in staining property between specimens of the same test and the difference in thickness of the thin section greatly hinder reliable specimen observation and quantitative analysis with an image analyzer. As a result, work efficiency such as re-manufacturing thin slices is reduced. Therefore, if a thin section suitable as a tissue sample can be easily selected from the thin sections obtained by a general sectioning method, when a thin section obtained by the selection is used to prepare a tissue sample, variations will occur. It is possible to prepare a tissue specimen without a tissue.

[0004]

In order to solve the above-mentioned problems, the inventors of the present invention have made diligent studies and as a result, as a result, when selecting a suitable thin section as a tissue sample having a uniform thickness, a biological microscope and a photographic apparatus are selected. The exposure time of each thin section is measured using the exposure meter of and the thin section within the range of the exposure time of a certain width is used as a sample, so that it is easy to observe the sample, the quality of the micrograph and the image analyzer. The present inventors have completed the present invention by finding that it is possible to produce a tissue sample with improved efficiency of sample evaluation such as quantitative analysis.
That is, the present invention is as follows. (1) A method for selecting a thin section suitable as a tissue sample. (2) The selection method according to (1), wherein the selection method uses a biological microscope and an exposure meter of a photographic device. (3) The selection method is such that the exposure time of a thin section is measured using a biological microscope and an exposure meter of a photographing device, and a thin section suitable as a tissue sample is selected according to the exposure time. ) The selection method described. (4) The selection method according to (3), wherein a thin section having an exposure time width within a range of 0.06 seconds is selected as a suitable thin section as a tissue sample. (5) The selection method according to (3), wherein a thin section having an exposure time width within the range of 0.05 seconds is selected as a suitable thin section as a tissue sample. (6) The selection method according to (3), wherein a thin section having an exposure time width within a range of 0.04 seconds is selected as a suitable thin section as a tissue sample.

According to the present invention, (a) it is possible to quantify a thin section having a delicate thickness, which was difficult to measure visually, as an exposure time,
(B) By measuring the exposure time of the section before staining, it is possible to estimate the shade of the section to some extent without using advanced technology, and it is possible to easily prepare a specimen with uniform shade of stainability. (C) By making a tissue sample with low density of staining, the visibility of the sample is improved, the quality of the micrograph and the efficiency of sample evaluation in quantitative analysis with an image analyzer are improved, and the work efficiency is improved from the above. Can contribute to the improvement of.

(Types of Tissue Specimens Usable for the Present Invention) The present invention can be applied to all potential tissue specimens in the field of pathology, but in general, uniform thin sections can be obtained. It is used when preparing tissue samples of organs that are difficult to obtain. Particularly, it is particularly useful in thin sections of tissue samples of organs such as liver, brain, spleen, heart, lung, kidney, pancreas and digestive tract of human or laboratory animals.

(Tissue thin sectioning method usable in the present invention) The tissue thin section usable in the present invention is not particularly limited as long as it is obtained by an ordinary method. Methods such as paraffin embedding and resin embedding,
Or paraffin block and resin block obtained by freezing using an embedding medium for frozen sections, or a method for thin sectioning a frozen block usually, for example, sliding microsomes, rotary microtome, cryostat and ultramicrotome. The thin slice obtained by the method is subjected to the selection method of the present invention.

(Method for preparing specimen) The thin section in the present invention is made into a tissue specimen by an ordinary method. For example, hematoxylin / eosin staining (HE staining), special staining and immunostaining (reference: " All '' (MEDICAL TECHNOLOGY
Tissue specimens can be obtained by various staining methods such as a separate volume, Ito Denshaku Publishing Co., Ltd.)).

(Types of Biological Microscope Usable in the Present Invention) The biological microscope used in the present invention may be any biological microscope that is actually used when observing a tissue specimen. However, preferably, it means a transmission optical microscope, and includes an upright microscope and an inverted microscope, but is not limited to the classification of the type. Specifically, ECLIPSE
For example, E800 (Nikon).

(Type of exposure meter that can be used in the present invention) Further, the exposure meter of the photographic apparatus used in the present invention may be any one as long as it can measure the exposure time at the time of photographing, but is preferable. Is only required to be capable of displaying with an accuracy of 1/100 second or more, and is specifically U-III (Nikon) or the like.

(Combining Method of Microscope and Exposure Meter Usable in the Present Invention) In the present invention, the biological microscope and the exposure meter of the photographic device are simply combined as in the case of ordinary tissue sample observation or sample photography. Therefore, no special combination is required. A suitable thin section can be selected as a tissue sample by measuring the exposure time of the thin section set on the stage of the microscope before making the tissue sample and selecting the thin section whose exposure time is within a certain range. .

(Calculation method of exposure time) Turn on the main power source of the biological microscope and the photographic device, and attach it to the slide glass.
One piece of the dried thin section before dyeing is placed on the stage of the microscope, and the light source is adjusted by the dimming dial of the optical microscope while observing the exposure meter of the photographing device. The appropriate amount of light source here means the amount of light source within the automatic exposure interlocking range. Specifically, it is desirable to select AUTO as the shooting mode and set the metering mode to average metering at the center 35% of the 35 mm film shooting range. At this time, it is desirable that the objective lens used has a low magnification, but since it varies depending on the sample, an appropriate magnification is used as needed. The exposure time of the central part of the section is measured with an exposure meter, and the exposure time of the central part of another section is measured under the same conditions.

In the present invention, it is preferable to use a thin section having an exposure time within a certain range in order to prepare a suitable tissue sample. The exposure time depends on the type of tissue, the type of tissue sample, etc. It can be appropriately selected. However, in order to select a suitable thin section as the tissue sample of the present invention, the width of the exposure time is within the range of 0.06 seconds, preferably within the range of 0.05 seconds, more preferably 0.04 seconds.
It is preferred to use thin sections within the range of seconds. By using the thin section selected according to the present invention by using these methods, it is possible to reduce variations in staining property, and to prepare a tissue sample that can be observed with certainty and quantitative analysis can be performed with an image analyzer. Therefore, the work efficiency can be improved.

[0014]

The present invention will be described below with reference to experimental examples and examples, but the present invention is not limited to these.

Experimental Example 1 (Method for embedding paraffin in tissue) According to a conventional method, a rat liver fixed with 10% neutral buffered formalin was used as a closed automatic fixed embedding device ETP-1208 (Sakura Seiki) and a paraffin block. Paraffin block was manufactured using Tissue Tech III (Miles Sankyo).

Experimental Example 2: (Method for preparing thin section with sliding microtome) The paraffin block prepared in Experimental example 1 was attached to the sample stand of sliding microtome IVS-400 (Sakura Seiki), and the thin section was rough-cut. A paratome section was sliced by adjusting the scale of the microtome to a thickness of 4 μm.

Experimental Example 3: (Measurement of exposure time of thin section) Optical microscope ECLIPSE E800M
(Nikon) and microphotographer U-III (Nikon) are turned on, and the thin section before staining attached to a slide glass and dried is placed on the stage of an optical microscope, and the objective lens is set to × 1. Exposure time of the center area is metering mode: 3
It was measured under the average photometric condition of 35% in the center of the 5 mm film photographing range.

Experimental Example 4: (Staining method for thin section: HE staining method) The paraffin section prepared in Experimental Example 2 was subjected to HE staining by a conventional method (reference: "all of the staining methods" (MEDICAL TECHNOLOGY separate volume, dentistry and medicine publication) Ltd.)). At this time, staining of a plurality of thin sections was performed strictly under the same conditions.

Experimental Example 5: (Evaluation Method of Tissue Specimen: Observation of HE Staining, Quantitative Analysis with Image Analyzer) The HE staining specimen was examined with a naked eye and a biological microscope to confirm the identity of staining.
In addition, the HE-stained specimen placed on the microscope stage is input as a color image to the image analyzer Luzex-F (Nireco) through the color chilled 3CCD camera C5810-01 (HAMAMATSU), and the image is processed by grayscale image processing. Quantitative analysis was performed by expressing the light and shade composition of R as a numerical value of the concentration histogram of R (red), G (green), and B (blue).

Example 1: (Exposure time of thin section prepared with sliding microtome as 4 μm, HE stained sample) Exposure of 20 thin sections prepared by cutting with sliding microtome as 4 μm according to Experimental Example 2 The time was measured by the method of Experimental Example 3. The result is shown in FIG. The 20 thin slices were HE-stained by the method of Experimental Example 4 to prepare specimens. The longest exposure time (0.58 seconds) and the shortest exposure time (0.37 seconds)
The state of the HE-stained sample prepared from the thin section (sec.) Is shown in FIGS. 2 and 3. Specimens with a long exposure time also stain HE stain deeply (Fig. 2), and specimens with a short exposure time stain HE stain lightly (Fig. 3).

Example 2: (Quantitative analysis of HE-stained sample by image analyzer:
Quantification of G (green)) The HE-stained sample obtained in Example 1 was subjected to quantitative analysis of image contrast with an image analyzer according to the method of Experimental Example 5. The shade of the HE-stained specimen is
Among the density histograms of density components R (red), G (green), and B (blue), G (green) is dominant. Therefore, fix the values of R (red) and B (blue) at 255,
The G (green) value of each sample was measured as a shade index. The result is shown in FIG. The value of G (green) was higher in the sections with shorter exposure time (lighter staining), and lower in the section with longer exposure time (darker staining). The exposure time is 0.4
The value of G (green) reached a ceiling for samples of 8 seconds or longer. The average value of G (green) values of each HE-stained sample (20 sheets) was “143”.

Experimental Example 3: (Method for determining optimum exposure time) In a quantitative analysis of a tissue sample with an image analyzer, a thin section having the same thickness and a sample having the same area stained under the same conditions were
Theoretically, the same area value is obtained when the density histogram is set to a constant value during the grayscale image processing. However, in the case of specimens with different thicknesses and / or stains of different shades, if the area is measured (binarization) with the same settings for grayscale image processing, it is recognized as the area of the specimen. In some cases, it may be impossible to recognize it, or it may be excessively recognized, resulting in a deviation from the true area value. Therefore, the value of G (green) in the density histogram at the time of grayscale image processing is set to the average value “143” of the second embodiment, and R (red) and B (blue) are set.
The values of were set to 255 as in the above, and the area of each sample was measured by an image analyzer to calculate the area ratio to the true area. The result is shown in FIG. The shorter the exposure time (the lighter the stain), the smaller the area ratio, and the longer the the exposure time (the darker the stain), the higher the area ratio. A sample having an area ratio of 1 ± 0.02 (0.98 to 1.02) is particularly suitable for quantitative analysis of a tissue sample by an image analyzer, and in that case, the exposure time of the thin section before sample preparation is It was 0.40 to 0.44 seconds.

[0023]

As is apparent from the above-mentioned Reference Examples and Examples, when a thin section is prepared by an ordinary gliding microtome, the tissue specimen prepared thereby produces considerable staining unevenness. By using a thin section selected by the method, a tissue sample without uneven staining can be obtained, and reliable sample observation and quantitative analysis with an image analyzer are possible, contributing to improving work efficiency. You can

[0024]

[Brief description of drawings]

FIG. 1 shows the exposure time of each thin section prepared as 4 μm on a gliding microtome. Numbers in parentheses mean the number of slices

FIG. 2 shows a state of a HE-stained sample (× 20 times) of a thin section having the longest exposure time (0.58 seconds).

FIG. 3 shows a state of a HE-stained sample (× 20 times) of a thin section with the shortest exposure time (0.37 seconds).

FIG. 4 shows the relationship between the exposure time of each thin section and the quantitative analysis (G (green) value) of the HE-stained sample by an image analyzer.

[Fig. 5] The average value (R (red) 25
5, G (green) 143, B (blue) 255) and the relationship between the area ratio to the true area and the exposure time when binarized under the same conditions are shown.

Claims (6)

[Claims] 1. A method for selecting a thin section suitable as a tissue specimen. 2. The selection method according to claim 1, wherein the selection method uses a biological microscope and an exposure meter of a photographic device. 3. The selection method is one in which the exposure time of a thin section is measured using an exposure meter of a biological microscope and a photographic device, and a thin section suitable as a tissue sample is selected according to the exposure time. Selection method described. 4. The selection method according to claim 3, wherein a thin section having an exposure time width within a range of 0.06 seconds is selected as a suitable thin section as a tissue sample. 5. The selection method according to claim 3, wherein a thin section having an exposure time width within a range of 0.05 seconds is selected as a suitable thin section as a tissue sample. 6. The selection method according to claim 3, wherein a thin section having an exposure time width within the range of 0.04 seconds is selected as a suitable thin section as a tissue sample.