JP2002202229A - Method of cutting-bio-sample, and device used therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method excellent in operability and having a satisfactory cut condition (sharpness) in laser beam cutting, in the method wherein a bio- sample is deposited on a film, wherein a target portion is cut, and wherein the film is separated thereafter to recover a cut piece. SOLUTION: In this method for cutting the bio-sample by irradiation of a beam, the bio-sample and the colored film having 3-6 μm of thickness are set on one side face of a supporting body, and the bio-sample is irradiated with the beam to cut the target portion of the bio-sample.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for cutting a biological sample and an apparatus used therefor. More specifically,
The present invention relates to a method for cutting a target portion of a biological sample by irradiating the biological sample with light, and an apparatus used for the method.

[0002]

2. Description of the Related Art In the microarray method, a DNA spotted on a slide glass is hybridized with a labeled probe prepared from RNA extracted from a tissue or a cell to be examined, and a signal is detected. This is a method for measuring the relative expression level of the gene corresponding to the spot. Advances in such microarray technology have made it possible to analyze the expression of thousands to tens of thousands of genes at once.
By this method, it is possible to comprehensively investigate what kind of gene expression abnormality occurs in a disease involving a plurality of gene abnormalities such as cancer and leukemia. In research aimed at elucidating the cause and pathogenesis of such diseases, it is important to extract only specific cells from a biological sample.

[0003] For example, in the molecular study of human tumors, it is necessary to quantitatively or qualitatively evaluate the expression of proteins or nucleic acids in human tumor cells. It is necessary to collect them separately, analyze the expression level of the target nucleic acid or target protein in each cell, and compare them.

However, in invasive tumor tissues, many cell types (eg, tumor cells, stromal cells, endothelial cells,
Normal epithelial cells and inflammatory cells). In order to accurately and quantitatively or qualitatively evaluate the expression of a protein or nucleic acid in tumor cells, it is necessary to collect only tumor cells from among many types of cells present in tumor tissues. Requires cutting the biological sample at the target location.

[0005] As a method for cutting a biological sample such as a biological tissue piece, a cell, a chromosome, and a microorganism at a target position and recovering the cut sample, a method of irradiating a sample with laser light to cut and recover (a laser The capture microdissection method is known (Emmert-Buck, MR et
al., Science 274, 998-1001, 1996; and Bonner, RR.
et al .: Science, 278, 1481-1482, 1997). In this method, a biological sample is attached to a film, a target site is cut, and then the film is peeled off to collect a cut piece. However, in these conventional methods,
Since the thickness of the film is thin, the operability is poor. In particular, there is a problem that it is difficult to handle the installation of the film on the support, and there is still room for improvement in the degree of cutting (sharpness) by laser light. .

[0006]

An object of the present invention is to provide a novel method for cutting a biological sample such as a biological tissue piece, a cell, a chromosome, and a microorganism at a target position and recovering the cut sample. It should be a task to be done. That is, the present invention relates to a method for collecting a cut piece by attaching a biological sample to a film, cutting a target site, and then peeling the film, which is excellent in operability and cutting condition by laser light ( The problem to be solved is to provide a method with good sharpness.

[0007]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, the use of a colored film having a thickness of 3 to 6 μm as a film to be attached to a biological sample has solved the above-mentioned problems. The inventors have found that an excellent method for cutting the solved biological sample at the target position can be provided, and have completed the present invention.

That is, according to the present invention, there is provided a method for cutting a biological sample by irradiating light, wherein the biological sample and a colored film having a thickness of 3 to 6 μm are provided on one surface of a support, and the light is applied to the biological sample. And cutting the target portion of the biological sample. The present invention also relates to a method for cutting and collecting a biological sample, further comprising collecting the cut sample.

According to another aspect of the present invention, there is provided an apparatus for cutting a biological sample, comprising a colored film having a thickness of 3 to 6 μm provided on one side of a support.

[0010]

Embodiments of the present invention will be described below in detail. In the method of cutting a biological sample by light irradiation according to the present invention, a biological sample and a colored film having a thickness of 3 to 6 μm are placed on one surface of a support, and the biological sample is irradiated with light to irradiate a target portion of the biological sample. It is characterized by cutting. In one example of the present invention, the target portion of the biological sample can be cut by irradiating light from the side of the support opposite to the surface holding the biological sample. In another example of the present invention, it is also possible to turn the support holding the biological sample upside down and irradiate light from the surface holding the biological sample to cut the target portion of the biological sample. That is, in the present invention, the direction of light irradiation is not particularly limited. The type of the biological sample used in the present invention is not particularly limited as long as it is derived from a living body, and examples thereof include biological tissue pieces, cells, chromosomes, and microorganisms. As the biological sample, for example, a biological sample containing a disease site (for example, cancer) can be used. In this case, the diseased cells (for example, cancer cells) are separated from normal cells by the method of the present invention and collected. can do.

The light used in the present invention is preferably laser light, because laser light is light that can be easily and effectively concentrated on a small area on a given surface. The wavelength of the laser light is usually 150 to 1
It is about 000 nm, particularly preferably an ultraviolet laser beam (a wavelength of 180 to 400 nm). In the case of laser light, the beam diameter needs to be appropriately set depending on the type, thickness, etc. of the biological sample.
It can be about 50 μm. The light intensity is
It can be appropriately determined depending on the type and thickness of the biological sample, the size of the cut piece, and the like. For example, when the biological sample is a tissue section, it is about 0.5 to 3.0 J / cm ² , when it is a cell, it is about 0.1 to 3.0 J / cm ² , and when it is a chromosome or microorganism, it is about 0.01 to 3.0 J / cm ^2. About 3.0 J / cm ² .

The type of the support used in the present invention is not particularly limited as long as it can transmit light, but it is preferably a transparent support, for example, a glass support (slide glass or cover glass) or polyethylene. And terephthalate supports.

The colored film used in the present invention has a thickness of 3 to
The thickness is 6 μm. If the thickness is less than 3 μm, the operability is inferior, and the operation of installing the support on the support becomes complicated, which is not preferable. If the thickness exceeds 6 μm, cutting by light becomes difficult, which is not preferable. The present invention is characterized in that a colored film is used instead of a transparent film, and the use of a colored film is advantageous in that an effect of facilitating cutting by light is obtained.

Specific examples of the colored film that can be used in the present invention include an aramid film having a yellow color. Specific examples of such an aramid film include polymers of terephthalic acid and paraphenylenediamine (paraphenylene terephthalamide, hereinafter also referred to as PPTA) (Kevlar (registered trademark), Twaron (registered trademark), Technora (registered trademark), etc. ).

A colored film such as an aramid film can be prepared by a known method. For example, an aramid polymer is dissolved in concentrated sulfuric acid and the dope is degassed. next,
From a die with a slit of appropriate size, cast to a tantalum belt polished to a mirror surface, blow air to make the casting dope optically isotropic, and guide it together with the belt into an aqueous sulfuric acid solution to coagulate . At this time, the film itself can be provided with cupping. Next, the coagulated film is peeled off from the belt and washed by running in warm water, an aqueous solution of sodium carbonate, and then in water at 25 ° C.
The washed film having a water content of about 280% is first uniaxially stretched in the machine direction (MD) at room temperature by using a difference in peripheral speed of a roll, and then put in a tenter and width direction (TD) near the entrance. The center of the tenter is heated to a constant length of 150 ° C and dried, and an infrared lamp is attached near the exit of the tenter and heat-treated at 400 ° C. Obtainable.

In the method of the present invention, a colored film having a thickness of 3 to 6 μm may be provided on one side of the support, and a biological sample may be provided thereon (in this case, the support, the support, Alternatively, a biological sample may be placed on one side of the support, and a colored film having a thickness of 3 to 6 μm may be placed on the biological sample (in this case, a lower portion in this case). From the side, a support, a biological sample, and a colored film are arranged in that order), preferably the former. According to the present invention, there is also provided an apparatus for cutting a biological sample, comprising a colored film having a thickness of 3 to 6 μm provided on one surface of a support.

In order to cut the target portion of the biological sample in the method of the present invention, it is preferable to stain the biological sample by a suitable method. The biological sample that can be used in the present invention can be stained with, for example, hematoxylin and eosin, which makes it possible to identify the position of a specific cell population under microscopic visualization, and to identify the target cells. It becomes possible to isolate and collect from the whole tissue.

In the method of the present invention, it is preferable to cut the target portion under a microscope. Specifically, the method for cutting a biological sample according to the present invention includes, for example, installing a colored film and a transparent support holding a biological sample (a biological sample containing target cells to be collected) on the upper surface on a microscope stage, The target area of the biological sample is determined via the transparent support while checking the image captured by the eyepiece or the CCD camera on the monitor. The determination of the target area can be performed by staining with a dye or immunostaining.

Next, light is generated by a light irradiating means such as a laser light (preferably, ultraviolet laser light) irradiating device, the beam diameter of the light is reduced, and a support is formed along the target cutting portion of the biological sample. The biological sample is irradiated with light from below to cut the biological sample. By focusing the laser on the optical center of the field of view of the optical microscope, the activation energy will be concentrated on the target area of the colored film in contact with the biological sample. In addition, the duration of the laser irradiation can be appropriately adjusted so that the required amount of energy is directed to the target cutting site.

The present invention further relates to a method for cutting and collecting a biological sample, which comprises cutting a biological sample by the above-described cutting method and collecting the cut sample. When sufficient energy of the laser light is absorbed by the surface of the colored film in contact with the target cells of the biological sample, an adhesive layer is formed between the colored film and the target cells. Thus, by removing and collecting the film, the target cells can be separated and collected from other biological samples.

The size of the sample to be separated and recovered can be changed by changing the diameter of light (preferably laser light) and the duration of a pulse. According to the method of the present invention, a small sample having a size of about 1 μm to 100 μm can be collected. Furthermore, since the laser beam can be radiated to a region smaller than the diameter of one cell, it is also possible to separate and collect a single target cell or only a part thereof.

The method of collecting the cut sample is not particularly limited. For example, instead of cutting the section by shifting the focus of the lens of the laser beam irradiated for cutting, expanding the diameter of the laser and dispersing the power, Then, the cut sample can be peeled off from the support and blown upward. The sample thus peeled from the support can be collected in a container such as a microtube cap. As an apparatus that can be used for carrying out the method for cutting a biological sample of the present invention, for example, a CRI-337 Laser Scissors ^™ 337/120 module (manufactured by Cell Robotech, USA) and the like can be mentioned.

The target sample cut and recovered as described above can be dissolved and stored, and can be subjected to target analysis. When analyzing DNA or RNA contained in target cells, the collected sample is subjected to polymerase chain reaction (PC).
R) The reaction can be followed by a conventional gene analysis method (for example, Southern blot, Northern blot, dot blot hybridization, or sequencing).

For example, mRNA can be extracted from the recovered sample using column chromatography on oligo dT. The mRNA recovered from the target cells can be further amplified by the RT-PCR method, which can be further analyzed.

When analyzing a specific protein or polypeptide contained in a target cell, the collected sample is
It can be used for an enzyme assay using a labeled substrate, an immunoassay using a labeled antibody, a biochemical assay, and the like. The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.

[0026]

EXAMPLES Example 1 Microdissection by the Method of the Present Invention (1) Preparation of Aramid Film An aramid film as a non-magnetic film was prepared by the following known method. PPTA with ηinh (logarithmic viscosity) of 5.5
0.005 of colloidal silica having an average particle size of 80 nm
The polymer was dissolved in concentrated sulfuric acid containing 1% by weight so as to have a polymer concentration of 11.5%, and the dope was degassed under vacuum while maintaining the temperature at about 70 ° C. From a die with a slit of 0.15mm x 300mm
At a discharge linear speed of 3.5 m / min, cast on a tantalum belt polished to a mirror surface (moving at 12 m / min), about 85% relative humidity
The casting dope was optically isotropic by blowing air at about 90 ° C., and was introduced together with a belt into a 15% by weight aqueous sulfuric acid solution at −5 ° C. for coagulation. At this time, the film itself can be provided with cupping. Next, the coagulated film was peeled off from the belt, and washed by running in a 1% aqueous solution of sodium carbonate in about 40 ° C. water and then in 25 ° C. water. The washed film having a water content of about 280% is subjected to uniaxial stretching at a room temperature by a factor of 1.2 in the machine direction (MD) at a room temperature using a difference in peripheral speed of a roll. The film is stretched 1.2 times in the width direction (TD), the center of the tenter is heated at a constant length of 150 ° C. and dried, and an infrared lamp is attached near the exit of the tenter.
After heat treatment at 00 ° C., the long film was wound up.
The obtained PPTA film is excellent in transparency and 3.8
It had a thickness of μm or 4.2 μm.

(2) Preparation of Cutting Sample A 2 cm square aramid film (obtained in Example 1, 3.8 μm or 4.2 μm in thickness) was placed on a slide glass (transmitting a laser beam having a wavelength of 377 nm). , Nail P
Bonded using olish. Mouse testis tissue was placed in a plastic cryocontainer (for example, Cryomond manufactured by MILES) and placed in an O.C. C. The T compound was placed and the sample tissue was embedded. Samples were frozen within about 30 to 40 seconds under liquid nitrogen. The frozen sample was sectioned at a thickness of about 8 μm using a cryosection preparation device (cryostat). The thin section was directly pasted on the slide glass prepared above on which the film was pasted.

The slide glass to which the frozen section was attached was prepared by a conventional method (“All of the staining methods”, Itoyaku Shuppan Publishing Co., Ltd., 19
Hematoxylin and Eosin (H
E) Staining was performed. For hematoxylin, a Mayer hematoxylin solution (manufactured by Wako Pure Chemical Industries, Ltd.) was used, and for eosin, a 1% eosin solution (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

(3) Cutting of Sample by Laser Observing the sample on the slide glass with a microscope, LS-33
7 The sample was cut at the target site using a Laser Scissors ^™ 337/120 module (the overall system configuration was CRI-337) (manufactured by Cell Robotech, USA) (microdissection). The conditions for microdissection are as follows. Nitrogen excitation pulse laser Wavelength 337 nm Output 1 to 120 μJ / pulse (variable) (at cutting: 65 to 80%, 100 pps, so about 80 μJ
/ Pulse)

Subsequently, by shifting the focal point of the lens of the laser beam downward to expand the diameter of the laser and disperse the power, the cut section is peeled off from the slide glass, and is blown upward to cut the cut section. Collected in tube cap.

FIG. 1 shows the state of the sample before and after the microdissection, and the sample at the target site which has been cut and collected. As shown in FIG. 1, the target site of the tissue was successfully cut and recovered by the method of the present invention.

Example 2: Comparison between the method of the present invention and a comparative example A biological sample was prepared in the same manner as in Example 1 except that the material and thickness of the film placed on the slide glass were changed as shown in Table 1 below. And microdissection was performed. Suitability of film to slide glass (operability), suitability for microdissection, cutting condition (sharp cut)
Table 1 shows the results of the evaluation of
Shown in

[0033]

[Table 1]

The results in Table 1 show that the method of the present invention is excellent.

[0035]

According to the present invention, there is provided a method for recovering cut pieces by attaching a biological sample to a film, cutting a target site, and then peeling the film. It has become possible to provide a method with good sharpness (sharpness).

[Brief description of the drawings]

FIG. 1 is a diagram showing the results when mouse testis samples were collected by microdissection.

Continued on the front page (72) Inventor Kazuko Hanai 2-12-1, Ogimachi, Odawara-shi, Kanagawa Prefecture Inside Fuji Photo Film Co., Ltd. (72) Inventor Hiroshi Arakatsu 210 Nakanakanuma, Minamiashigara-shi, Kanagawa Prefecture Fuji Photo Film Co., Ltd. F term (reference) 2G045 BA14 BB17 BB60 CB01 CB21 CB30 FA27 HA16 2G052 AA28 DA05 EB08 EC17 ED07 FA08 FD09 JA16

Claims (13)

[Claims] 1. A method for cutting a biological sample by light irradiation, wherein the biological sample and a thickness of 3 to 6 μm are formed on one surface of a support.
m, and irradiating the biological sample with light to cut a target portion of the biological sample. 2. The cutting method according to claim 1, wherein the target portion of the biological sample is cut by irradiating light from a side of the support opposite to a surface holding the biological sample. 3. The cutting method according to claim 1, wherein the biological sample is a biological tissue piece, a cell, a chromosome, or a microorganism. 4. The method according to claim 1, wherein the light is laser light.
The cutting method according to any one of the above. 5. The method of claim 1, wherein the light is an ultraviolet laser light.
5. The cutting method according to any one of items 1 to 4. 6. The support according to claim 1, wherein the support is a glass support.
6. The cutting method according to any one of claims 1 to 5. 7. The cutting method according to claim 1, wherein the colored film is an aramid film. 8. The cutting method according to claim 1, wherein a colored film having a thickness of 3 to 6 μm is provided on one surface of the support, and a biological sample is provided thereon. 9. The cutting method according to claim 1, wherein the cutting of the target portion is performed under a microscope. 10. A method for cutting and collecting a biological sample, comprising cutting a biological sample by the cutting method according to claim 1, and collecting the cut sample. 11. An apparatus for cutting a biological sample, comprising a colored film having a thickness of 3 to 6 μm provided on one side of a support. 12. The method according to claim 1, wherein the support is a glass support.
An apparatus according to claim 1. 13. The device according to claim 11, wherein the colored film is an aramid film.