JPH0462032B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to autoradiographic measurements.

[Background of the Invention] After a substance to which a radioactive label has been given is administered to a living organism, a part of the tissue of the living organism or the body object is used as a sample, and this sample is combined with a photographic material such as a radiation film (for example, Autoradiography is an autoradiography process that consists of exposing (or exposing) a high-sensitivity X-ray film to light by overlapping the film with light for a certain period of time, and obtaining positional information of the radiolabeled substance in the sample from the exposed area. The autoradiographic measurement method (also called radioautography) is known from the prior art. This autoradiography is used to study in detail the metabolism, absorption, and excretion routes and conditions of administered substances in living organisms.
The details are described in the following documents, for example.

Biochemistry Experiment Course 6 Tracer Experiment Method (1)
pp. 271-289, "8. Autoradiography" Toru Sueyoshi, Akiyo Shigematsu (1977, published by Tokyo Kagaku Doujin Co., Ltd.) In recent years, autoradiography has been used to analyze biological materials such as proteins and nucleic acids. After attaching a radioactive label to a polymeric substance, the radiolabeled polymeric substance, its derivatives, or its decomposition products are separated and developed by gel electrophoresis, etc., and the positional information of the radioactively labeled substance on the support medium is obtained. It is also used effectively to obtain Methods for separating and identifying polymeric substances, or evaluating their molecular weights and properties based on the positional information have also been developed and are in actual use.

Particularly in recent years, autoradiography has been effectively used to determine the base sequence of nucleic acids such as DNA, and has therefore become an extremely useful tool for determining the structure of polymeric substances derived from living organisms. There is.

However, there are several problems when actually utilizing such useful autoradiography.

The first is to obtain an autoradiograph of a radiolabeled substance separated and developed on a support medium.
A support medium and a radiation film are overlapped for a certain period of time to expose the film to light, but this exposure operation is carried out at low temperatures (for example, 0
℃ to -80℃) for a long period of time (several hours to several days). This is because the radiolabeled substances that are measured by autoradiography generally do not have high radioactivity;
At a relatively high temperature such as room temperature, a latent image in the silver salt of the film is formed by exposure to radiation emitted from a radiolabeled substance, or, if an intensifying screen is used, to fluorescence from the intensifying screen. This is because the silver salt tends to deteriorate and become an undevelopable image, and harmful components tend to migrate from the support medium to the silver salt, resulting in the formation of chemical fog.

Second, in order to prevent deterioration of image quality due to chemical fog or the like, the support medium containing the radiolabeled substance must be exposed in a dry state while superimposed on the radiation film. For this reason, the support medium is usually dried or wrapped with a synthetic resin film or the like.

If such fogging occurs in images obtained by autoradiography, the accuracy of positional information of radiolabeled substances will be significantly reduced. For the reasons mentioned above, the operation of autoradiography has become complicated.

Thirdly, radiation film has the disadvantage that it is easily affected by physical stimuli associated with operations such as its movement and installation, resulting in physical fog. In order to avoid such physical fogging of the radiation film, a high degree of skill and care is required in handling the radiation film. In addition, since conventional autoradiography involves long-time exposure operations as described above, it is also exposed to natural radioactivity contained in the sample in addition to the radiolabeled substance, and the resulting position of the radiolabeled substance is There is a problem in that it reduces the accuracy of information. In order to eliminate such interference due to natural radioactivity, attempts have been made, for example, to conduct parallel experiments using control samples and to optimize the exposure time, but as the number of experiments increases, the overall operation becomes complicated. There are drawbacks to it.

Furthermore, in conventional autoradiography, in order to obtain the necessary information from an imaged autoradiograph, it is necessary to perform the simple task of visually reading the position information over a long period of time. It was hot.

The present applicant has solved the above-mentioned problems by using a stimulable phosphor sheet containing a stimulable phosphor instead of a conventional photosensitive material such as a radiation film in an autoradiographic measurement method. An application has already been filed for an invention that reduces defects (Japanese Patent Application No. 193418/1982).

In the above, the stimulable phosphor sheet is also called a radiation image conversion panel, and an example thereof is described in Japanese Patent Application Laid-Open No. 12145/1983, and its general configuration is already known.

In other words, the stimulable phosphor sheet absorbs the radiation energy transmitted through the subject or the radiation energy emitted from the subject into the stimulable phosphor of the panel, and then exposes the stimulable phosphor to visible light and infrared rays. By exciting the stimulable phosphor in a time-series manner using electromagnetic waves (excitation light), the radiation energy stored in the stimulable phosphor is released as fluorescence, and this fluorescence is read photoelectrically to obtain an electrical signal. This electrical signal is reproduced as a visible image on a recording material such as a radiation film, a display device such as a CRT, or expressed as numerical or symbolic position information.

According to the autoradiographic measurement method using the above-mentioned stimulable phosphor sheet, not only can the exposure time be significantly shortened, but also the exposure can be carried out at or near ambient temperature. , the accuracy of the obtained position information will not decrease. Therefore, the exposure operation, which was conventionally carried out over a long period of time under cooling, is significantly simplified, and the autoradiography operation is simplified.

Furthermore, by using the above-mentioned stimulable phosphor sheet in the autoradiographic measurement method, chemical fog and physical fog, which have traditionally been a major problem when using radiation films, can be virtually eliminated. This has a very advantageous effect on improving the accuracy of location information and on workability. In addition, the decrease in accuracy due to radioactivity of impurities contained in the sample or natural radioactivity, etc.
By electrically processing the position information stored and recorded on the stimulable phosphor sheet, it is possible to easily reduce or eliminate the problem.

Furthermore, when a stimulable phosphor sheet is used, there is no need for special imaging to obtain positional information of the radiolabeled substance accumulated and recorded on the stimulable phosphor sheet, and the stimulable phosphor sheet is By scanning with excitation light, it is possible to read out the above position information and convert the position information into any form such as an image, symbol and/or numerical value, or a combination thereof. This image information can also be obtained in various desired forms by further processing via electrical means, i.e., as other information obtained by signal processing of electrical or digital signals containing the image information. It is.

As mentioned above, autoradiography, which utilizes a radiation image conversion method using a stimulable phosphor sheet, is a very useful method, but so far, autoradiography has been limited to conventional radiography. Currently, it is desired to obtain the positional information of the radiolabeled substance in the form of an image so that it can be directly compared with the visible image obtained by this conventional method. Therefore, in terms of storage and management of the obtained position information, it is desired to store and manage the obtained position information in the form of images as well.

However, the above method requires special equipment to image the positional information of the radiolabeled substance, and in particular, the obtained image is in a form that can be easily compared with other visible images and can be stored. has the disadvantage that the device is necessarily complex.

[Summary of the Invention] In autoradiography for obtaining positional information of a radiolabeled substance separated and developed on a support medium, the present inventor has developed a stimulable fluorescence that absorbs and accumulates radiation energy emitted from a radiolabeled substance. A conventional photosensitive material is superimposed on a body sheet and irradiated with excitation light, and the photosensitive material is sensitized with the photostimulated light emitted from the stimulable phosphor sheet. We have discovered that graphs can be directly visualized, and have arrived at the present invention. In other words, by using the function of the stimulable phosphor sheet as a sensitizing function, it is possible to directly obtain positional information of radiolabeled substances under significantly relaxed conditions (temperature, time, etc.) than when using conventional radiography. It has been discovered that the autoradiography process can be obtained as a visible image, thereby simplifying the autoradiography operation.

Furthermore, in addition to creating an autoradiograph image on a photographic light-sensitive material using a stimulable phosphor sheet as described above, the present inventor has also developed a method for forming a sheet by irradiating the same stimulable phosphor sheet with excitation light again. We have discovered that it is also possible to photoelectrically detect (read out) the photostimulated light from. The present invention also includes this. That is, by using a single stimulable phosphor sheet, it is possible to obtain positional information of a radiolabeled substance as digital data and as a direct visible image, thus obtaining qualitatively and quantitatively rich positional information. be able to.

That is, the present invention provides an autoradiographic measurement method for obtaining one-dimensional or two-dimensional positional information of a living body-derived substance to which a radioactive label has been applied and which has been separated and developed on a support medium. A step of overlapping a support medium and a stimulable phosphor sheet containing a stimulable phosphor for a certain period of time, thereby causing the sheet to absorb at least a portion of the radiation energy emitted from the radiolabeled substance in the support medium. , and 2, after overlapping the stimulable phosphor sheet and the photographic light-sensitive material, irradiating the stimulable phosphor sheet with excitation light to release the radiation energy stored in the sheet as photostimulated light, and The present invention provides an autoradiographic measuring method characterized by comprising the step of: obtaining positional information of a radiolabeled substance as an image on a photosensitive material by exposing a photographic light-sensitive material to the photosensitivity light.

The present invention also provides an autoradiographic measurement method for obtaining one-dimensional or two-dimensional positional information of a biologically-derived substance to which a radioactive label has been applied and which has been separated and developed on a support medium. A step of overlapping a support medium and a stimulable phosphor sheet containing a stimulable phosphor for a certain period of time, thereby causing the sheet to absorb at least a portion of the radiation energy emitted from the radiolabeled substance in the support medium. 2. After overlapping the stimulable phosphor sheet and the photographic light-sensitive material, the stimulable phosphor sheet is irradiated with excitation light to release the radiation energy stored in the sheet as photostimulated light, and the stimulable phosphor sheet is released as stimulated light. 3. Obtaining positional information of the radiolabeled substance as an image on the photosensitive material by sensitizing the photographic light-sensitive material by exposure to light; and 3. Scanning the stimulable phosphor sheet with excitation light so that the stimulable phosphor sheet is accumulated on the sheet. A step of emitting radiation energy as photostimulated light and photoelectrically detecting the stimulated light to obtain positional information of the radiolabeled substance as an electrical signal, and the step of 3) above is combined with the step of 2) above. The present invention also provides an autoradiographic measurement method that is characterized in that it can be carried out before or after the process.

In addition, in the present invention, "position information" of a radiolabeled substance that is separated and developed on a support medium refers to various information centered on the position of a radiolabeled substance or an aggregate thereof, such as information that exists in the support medium. Refers to various types of information obtained as one or any combination of information such as the location and shape of an aggregate of radioactive materials, the concentration and distribution of radioactive materials at that location, etc.

[Effects of the Invention] According to the method of the present invention, conventional methods have been carried out by directly overlapping a support medium in which a radiolabeled substance has been separated and developed and a photographic light-sensitive material at a low temperature for a long period of time and exposing them to light. Autoradiographic imaging can be performed under very relaxed conditions by using a stimulable phosphor sheet. In other words, when a stimulable phosphor sheet, in which an autoradiograph of a radiolabeled substance is recorded as an accumulated image of radiation energy, is superimposed on a photographic light-sensitive material and the sheet is irradiated with appropriate excitation light, this sheet By sensitizing a photosensitive material with the stimulated light emitted from the photosensitive material, sensitization can be carried out at room temperature in a short time. Therefore, fogging does not occur in the image obtained as in the conventional case.

In addition, since the stimulable phosphor sheet can be irradiated with appropriate excitation light and the accumulated radiation energy can be instantly released as photostimulated light, it is possible to perform sensitizing exposure using a conventional intensifying screen. It is possible to sensitize a photosensitive material under considerably relaxed conditions compared to the conventional method. Furthermore, since the stimulable phosphor sheet is used, the photosensitive operation does not need to be performed immediately after the sheet absorbs radiation energy, and there is no time restriction. Furthermore, it is possible to expose a plurality of photosensitive materials using the same stimulable phosphor sheet. Autoradiography operations can therefore be significantly facilitated.

Compared to the case where images are created from electrical or digital signals obtained by photoelectrically reading out stimulable phosphor sheets, visible images of autoradiographs do not require the use of special equipment such as an image reproduction device. can be obtained easily and at low cost.

In addition, since the images obtained in this way are directly visualized autoradiographs of radiolabeled substances, as in the case of conventional radiography, they are different from other images obtained by conventional methods. (autoradiographic image). Furthermore, since images obtained by the method of the present invention are obtained by exposing a photographic light-sensitive material and a stimulable phosphor sheet in close contact with each other, image distortion does not occur and image registration is automatic. It is something that can be taken as a target.

In particular, as a result of research, the present inventor has discovered that the photographic light-sensitive material may be placed on the excitation light irradiation side of the stimulable phosphor sheet. That is, the stimulated light emitted from the stimulable phosphor sheet is absorbed by the photosensitive substance in the photosensitive material, thereby sensitizing the photosensitive material and contributing to image formation. put it here,
Because the excitation light wavelength range for exciting the photostimulable phosphor contained in the stimulable phosphor sheet is different from the wavelength range of the photostimulable light emitted from this phosphor,
It has been found that a photographic light-sensitive material can be exposed to stimulated light without being exposed to excitation light, and a desired image can be formed on the light-sensitive material.

Note that the photographic light-sensitive material is not sensitive to excitation light here, meaning that the sensitivity at the excitation light wavelength is significantly lower than the sensitivity in the maximum sensitivity wavelength region of the photographic light-sensitive material. This does not mean that the material is completely unsensitized by the excitation light.

Separately, by irradiating the stimulable phosphor sheet with excitation light and photoelectrically reading out the stimulated light,
Positional information on the radiolabeled substance can also be obtained as an electrical signal. In other words, by appropriately irradiating a stimulable phosphor sheet with excitation light in stages to efficiently release the radiation energy accumulated in the sheet, it is possible to create an image on a photosensitive material using a portion of the stimulated light. The remaining part of the stimulated light is detected to obtain an electrical signal. In the present invention, either the photosensitive operation of the photographic light-sensitive material or the photoelectric readout operation of the stimulated light may be carried out first.

Therefore, on the one hand, the positional information of the radiolabeled substance separated and developed on the support medium is obtained as an electrical signal;
On the other hand, it can be obtained as an image on a photographic material. This also means that the positional information of the radiolabeled substance can be recorded and stored on a magnetic tape, etc. in the form of an electrical signal or an A/D-converted digital signal, and at the same time, it can also be stored in the form of an image recorded on a photosensitive material. This means that it can be saved.

Furthermore, in the method of the present invention, it is possible to read out the sheet or expose the photographic light-sensitive material with the support medium for electrophoresis in close contact with the stimulable phosphor sheet, and in this case, the above-mentioned method can be used. In addition to the advantages, it is also possible to overlap the support medium and the stimulable phosphor sheet and perform the exposure operation and then perform the readout operation without separating them, or to further overlap the photosensitive materials and perform the exposure operation. can be done. In particular, if the support medium and stimulable phosphor sheet are integrated, the support medium such as gel may be scraped off from the stimulable phosphor sheet before the readout and exposure steps, or an appropriate solvent may be added to the stimulable phosphor sheet. The autoradiography procedure can be simplified without the need for washing and washing.

In particular, if the readout device (which can also serve as a photosensitive device) for reading out the position information of the radiolabeled substance accumulated and recorded on the stimulable phosphor sheet is light-shielded, a special dark place should be provided. There is no need to perform an exposure operation. Therefore, it is possible to perform the exposure operation of a stimulable phosphor sheet using a support medium containing a radiolabeled substance and the reading operation of the stimulable phosphor sheet or the exposure operation of a photographic light-sensitive material in one continuous process. It is.

[Detailed Description of the Invention] The stimulable phosphor sheet used in the present invention has a basic structure consisting of a support and at least one phosphor layer provided on one side of the support. The phosphor layer consists of a stimulable phosphor and a binder that contains and supports the stimulable phosphor in a dispersed state. Note that a transparent protective film is generally provided on the surface of this phosphor layer opposite to the support (the surface not facing the support), and the phosphor layer is protected from chemical or physical alteration. Protects from physical impact.

The stimulable phosphor sheet having the above structure can be manufactured, for example, by the method described below.

The support can be appropriately selected from various materials used as supports for intensifying screens in conventional radiography or supports for known stimulable phosphor sheets. Examples of such materials include films of plastic materials such as cellulose acetate and polyethylene terephthalate;
Examples include metal sheets such as aluminum foil, ordinary paper, baryta paper, and resin-coated paper. Note that the surface of the support on which the phosphor layer is provided may be provided with an adhesion-imparting layer, a light-reflecting layer, a light-absorbing layer, etc.
As disclosed in Publication No. 200200, fine irregularities may be uniformly formed (these irregularities are
When an adhesion-imparting layer, a light-reflecting layer, a light-absorbing layer, etc. are provided on the surface of the support on the phosphor layer side,
formed on its surface).

A phosphor layer containing and supporting a stimulable phosphor in a dispersed state is provided on this support.

As mentioned above, a stimulable phosphor is a phosphor that exhibits stimulated luminescence when it is irradiated with radiation and then irradiated with excitation light. It is required that the wavelength range is in a wavelength range that does not sensitize the photosensitive material such as silver halide contained in the photographic light-sensitive material, and that the stimulated emission wavelength range is in a wavelength range that sensitizes the photosensitive material. Ru. From a practical standpoint, the wavelength is 600 ~
350-500nm with excitation light in the range of 830nm
It is desirable that the phosphor exhibits stimulated luminescence in the wavelength range of . The stimulable phosphor used in the stimulable phosphor sheet used in the present invention is preferably a divalent europium-activated alkaline earth metal fluorohalide phosphor, but is not limited thereto. isn't it.

Other examples of stimulable phosphors include those described in U.S. Pat. No. 3,859,527.
Phosphors expressed by composition formulas such as SrS:Ce, Sm, SrS:Eu, Sm, ThO ₂ :Er, and La ₂ O ₂ S:Eu, Sm, as described in JP-A-55-12142.
ZnS: Cu, Pb, BaO・xAl ₂ O ₃ : Eu [however, 0.8
≦x≦10], and M〓0・xSiO ₂ :A [however,
M〓 is Mg, Ca, Sr, Zn, Cd, or Ba,
A is Ce, Tb, Eu, Tm, Pb, Tl, Bi, or
A phosphor represented by a composition formula such as Mn, and x is 0.5≦x≦2.5] is described in JP-A-55-12143 (Ba _1-xy , Mg _x , Ca _y ) FX: aEu ²⁺ [However, X
is at least one of Cl and Br,
A phosphor represented by the composition formula: x and y are 0<x+y≦0.6 and xy≠0, and a is 10 ^-6 ≦a≦5×10 ^-2 JP-A-12144-1987 stated in the issue
LnOX: xA [However, Ln is La, Y, Gd, and
At least one of Lu, X is at least one of Cl and Br, A is at least one of Ce and Tb, and x is 0<x<0.1
], and (Ba _1-x , M〓 _x )FX:yA [where M〓 is Mg,
At least one of Ca, Sr, Zn, and Cd, X is at least one of Cl, Br, and I, A is Eu, Tb, Ce, Tm, Dy, Pr, Ho,
At least one of Nd, Yb, and Er, x is 0≦x≦0.6, and y is 0≦y≦0.2.

First, stimulable phosphor particles and a binder are added to a suitable solvent (for example, lower alcohol, chlorine atom-containing hydrocarbon, ketone, ester, ether),
These are thoroughly mixed to prepare a coating solution in which the stimulable phosphor is uniformly dispersed in the binder solution.

Examples of binders include proteins such as gelatin, synthetic polymeric substances such as polyvinyl acetate, nitrocellulose, polyurethane, polyvinyl alcohol, polyalkyl (meth)acrylate, linear polyester, etc. can be mentioned.

The mixing ratio of the binder and the stimulable phosphor in the coating solution is usually selected from the range of 1:8 to 1:40 (weight ratio).

Next, this coating liquid is uniformly applied to the surface of the support to form a coating film of the coating liquid, and then this coating film is dried to complete the formation of the phosphor layer on the support. The thickness of the phosphor layer is generally 50 to 50 mm.
It is 500μm.

Furthermore, a transparent protective film for physically and chemically protecting the phosphor layer may be provided on the surface of the phosphor layer opposite to the side in contact with the support. Examples of materials used for the transparent protective film include cellulose acetate, polymethyl methacrylate, polyethylene terephthalate, and polyethylene. The thickness of the transparent protective film is usually about 0.1
The thickness is between 20 μm and 20 μm.

The surface of the stimulable phosphor sheet produced in this manner may be subjected to various surface treatments in order to improve the adhesion to the separation and development support medium superimposed thereon. For example, by performing surface activation treatment such as glow discharge treatment or roughening treatment on the surface of the protective film (or surface of the support),
Hydrophilicity may be imparted. A stimulable phosphor sheet subjected to a hydrophilic treatment is described in Japanese Patent Application No. 1983-30605 filed by the present applicant.

Next, support media for separating and developing radioactively labeled substances derived from living organisms are available for various types of separation and development that have been utilized in conventional autoradiography techniques or have been proposed for use in conventional autoradiography techniques. Any support medium can be selected. Examples of such support media for separation development include gel support media, polymer moldings such as acetate membranes, support media for electrophoretic separation in the form of various support media such as filter paper, and thin layers such as silica gel. Mention may be made of support media for chromatography. These support media for development and separation are usually used in a dry state, but if desired, they may be impregnated with, for example, a solvent for separation and development. Furthermore, these supporting media for separation and development may be provided with supporting aids such as glass plates, plastic sheets, etc.

Note that the support medium for separation and development is not limited to the support media exemplified above, and any support medium can be used as long as it can be used for separation and development of a sample in autoradiography technology.

Further, the support medium for separation and development may be attached to the stimulable phosphor sheet from the beginning to form an integrated structure. In the case of an integral type, the support medium is a radiation emitted from a radiolabeled substance in the support medium (α
Since the intensity of rays, β rays, etc.) is weak, it is usually attached to the surface of the phosphor layer of the stimulable phosphor sheet (or the surface of the protective film if a protective film is provided).

For details on the support medium for separation and development and the stimulable phosphor sheet described above, please refer to Japanese Patent Application No. 1934-1988 and Japanese Patent Application No. 30604-1988 filed by the present applicant as separate and integrated measurement kits, respectively. It is stated in the specification.

The basic structure of the photographic material used in the present invention is a support and a photographic emulsion layer. The photographic emulsion layer consists of a binder, such as gelatin, containing and supporting silver halide in a dispersed state. A photosensitive material is one in which a transparent sheet such as polyethylene terephthalate is used as a support, and the above-mentioned photographic emulsion layer is provided on this sheet, and an example thereof is a radiation film such as a high-sensitivity X-ray film. I can do it.

The autoradiographic operation of the present invention will now be described.

A sample to be subjected to separation and development in the present invention,
That is, examples of biologically derived substances having radioactive labels include polymeric substances such as proteins, nucleic acids, derivatives thereof, and decomposition products thereof. Note that the biologically derived substances to be measured by the autoradiography of the present invention are not limited to the above-mentioned polymeric substances. Radiolabeled substances can be obtained by allowing these substances to retain radioactive elements in an appropriate manner. The radioactive elements used in the present invention include radiation (α rays, β rays,
Any nuclide may be used as long as it emits rays, gamma rays, neutron rays, X-rays, etc.), but typical examples include ³² P, ¹⁴ C, ³⁵ S, ³ H, ¹²⁵ I and so on.

In addition, separation and development methods using the various support media for separation and development as described above, such as methods of performing electrophoresis and forming separation and development arrays of samples on the support medium, are already well known. I won't touch on it in particular.

Next, the support medium on which the sample has been separated and developed and the stimulable phosphor sheet are placed on top of each other for a certain period of time, preferably in a dark place or dark box, and an exposure operation is performed. Generally, the intensity of radiation emitted from a radiolabeled substance in a support medium is weak, so stimulable phosphor sheets are preferably stacked so that the surface of the phosphor layer (or the surface of the protective film) is in contact with the support medium.

By causing the stimulable phosphor sheet to absorb at least a portion of the radiation emitted from the radiolabeled substance in the support medium during the exposure operation, an autoradiograph of the radiolabeled substance is recorded on the sheet as an image of accumulated radiation energy. be done.

This exposure time varies depending on the strength of the radioactivity of the radiolabeled substance contained in the sample, the concentration and density of the substance, or the sensitivity of the stimulable phosphor sheet. However, when a stimulable phosphor sheet is used in accordance with the present invention, the exposure time is significantly reduced compared to that required for radiography using conventional radiation films.

Although there is no particular restriction on the temperature at which the exposure operation is performed, autoradiography using the stimulable phosphor sheet of the present invention can be performed particularly at an environmental temperature of 10 to 35°C. However, the exposure operation may be performed at a low temperature (eg, around 5° C. or lower) as used in conventional autoradiography.

As will be described later, when reading out (and exposing the photographic light-sensitive material) with the support medium for separation and development in close contact with the stimulable phosphor sheet, if the reading device for the stimulable phosphor sheet is light-shielding, After these are superimposed in a bright place, exposure can be performed within the reading device.

In addition, if the support medium is an integrated structure attached to the stimulable phosphor sheet, there is no need to overlap the two before performing the above exposure operation, but it is necessary to irradiate the two with appropriate light, heat, etc. By doing so, the radiation energy accumulated in the stimulable phosphor sheet during the separation and development process of the sample is emitted as fluorescence. In other words, during the separation and development process, the stimulable phosphor sheet is exposed to radioactivity such as impurities in the sample and to radiation emitted from the moving radiolabeled substance, so this is the radiation energy that produces the desired autoradiograph. This becomes noise in the accumulated image. Therefore,
It is desirable to eliminate noise before forming an autoradiograph to be measured on a stimulable phosphor sheet as an image of accumulated radiation energy.

Note that the above-mentioned noise erasing operation can be performed with the support medium on which the sample is separated and developed as it is, or after it has been subjected to any desired treatment such as drying or fixation of the separated and developed product. .

Next, with the support medium for separation and development in close contact with the stimulable phosphor sheet, or after separating the support medium from the stimulable phosphor sheet, the photographic light-sensitive material is superimposed on the stimulable phosphor sheet,
The process begins in which the autoradiograph stored and recorded on the sheet is converted into an image on a photosensitive material. Depending on the purpose, the support medium can be easily removed from the stimulable phosphor sheet by, for example, peeling or scraping the support medium, or washing it away using a solvent such as water.

The photographic light-sensitive material is not necessarily superimposed on the stimulable phosphor sheet after the exposure operation, but may be done before the exposure. However, if an erasing operation is required, it must be done after erasing.

Typical embodiments of the superposition of two materials consisting of a stimulable phosphor sheet and a photographic light-sensitive material in the photosensitive process, and the superposition of three materials consisting of a stimulable phosphor sheet, a support medium for separation and development, and a photographic light-sensitive material. Shown in Figure 1.

FIG. 1-(1) is a sectional view showing a state in which a photographic material 1b is superimposed on the phosphor layer _a2 side of the stimulable phosphor sheet 1a.

FIG. 1-(2) is a sectional view showing a state in which a photographic material 1b is superimposed on the support a ₁ side of a stimulable phosphor sheet 1a.

Figure 1-(3) shows a state in which the support medium 1c for separation and development is stacked on the phosphor layer a ₂ side of the stimulable phosphor sheet 1a, and the photographic light-sensitive material 1b is stacked on the support a ₁ side of the sheet. FIG.

Figure 1-(4) shows a state in which the photographic material 1b is stacked on the phosphor layer _a2 side of the stimulable phosphor sheet 1a, and the support medium 1c for separation and development is stacked on the support _a1 side of the sheet. FIG.

Here, 1a: stimulable phosphor sheet (a ₁ : support, a ₂ : phosphor layer) 1b: photographic light-sensitive material (b ₁ : support, b ₂ : photographic emulsion layer) 1c: support medium for separation and development It represents.

However, the superposition used in the present invention is not limited to the embodiments shown in FIG. , any superposition can be used as long as the sheet allows exposure of the photographic light-sensitive material. Further, as will be described later, the stimulable phosphor sheet and the photographic light-sensitive material do not necessarily have to be in close contact with each other, but may be superimposed closely together.

In the above superposition, the radiation emitted from the radiolabeled substance in the support medium for separation and development is absorbed and accumulated in the phosphor layer of the stimulable phosphor sheet, and also shines from the phosphor layer of the sheet by irradiation with excitation light. Since exhaust light is emitted, when a stimulable phosphor sheet and a photographic light-sensitive material are superimposed, the phosphor layer side of the sheet and the emulsion layer side of the photographic light-sensitive material should face each other. It is preferable to do so [Figure 1-(1)].
In addition, in the case of stacking a stimulable phosphor sheet, a support medium for separation and development, and a photographic light-sensitive material, the support medium for separation and development is stacked on the phosphor layer side of the sheet, while the support medium for separation and development is stacked on the support side. It is preferable that the photographic materials are stacked so that they are in contact with the emulsion layer side [Fig. 1-(3)].

The photosensitive operation of the photographic light-sensitive material for imaging the positional information of the radiolabeled substance contained in the autoradiograph stored and recorded on the stimulable phosphor sheet can be carried out, for example, as follows.

By scanning the entire surface of the stimulable phosphor sheet with a laser beam from a minute spot similar to that used in the readout operation described later, the radiation energy stored in the sheet is released in a time-series manner as photostimulated light. The photographic light-sensitive material closely adhered to the stimulable phosphor sheet is sensitized by stimulating light. In this case,
The laser beam may be scanned from the stimulable phosphor sheet (or the support medium if the support medium for separation and development is overlapped) or from the photographic material side. However, the wavelength range of the laser light must be selected within a wavelength range that does not overlap with the main wavelength range of stimulated luminescence emitted from the stimulable phosphor sheet and to which the photosensitive material is not sensitive. Therefore, usable laser light varies depending on the phosphor in the stimulable phosphor sheet and the photosensitive material in the photosensitive material, but preferably has a wavelength in the red region. Note that this photosensitive operation by laser beam scanning can be performed using the same device (readout device) used in the readout operation.

Alternatively, the photosensitive material can be exposed by scanning the entire surface of the sheet with a wide scanning light spot while the stimulable phosphor sheet and the photosensitive material are in close contact with each other. Alternatively, the entire surface of the stimulable phosphor sheet may be uniformly exposed (flattened) to excitation light using a lamp or the like.

Another method is to place the sheet and the photosensitive material in close proximity so that the surface of the stimulable phosphor sheet forms a lens image on the surface of the photosensitive material, and scan the entire surface of the sheet with a wide scanning light spot. The light-sensitive material can be sensitized by applying a uniform exposure to light (lens imaging method).

Next, the photographic material, which has been exposed to photostimulated light emitted from the stimulable phosphor sheet under excitation light to form a latent image, is separated from the stimulable phosphor sheet and then developed. , a visible image corresponding to an autoradiograph of the radiolabeled substance in the support medium is obtained.

In this way, images similar to autoradiographic images obtained by conventional radiography methods can be obtained.

Furthermore, in the present invention, by suitably adjusting the irradiation of excitation light in the above photosensitive operation,
In other words, by emitting only a portion of the radiation energy stored in the stimulable phosphor sheet, the stimulable phosphor sheet is irradiated with excitation light again, and the autoradiation stored and recorded on the stimulable phosphor sheet is activated. The positional information of the radiolabeled substance included in the graph can be obtained as an electrical signal.

A method for photoelectrically reading out the positional information of a radiolabeled substance accumulated and recorded on a stimulable phosphor sheet will be briefly described below with reference to the example of the reading device (or reading device) shown in FIG. .

FIG. 2 shows a schematic diagram of an example of a reading device for reading out one-dimensional or two-dimensional positional information of a radiolabeled substance stored and recorded on the stimulable phosphor sheet 1.

In the reading device, the following reading operation is performed.

The laser beam 3 generated from the laser light source 2 passes through the filter 4, so that a portion of the wavelength region corresponding to the wavelength region of stimulated luminescence generated from the stimulable phosphor sheet 1 in response to excitation by the laser beam 3 is filtered. It is cut. The laser beam 3 that has passed through the filter 4 then passes through the beam expander 5
The size of the beam diameter is precisely adjusted. Next, the laser beam passes through an optical deflector 6 such as a galvano mirror.
After being deflected by the plane reflecting mirror 7, the light is one-dimensionally deflected and incident on the stimulable phosphor sheet 1. Note that an fθ lens 8 or the like is arranged between the optical deflector 6 and the plane reflecting mirror 7, so that when the deflected laser beam scans the stimulable phosphor sheet 1, a uniform beam velocity is always maintained. has been done.

The laser light source 2 used here is selected so that the wavelength range of its laser light 3 does not overlap with the main wavelength range of stimulated luminescence emitted from the stimulable phosphor sheet 1.

The stimulable phosphor sheet 1 is transported in the direction of the arrow 9 under irradiation with the above-mentioned polarized laser light.
Therefore, the polarized laser beam is applied to the stimulable phosphor sheet 1.
The entire surface of the area will be irradiated.

When the stimulable phosphor sheet 1 is irradiated with the laser light as described above, it exhibits stimulated luminescence with an amount of light proportional to the accumulated radiation energy, and this light enters the light guide sheet 10 . The light guide sheet 10 has a linear incident surface and is placed close to the scanning line on the stimulable phosphor sheet 1 so as to face the scanning line, and its exit surface forms a ring and is used for photoelectron multiplication. It is connected to the light receiving surface of a photodetector 11 such as a tube. The light guide sheet 10 is made by processing a transparent thermoplastic resin sheet such as acrylic synthetic resin, and allows light incident from the incident surface to be transmitted to the exit surface while being totally reflected inside. It is configured to The stimulated luminescence from the stimulable phosphor sheet 1 is guided through the light-guiding sheet 10 and reaches the exit surface, is emitted from the exit surface, and is received by the photodetector 11. Photodetector 11
A filter is attached to the light-receiving surface of the sensor, which transmits only light in the wavelength range of stimulated luminescence and cuts out light in the wavelength range of excitation light (laser light), so that only stimulated luminescence can be detected. There is. The stimulated luminescence detected by the photodetector 11 is converted into an electrical signal, and the amplifier 13 whose sensitivity is set according to the amplification factor setting value a output from the control circuit 12 amplifies the electrical signal to an appropriate level.

The obtained electrical signal is then input to the A/D converter 14. The A/D converter 14 converts the digital signal into a digital signal using a scale factor suitable for the signal fluctuation range according to the recording scale factor setting value b output from the control circuit 12, and inputs the digital signal to the signal processing circuit 15. In the signal processing circuit 15, the digital signal is subjected to suitable signal processing and outputted as digital data.Then, if necessary, the signal is sent to a recording device (not shown) via a storage means such as a magnetic tape.
transmitted to.

Note that the amplification factor setting value a and the recording scale factor b output from the control circuit 12 are based on accumulated record information obtained by performing a preliminary read operation (pre-read operation), for example, before the above read operation. Depending on the situation, the above factors a and b can be set to obtain a signal at an appropriate level, or if the content of radioactive substances in the sample is known in advance, the fluorophore for that sample can be set. These factors can also be set empirically depending on the exposure time of the sheet.

In the signal processing circuit 15, the input digital signal is subjected to calculation processing such as analyzing the distribution site of the radiolabeled substance and its radiation intensity, and the positional information of the radiolabeled substance is converted into symbols and/or numerical values. Obtained as digital data. A signal processing method for obtaining positional information of a radiolabeled substance distributed one-dimensionally in the form of symbols and/or numerical values is described, for example, in Japanese Patent Application No. 1327/1983 filed by the present applicant. That is, after obtaining the positional information of a radiolabeled substance (for example, a cleavage product of radiolabeled DNA) separated and developed one-dimensionally on a support medium as a digital signal as described above, the digital The signal is subjected to signal processing consisting of determining the one-dimensional distribution direction of the radiolabeled substance and then detecting the distribution points of the radiolabeled substance along this distribution direction, thereby obtaining positional information of the radiolabeled substance (e.g. , DNA base sequence) as desired symbols and numerical values.

The above specification also describes a method of converting the electrical signal or digital signal obtained as described above into an image using a reproducing/recording device in order to obtain positional information of a radiolabeled substance in the form of an image. There is. In the present invention as well, by inputting the reproduced image processing condition setting value c from the control circuit 12 to the signal processing circuit 15, the digital camera can be used to obtain a visible image with appropriate density and contrast and excellent observation and interpretation performance. signal (or A/D
Suitable image processing may be performed on the electrical signal (before conversion). Examples of image processing include spatial frequency processing, gradation processing, and subtraction processing.

Examples of recording devices include those that optically record by scanning a photosensitive material with a laser beam or the like;
What is displayed electronically on a CRT, etc., or the numbers, symbols, or radiation images displayed on a CRT, etc.
Recording devices based on various principles can be used, such as those that record on a printer or the like, and those that record on a heat-sensitive recording material using heat rays.

In addition, in the above readout operation, the case where only the stimulable phosphor sheet was used was explained.
Stimulated light can also be detected on stimulable phosphor sheets overlaid on a support medium for separation and development. In this case, irradiation with laser light (excitation light) and detection of stimulated luminescence are preferably performed from the phosphor layer side of the stimulable phosphor sheet.

Furthermore, as a method for reading out the positional information of the radiolabeled substance in the sample that has been transferred and accumulated on the stimulable phosphor sheet in the present invention, it is of course possible to use an appropriate method other than those exemplified above. .

For example, when trying to obtain positional information of a radiolabeled substance that has been separated and developed one-dimensionally on a support medium (for DNA base sequencing, etc.), a preliminary reading operation is first performed, and the resulting digital After determining the one-dimensional distribution direction of the radiolabeled substance based on the signal, in the main reading operation, only a certain region along this distribution direction may be scanned with excitation light. Details of such a signal detection method are described in Japanese Patent Application No. 58-57417 filed by the present applicant. According to this method, there is no need to scan the entire surface of the stimulable phosphor sheet with excitation light, and only the digital signal having the desired position information is efficiently stored in the memory in the signal processing circuit, so that it is not necessary to scan the entire surface of the stimulable phosphor sheet. Time can be shortened, high precision scanning is not required, and the readout device can therefore be simplified and inexpensive.

Alternatively, if a separation and development column (for example, an electrophoresis column) formed by one-dimensionally separating and developing a radiolabeled substance is formed in a straight line and almost parallel on a support medium, the excitation in the main reading operation By setting the light scanning conditions (scanning direction, scanning width, etc.) in advance, the above preliminary operation can be omitted, and the reading operation can be further simplified.

In addition, although the case where the reading operation of the stimulable phosphor sheet is carried out after carrying out the photosensitive operation of the photographic light-sensitive material has been described above, it is also possible to carry out the reading operation and then the photosensitive operation in the reverse order. You can also do that.

By directly comparing the digital data regarding the position information of the radiolabeled substance obtained in this way with the autoradiographic image visualized on the photosensitive material, confirmation and further analysis of the obtained position information can be carried out. can be done. Moreover, this autoradiographic image and image-processed digital data (image) can also be compared.

Therefore, using a single stimulable phosphor sheet,
It is possible to image an autoradiograph having the positional information of the radiolabeled substance stored and recorded on the sheet and to read out the positional information. especially,
If exposure and readout are performed while the support medium for separation and development is also stacked together, autoradiographic measurement essentially involves the separation and development process of the sample on the support medium and the exposure of the stimulable phosphor sheet. The process can be simplified into three steps: a photosensitive step including 1, and a readout step. Furthermore, if the exposure step and the readout step are performed in the same device, the process can be simplified to two steps.

Next, an embodiment of the autoradiography of the present invention will be described using the initial operation of a DNA base sequencing method as an example.

The support medium for separation and development used in the following examples was a slab gel (1.5 mm
This is a support medium for electrophoresis consisting of 200mm x 200mm). Further, the stimulable phosphor sheet was prepared as follows.

Methyl ethyl ketone was added to a mixture of photostimulable divalent europium-activated barium fluoride bromide phosphor (BaFBr: Eu ²⁺ ) particles and linear polyester resin, and further nitrocellulose with a nitrification degree of 11.5% was added. A dispersion containing phosphor particles in a dispersed state was prepared. Next, tricresyl phosphate, n-butanol, and methyl ethyl ketone were added to this dispersion, and then thoroughly stirred and mixed using a propeller mixer to ensure that the phosphor particles were uniformly dispersed and that the viscosity was 25 to 35 PS (25 ℃) coating solution was prepared.

This coating solution was uniformly applied using a doctor blade onto a carbon black kneaded polyethylene terephthalate sheet (support, thickness: 250 μm) placed horizontally on a glass plate. After coating, the support on which the coating film has been formed is placed in a dryer, and the temperature inside the dryer is gradually raised from 25°C to 100°C to dry the coating film. A phosphor layer with a layer thickness of 300 μm was formed on.

Next, after applying a polyester adhesive to one side of a transparent polyethylene terephthalate film (thickness: 12 μm), a protective film was formed by adhering it to the phosphor layer with the adhesive layer side facing down. A stimulable phosphor sheet composed of a support, a phosphor layer, and a protective film was prepared.

[Example 1] Isolation and radioactive labeling of DNA to be sequenced Escherichia coli plasmid DNA (pBR322) was prepared using a conventional method.
After cutting with the restriction enzyme Hind-, the 5'-
Double-stranded DNA ( ^32P -labeled product) by labeling the ends with ^32P
1 μg was obtained.

5mM magnesium chloride prepared separately and
Add the above double strand to 20 μl of 20 mM Tris [tris(hydroxymethyl)aminomethane]/hydrochloric acid buffer (PH7.4) containing 1 mM dithiothreitol.
Add 1 μg of DNA and about 1 unit of restriction enzyme Hae,
A specific decomposition reaction was carried out at 37°C for 1 hour to obtain a decomposition mixture solution containing decomposition products of the above fragments.

Using this decomposition mixture solution as a sample and using the above-mentioned electrophoresis support medium and 50mM Tris-borate buffer (PH8.3) containing 1mM EDTA as an electrode solution, the sample was placed on a slab gel support medium at a voltage of 500V. Electrophoresis operation was carried out. The electrophoresis was stopped when the marker dye previously added to the sample reached the lower end of the gel support medium, and the origin of the coordinate axes was marked with ³² P-containing ink.

Next, the gel support medium and the stimulable phosphor sheet were stacked one on top of the other, and exposed at room temperature (approximately 25° C.) for 12.5 minutes.

The gel support medium is then separated from the stimulable phosphor sheet and replaced with X-ray film (RX type,
Fuji Photo Film Co., Ltd.) was placed on the protective film side of the stimulable phosphor sheet, and then introduced into a reading device as shown in Figure 2, excitation light (He-Ne laser light) was applied from the X-ray film side. , wavelength: 633 nm, light energy: 7 × 10 ^-4 J/cm ² ), and sensitize the X-ray film by stimulated emission of divalent europium-activated barium fluoride bromide phosphor (peak wavelength: 390 nm). I let it happen.

After separating the stimulable phosphor sheet and the X-ray film, the X-ray film was developed by a conventional method. On the film, the electrophoretic pattern of the decomposition product with the ³² P label was imaged.

Next, by introducing the sheet into the same device as the stimulable phosphor sheet and scanning with excitation light in the same manner as above, ^{the 32P} -labeled fragments are decomposed using the position marked with ^32P -containing ink as the origin of the label axis. Position information indicating the migration position of the product was read out.

According to the obtained positional information, a gel portion containing a ³² P-labeled decomposition product was cut out of the slab gel support medium using a thin razor, and this was transferred to a test tube. For confirmation, the remaining gel support medium from which the above partial cutting operation was performed was superimposed on the stimulable phosphor sheet again, and the remaining decomposition products with the ^32P label were detected using a readout device. When the presence or absence was examined, it was found that the entire amount of decomposition products having ^32P labels had been removed.

That is, it was confirmed that the positional information of the decomposition product having the ³² P label obtained by reading out the stimulable phosphor sheet provided with the support medium was highly accurate.

[Brief explanation of drawings]

Figure 1-(1) to (4) show the state in which the stimulable phosphor sheet and the photosensitive material are stacked [(1) and (4), respectively.
(2)] and a state in which the support medium for separation and development, the stimulable phosphor sheet, and the photographic light-sensitive material are stacked [(3) and (4)]. 1a... stimulable phosphor sheet (a ₁ ... support,
a ₂ ... phosphor layer), 1b ... photographic material (b ₁ ...
...Support, b ₂ ... Photographic emulsion layer) Figure 2 shows a reading device (or reading device) for reading the positional information of the radioactive labeling substance accumulated and recorded on the stimulable phosphor sheet in the present invention. This is an example. DESCRIPTION OF SYMBOLS 1... stimulable phosphor sheet of photographic light-sensitive material superimposed, 2... laser light source, 3... laser light, 4... filter, 5... beam expander, 6... light deflector, 7... ...Flat reflector,
8...fθ lens, 9...transfer direction, 10...light guide sheet, 11...photodetector, 12...control circuit, 13...amplifier, 14...A/D converter, 1
5...Signal processing circuit.

Claims (1)

[Scope of Claims] 1. An autoradiographic measurement method for obtaining one-dimensional or two-dimensional positional information of a biological substance to which a radioactive label has been applied and which has been separated and developed on a support medium, comprising: 1 By overlapping this support medium and a stimulable phosphor sheet containing a stimulable phosphor for a certain period of time, at least a portion of the radiation energy emitted from the radiolabeled substance in the support medium is absorbed by the sheet. Step 2: After overlapping the stimulable phosphor sheet and the photosensitive material, irradiating the stimulable phosphor sheet with excitation light to release the radiation energy stored in the sheet as photostimulated light; An autoradiographic measuring method comprising the steps of: and sensitizing a photographic light-sensitive material with the stimulated light to obtain positional information of a radiolabeled substance as an image on the light-sensitive material. 2. The autoradiographic measurement method according to claim 1, wherein step 2) is performed by irradiating the stimulable phosphor sheet with excitation light and scanning with a laser beam. 3. The autoradiographic measurement method according to claim 1, wherein in step 2), the stimulable phosphor sheet is irradiated with excitation light by uniform exposure. 4. Claim 1, wherein the stimulable phosphor sheet has a support, a phosphor layer made of a stimulable phosphor dispersed in a binder, and a protective film.
The autoradiographic measurement method as described in any one of Items 1 to 3. 5. The autoradio according to claim 1, wherein the biologically-derived substance to which a radioactive label has been applied is a biopolymer substance to which a radioactive label has been applied, a derivative thereof, or a decomposition product thereof. Graph measurement method. 6. The autoradiographic measurement method according to claim 5, wherein the biopolymer substance is a nucleic acid, a derivative thereof, or a decomposition product thereof. 7 In an autoradiographic measurement method for obtaining one-dimensional or two-dimensional positional information of a biological substance to which a radioactive label has been applied and which has been separated and developed on a support medium, 1. a step of overlapping a stimulable phosphor sheet containing a stimulable phosphor for a certain period of time, thereby causing the sheet to absorb at least a portion of the radiation energy emitted from the radiolabeled substance in the support medium; 2. the stimulable phosphor sheet; After overlapping the phosphor sheet and the photographic light-sensitive material, the stimulable phosphor sheet is irradiated with excitation light to release the radiation energy stored in the sheet as photostimulated light. and (3) scanning the stimulable phosphor sheet with excitation light to radiate the radiation energy stored in the sheet. A step of emitting the stimulated light and photoelectrically detecting the stimulated light to obtain position information of the radiolabeled substance as an electrical signal, and the step of 3) above is performed before the step of 2) above. , or an autoradiographic measurement method characterized by being carried out afterward. 8. The autoradiographic measurement method according to claim 7, wherein in the step 2), the stimulable phosphor sheet is irradiated with excitation light by uniform exposure. 9. The autoradiographic measurement method according to claim 7, wherein in step 2), the stimulable phosphor sheet is irradiated with excitation light by scanning with laser light. 10. The autoradiographic measurement method according to claim 7, wherein in the step 3), a laser beam is used as excitation light. 11 In the step 3) above, a laser beam is used as the excitation light, and this laser light is the same laser light as the laser light used in the step 2) above, Claim 9
Autoradiographic measurement method described in section. 12 Claim 7, characterized in that the electrical signal obtained in step 3) above is converted into a digital signal and then subjected to signal processing to obtain the positional information of the radiolabeled substance as a symbol and/or numerical value. Autoradiographic measurement method described in section. 13. Claims 7 to 12, characterized in that the stimulable phosphor sheet has a support, a phosphor layer made of a stimulable phosphor dispersed in a binder, and a protective film. The autoradiographic measurement method described in any of the sections. 14. The autoradio according to claim 7, wherein the biologically-derived substance to which a radioactive label has been applied is a biopolymer substance to which a radioactive label has been applied, a derivative thereof, or a decomposition product thereof. Graph measurement method. 15. The autoradiographic measurement method according to claim 14, wherein the biopolymer substance is a nucleic acid, a derivative thereof, or a decomposition product thereof.