JPH0465997B2 - - Google Patents

Description

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

[Background of the Invention] After administering a substance to which a radioactive label has been given to an organism, the organism or a part of the tissue of the organism is used as a sample, and this sample is combined with a radioactive film such as a high-sensitivity X-ray film. Autoradiography (also known as radioautography) consists of exposing (or exposing) the film to light by overlapping them for a certain period of time, and obtaining positional information of the radiolabeled substance in the sample from the exposed area. (called autoradiographic measurements) are 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.
For example, it is described in the following documents.

Biochemistry Experiment Course 6 Tracer Experiment Method (Part 1) 271
~289 pages, “8. Autoradiography” Toru Sueyoshi,
Akiyo Shigematsu (1977, published by Tokyo Kagaku Dojin Co., Ltd.) In recent years, autoradiography has been used to attach radioactive labels to polymeric substances derived from living organisms, such as proteins and nucleic acids, and then analyze the high It is also effectively used to obtain positional information of a radiolabeled substance on a support medium obtained by separating and developing a molecular substance, its derivative, or its decomposition product by gel electrophoresis or the like. 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 relatively high temperatures, such as room temperature, the latent image in the silver salt on the film formed by exposure to radiation or fluorescence tends to regress, resulting in an undevelopable image, and the supporting medium may be harmful to the silver salt. This is because chemical components tend to migrate and form 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 the accuracy of information decreases. 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 proposed to solve the above-mentioned problems or reduce the drawbacks by using a stimulable phosphor sheet containing a stimulable phosphor instead of a radiation film as a photosensitive material in an autoradiographic measurement method. An application has already been filed for an invention that realizes the following (Japanese Patent Application No. 193418/1983).

In the above, the stimulable phosphor sheet is also called a radiation image conversion panel, an example of which is JP-A-55-
It is described in Publication No. 12145, etc., and is already known as a general welfare measure.

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 photosensitive film, a display device such as a CRT, or expressed as position information converted into a numerical value or signal.

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, becomes extremely simple, and the autoradiography operation is simplified.

Furthermore, by using the above-mentioned stimulable phosphor sheet as a photosensitive material in the autoradiographic measurement method, chemical fog and physical fog, which have traditionally been a major problem when using radiation film, are virtually eliminated. This has a very advantageous effect on improving the accuracy of the obtained position information and on workability. In addition, deterioration in accuracy due to radioactivity of impurities contained in the sample or natural radioactivity can be easily reduced by electrically processing the position information stored and recorded on the stimulable phosphor sheet. Or it can be resolved.

Furthermore, when a stimulable phosphor sheet is used as a photosensitive material, there is no need for special imaging to obtain positional information of the radiolabeled substance accumulated and recorded on the stimulable phosphor sheet. The above position information is read out by scanning with excitation light such as a laser, and the position information is converted into an image.
It is possible to extract the information in any form such as a symbol and/or a numerical value, or a combination thereof. This image information can be obtained in various desired forms by further processing via electrical means, that is, an electric signal having the image information, or a signal processing of an A/D converted digital signal. It is also possible to obtain this information. For example, it is also possible to obtain information regarding the target biological system by analyzing, using a computer or the like, an electrical signal or digital signal having positional information of a radiolabeled substance obtained by reading out a stimulable phosphor sheet.

The present applicant has also already applied for a signal processing method for obtaining positional information of a radiolabeled substance obtained as a digital signal in the form of symbols, numerical values, etc. (Japanese Patent Application No. 1327/1984, etc.). That is, after obtaining the positional information of a radiolabeled substance (for example, a cut and degraded product of radiolabeled DNA) that has been separated and developed one-dimensionally on a support medium as a digital signal, the digital signal is subjected to signal processing. By applying
This method consists of obtaining the base sequence (base sequence) as desired symbols and numerical values. The above specification also describes a method of converting the obtained electrical signal or digital signal into an image using a reproducing/recording device in order to obtain the image in the form of an image. By obtaining the positional information of the radiolabeled substance as a visible image in this way, the information obtained as symbols and numerical values can be compared with the image. It also allows comparison with other visualized autoradiographs.

In particular, until now autoradiography has exclusively used conventional radiographic methods, and the current method is to directly compare the positional information of radiolabeled substances with the visible images obtained by this conventional method. It is desired to obtain the information in the form of an image so that it can be used. Therefore, 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 equipment tends to be complicated.

[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. Irradiate the body sheet with excitation light and detect (read out) the photostimulated light emitted from the stimulable phosphor sheet.
In some cases, by superimposing a conventional photographic light-sensitive material on a stimulable phosphor sheet, on the one hand, the radioactive label is placed on the photographic light-sensitive material by the stimulated light emitted from the stimulable phosphor sheet. The inventors have discovered that it is possible to obtain position information of a substance as an image and, at the same time, to obtain an electrical signal by photoelectrically detecting this stimulated light, and have arrived at the present invention. In other words, by simultaneously using the stimulable phosphor sheet as a conventional intensifying screen (radiation intensifying screen), it is possible to obtain positional information of a radiolabeled substance as an electrical signal and directly as a visible image. It has been found that the autoradiography can be carried out simultaneously, thus further simplifying the autoradiographic operation.

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 is attached and which is 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) separating the stimulable phosphor sheet and overlaying it on a photographic light-sensitive material; scanning the stimulable phosphor sheet with excitation light to emit the radiation energy stored in the sheet as photostimulated light; By sensitizing a photographic light-sensitive material with the stimulated light, positional information of the radioactively labeled substance is obtained as an image on the light-sensitive material, and on the other hand, by photoelectrically detecting the stimulated light, the location information of the radioactively labeled substance is obtained. The present invention provides an autoradiographic measurement method characterized by comprising the steps of: obtaining position information as an electrical signal.

The present invention also 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 is attached and which is separated and developed on a support medium. ) absorbing at least a portion of the radiation energy emitted from the radiolabeled substance in the support medium into a stimulable phosphor sheet containing a stimulable phosphor superimposed on the support medium;
(The support medium and the storage phosphor sheet may be overlapped before the separation and expansion operation is performed on the support medium, or they may be overlapped after the separation and expansion operation is completed.) (2 ) After superimposing the photographic light-sensitive material on the surface of the stimulable phosphor sheet opposite to the support medium side, the stimulable phosphor sheet is inspected with excitation light to detect the radiation energy stored in the sheet. The positional information of the radiolabeled substance is obtained as an image on the photosensitive material by emitting it as photostimulated light and exposing a photographic light-sensitive material to the photosensitivity, and on the other hand, the photostimulated light is photoelectrically detected. The present invention also provides an autoradiographic measurement method comprising the steps of: obtaining positional information of a radiolabeled substance as an electrical signal.

The former measurement method is a method in which reading is performed with the stimulable phosphor sheet and the photographic material superimposed, while the latter measurement method is a method in which the stimulable phosphor sheet, the photographic material, and the support medium for separation and development are read out. This is a method of reading data in an overlapping state. In the latter measurement method, the stimulable phosphor sheet and the support medium for separation and development may be superimposed before the radiolabeled substance is separated and developed on the support medium, or the radioactive energy from the radiolabeled substance may be overlaid. It may be done immediately before being absorbed into the stimulable phosphor sheet.

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, when reading out the stimulable phosphor sheet in which the positional information of the radiolabeled substance on the support medium is stored and recorded as radiation energy by irradiating excitation light, By keeping the photographic light-sensitive material in close contact with each other, the photostimulated light emitted from the stimulable phosphor sheet upon irradiation with excitation light is photoelectrically detected, and at the same time, the photographic light-sensitive material is sensitized by this photosensitivity. be able to. In other words, part of the stimulated light enters a photodetector such as a photomultiplier tube placed close to the sheet and is photoelectrically detected and converted into an electrical signal. In this method, a photosensitive material adhered to a sheet is exposed to light to form an image on the photosensitive material.

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 first enters the photographic light-sensitive material, and a portion of it is absorbed by the light-sensitive substance in the light-sensitive material, thereby sensitizing the light-sensitive material and contributing to image formation. Thereafter, the photostimulated light that has passed through the remaining photosensitive material enters a photodetector and is photoelectrically detected to obtain an electrical signal. Here, since the excitation light wavelength range for exciting the stimulable phosphor contained in the stimulable phosphor sheet is different from the wavelength range of the stimulable light emitted from this phosphor, the photographic light-sensitive material cannot be excited. It has been found that a desired image can be formed on a photosensitive material by being exposed only to stimulated light without being exposed to light. On the other hand, by adjusting the reading gain to an appropriate value in the readout (reading) device, the remaining photostimulated light after exposing the photographic light-sensitive material can be processed without being affected by variations in sensitivity of the light-sensitive material. It has been found that this can be obtained as an electrical signal with sufficient accuracy.

In addition, in the present invention, the meaning that a photographic light-sensitive material is not sensitive to excitation light means that the sensitivity at the excitation light wavelength is significantly lower than the sensitivity at the highest sensitivity wavelength region of the photographic light-sensitive material, This does not mean that the photographic material is not sensitive to excitation light at all.

Therefore, the position information of the radiolabeled substance separated and developed on the support medium can be obtained as digital data, and at the same time, it can be obtained as an image on the photographic material without using a special device such as an image reproduction device. In other words, autoradiography operations can be simplified and costs can be reduced.

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. Comparison with images (autoradiographic images) becomes easier.
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. This also means that the position information of the radiolabeled substance can be recorded and stored in the form of digital data on a magnetic tape or the like, and at the same time, it can also be stored in the form of an image recorded on a photosensitive material.

The second method of the present invention, that is, a photographic material,
According to the method of performing reading with the stimulable phosphor sheet and the support medium for separation and development in close contact with each other, in addition to the above-mentioned advantages, there are also advantages that the support medium for separation and development and the stimulable phosphor sheet are After superimposing the two materials and performing an exposure operation, it is possible to further superpose the photosensitive materials and subject them to a reading process without separating the two. 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 or a suitable solvent may be used before the reading process. No rinsing is required, simplifying autoradiography operations.

Furthermore, if the reading device for reading out the positional information of the radiolabeled substance accumulated and recorded on the stimulable phosphor sheet is light-shielding, there is no need to provide a special dark place to perform the exposure operation. That is, if a stimulable phosphor sheet on which a support medium and a photographic light-sensitive material are superimposed is left still in a reading device for a certain period of time, the stimulable phosphor sheet is exposed to light, and then the light-sensitive material is exposed and read out. It's good. Therefore, it becomes possible to carry out the exposure operation of the stimulable phosphor sheet using a support medium containing a radiolabeled substance and the readout operation of the stimulable phosphor sheet, which also serves as the exposure of the photographic light-sensitive material, in one continuous process. It is something.

[Detailed Description of the Invention] The stimulable phosphor sheet used in the present invention basically consists 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 not subject to chemical alteration or physical alteration. Protects from physical impact.

A stimulable phosphor sheet having the above-mentioned properties 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 (or 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, metal sheets such as aluminum foil, ordinary paper, baryta paper, resin-coated paper, and the like. Note that an adhesion-imparting layer, a light-reflecting layer, a light-absorbing layer, etc. may be provided on the surface of the support on which the phosphor layer is provided. As shown in the figure, fine irregularities may be uniformly formed (these irregularities are caused by the fact that 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). (in some cases, it forms 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. be done. 600~830n from a practical point of view
350-500nm depending on the excitation light in the wavelength range of m
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〓O・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] (Ba _1-xy , Mg _x , Ca _y ) is described in JP-A-12143-1983. 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 500 μm.

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 transparent protective films include:
Mention may be made of cellulose acetate, polymethyl methacrylate, polyethylene terephthalate and polyethylene. The thickness of the transparent protective film is usually approx.
It is 0.1 to 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 samples in autoradiography technology. .

Further, the support medium for separation and development may have an integral structure attached to the stimulable phosphor sheet from the beginning. 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).

Details of the support medium for separation and development and the stimulable phosphor sheet described above are disclosed in Japanese Patent Application Nos. 57-193419 and 1983-30604 filed by the present applicant as separate and integrated measurement kits, respectively. It is written in the book.

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. Examples include radiation films such as high-sensitivity X-ray films. 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 There are l, etc.

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 a 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 stacked so that the surface of the phosphor layer (or the surface of the protective film) is in contact with the support medium. However, it is also possible to superpose a support medium for separation and development on the support side of the stimulable phosphor sheet.

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 is recorded on the sheet as an image of accumulated radiation energy.

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. In addition, in the operation of reading the positional information of the radiolabeled substance on the support medium that has been transferred and accumulated from the support medium to the stimulable phosphor sheet by exposure, the strength and distribution of the energy stored in the stimulable phosphor sheet,
Since the state of the obtained position information can be changed by performing various electrical processes depending on the desired information, there is no particular need to strictly control the exposure time during the exposure operation.

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 (for example, around 5° C. or lower) as used in conventional autoradiography.

As will be described later, when reading is performed with the support medium for separation and development, the stimulable phosphor sheet, and the photographic light-sensitive material in close contact with each other, if the readout device for the stimulable phosphor sheet is light-shielding, these three can be read out. After superposition in the bright light, exposure can be carried out in the readout device.

In addition, if the support medium is an integrated structure attached to the stimulable phosphor sheet, there is no need to overlay 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, the stimulable phosphor is produced by the natural radioactivity contained in the sample, or by the radiation emitted from the moving radiolabeled substance during the separation and development of the radiolabeled sample on the support medium. When the sheet is exposed, a radiation energy accumulation image other than the one to be measured is formed on the stimulable phosphor sheet, which becomes noise to the radiation energy accumulation image containing the desired autoradiograph. Therefore, if the influence of the noise is not negligible,
It is desirable to eliminate the noise before forming a radiation energy accumulation image with the desired autoradiograph on the stimulable phosphor sheet.

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 autoradiograph stored and recorded on the stimulable phosphor sheet is read out. The support medium can be easily removed from the stimulable phosphor sheet by, for example, peeling or scraping the support medium, washing it away using a solvent such as water, etc., depending on the purpose.

The overlay of the photographic light-sensitive material on the stimulable phosphor sheet does not necessarily have to be carried out after the exposure operation, but may be carried out before the exposure. is preferable. However, when performing an erasing operation, it must be done after erasing.

Superposition of two components consisting of a stimulable phosphor sheet and photographic material in the readout process (exposure and readout process), and superposition of three components consisting of a stimulable phosphor sheet, a support medium for separation and development, and a photographic material. A typical embodiment is shown in FIG.

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 a 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.

FIG. 1-3 is a cross-sectional view showing 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. It is a diagram.

Figure 1-4 is a cross section showing 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. It is a diagram.

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 FIGS. Any superposition can be used as long as the exposure of the photographic material and the readout of the sheet are possible.

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 side of the support medium. It is preferable that the photographic light-sensitive materials are superimposed so that they are in contact with the emulsion layer side [Fig. 1-3].

Since this overlapping state needs to be maintained during the readout operation, it is preferable that the photographic light-sensitive material, the stimulable phosphor sheet, and, if necessary, the supporting medium for separation and development be fixed so that they do not shift. For example, photographic materials (and supporting media)
One or both of the stimulable phosphor sheets may be mechanically processed so that they are fixed and superimposed on the stimulable phosphor sheet.

Regarding a method for imaging and reading out the positional information of a radiolabeled substance contained in an autoradiograph stored on a stimulable phosphor sheet,
A brief description will now be given with reference to an example of the reading device (or reading device) shown in FIG.

Figure 2 shows the primary state of radiolabeled substances accumulated and recorded on the overlapping stimulable phosphor sheet 1 of photographic light-sensitive material [Figure 1-1, 1a: stimulable phosphor sheet, 1b: photographic light-sensitive material]. 1 shows a schematic diagram of an example of a reading device for reading original or two-dimensional position information; FIG.

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

The laser light 3 generated from the laser light source 2 passes through the filter 4, and the stimulable phosphor sheet 1a is stimulated by the laser light 3.
A portion of the wavelength range corresponding to the wavelength range of stimulated luminescence generated from the photoluminescence is cut out. The beam diameter of the laser beam 3 that has passed through the filter 4 is then precisely adjusted by a beam expander 5.
Next, the laser beam is deflected by a light deflector 6 such as a galvano mirror, reflected by a flat reflecting mirror 7, and then the photographic light-sensitive material 1b (hereinafter referred to as "photosensitive material") of the stimulable phosphor sheet 1 on which the photographic light-sensitive materials are overlapped. (sometimes abbreviated as )) is deflected one-dimensionally and incident on the surface. Note that an fθ lens 8 or the like is arranged between the optical deflector 6 and the plane reflecting mirror 7, and the photosensitive material 1
When the deflected laser beam scans over b, a uniform beam velocity is always maintained.

The laser light source 2 used here is selected so that the wavelength range of the laser light 3 does not overlap with the main wavelength range of stimulated luminescence emitted from the stimulable phosphor sheet 1a and does not sensitize the photosensitive material 1b. . Although it depends on the phosphor in the stimulable phosphor sheet and the photosensitive substance in the photosensitive material, a preferable laser beam has a wavelength in the red region.

The stimulable phosphor sheet 1 made of superimposed photosensitive materials is transported in the direction of arrow 9 under irradiation with the above-mentioned polarized laser light. Therefore, the polarized laser beam passes through the photosensitive material 1b and irradiates the entire surface of the stimulable phosphor sheet 1a.

When the stimulable phosphor sheet 1a 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 a part of this light is absorbed by the photosensitive material 1b. An image (latent image) is formed on the photosensitive material 1b.

On the other hand, the light transmitted through the photosensitive material 1b 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 photosensitive material 1b, and has an exit surface that forms a ring and is arranged such as a photomultiplier tube, etc. The light receiving surface of the photodetector 11 is connected to the light receiving surface of the photodetector 11. 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 Stimulated luminescence from the stimulable phosphor sheet 1a is guided through the light guide sheet 10, reaches the exit surface, and is emitted from the exit surface to the photodetector 11.
The light is received by the A filter is attached to the light-receiving surface of the photodetector 11, which transmits only light in the wavelength region of stimulated luminescence and cuts out light in the wavelength region of excitation light (laser light), so that only stimulated luminescence can be detected. It is like that. The stimulated luminescence detected by the photodetector 11 is converted into an electrical signal, the sensitivity is set according to the amplification factor setting value a outputted from the control circuit 12, and the signal is amplified by the amplifier 13 to an electrical signal of an appropriate level.

The obtained electrical signal is then input to the A/D converter 14. The A/D converter 14 converts the signal fluctuation range into a digital signal using an appropriate scale factor 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 in one dimension as symbols and/or numerical values is described, for example, in the above-mentioned Japanese Patent Application No. 1327/1983. In addition, by inputting the reproduced image processing condition setting value c from the control circuit 12 to the signal processing circuit 15, the digital signal is Suitable image processing may also be performed. 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.

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, in the above, a method was described in which laser light (excitation light) is irradiated and stimulated luminescence is detected from the photographic light-sensitive material side. Alternatively, the detection of stimulated luminescence may be performed from the stimulable phosphor sheet side.

On the other hand, a photographic light-sensitive material in which a latent image has been formed by exposure to photostimulant light is separated from a stimulable phosphor sheet and then developed, resulting in a visible image corresponding to an autoradiograph of a radiolabeled substance in a support medium. is obtained.

In the above, the reading operation has been explained with reference to the embodiment shown in FIG. 1-1, but the same applies to other embodiments as well. By irradiating the photosensitive material with excitation light, it is possible to detect photostimulated light and sensitize the photographic light-sensitive material. It is preferable that the irradiation of excitation light and the detection of photostimulated light be performed from the phosphor layer side of the stimulable phosphor sheet.

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, the obtained position information can be confirmed and further analyzed. can be done. Moreover, this autoradiographic image and image-processed digital data (image) can also be compared.

Therefore, it is possible to simultaneously image and read out an autoradiograph having positional information of a radiolabeled substance stored and recorded on a stimulable phosphor sheet. In particular, when readout is performed while the support medium for separation and development is also stacked together, autoradiographic measurement essentially consists of the separation and development process of the sample on the support medium and the readout including exposure, sensitization, etc. It is possible to simplify 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 the substrate.

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 consisting 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.

The above duplex was added to 20μ of 20mM Tris[tris(hydroxymethyl)aminomethane]/hydrochloric acid buffer (PH7.4) containing 5mM magnesium chloride and 1mM dithiothreitol prepared separately.
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, using the above-mentioned support medium for electrophoresis and using 50mM Tris-borate buffer (PH8.3) containing 1mM EDTA as an electrode solution, apply it to 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 seal were superimposed and kept at room temperature (approximately 25° C.) for 12.5 minutes to perform an exposure operation.

The gel support medium is then separated from the stimulable phosphor sheet and replaced with X-ray film (RX type,
After overlapping the X-ray film (manufactured by Fuji Photo Film Co., Ltd.) on the protective film side of the stimulable phosphor sheet, a reading device as shown in Figure 2 was introduced and a mark was made from the X-ray film side using ^32P -containing ink. Position information indicating the migration position of the degradation product of the ³² P-labeled fragment was read out using the position as the origin of the coordinate axis. For reading, a He-Ne laser beam (wavelength: 633 nm, light energy: 7 x 10 ^-4 J/cm ² ) was used as excitation light, and a divalent europium-activated barium fluoride bromide phosphor (peak wavelength:
390 nm) was detected.

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 attached with the support medium was highly accurate.

On the other hand, the developed X-ray film showed an image of the electrophoretic pattern of the decomposition product having the ³² P label.

[Brief explanation of drawings]

Figures 1 to 4 show the state in which a stimulable phosphor sheet and a photographic light-sensitive material are superimposed [1 and 2], and the state in which a support medium for separation and development, a stimulable phosphor sheet and a photographic light-sensitive material are superimposed. FIG. 4 is a cross-sectional view showing states [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) FIG. 2 shows an example of a reading device (or reading device) for reading the positional information of the radiolabeled substance accumulated and recorded on the stimulable phosphor sheet in the present invention. 1: stacked stimulable phosphor sheets of photographic light-sensitive material, 2: laser light source, 3: laser light,
4: Filter, 5: Beam expander,
6: optical deflector, 7: plane reflector, 8: fθ lens,
9: Transfer direction, 10: Light guide sheet, 11: Photodetector, 12: Control circuit, 13: Amplifier, 14:
A/D converter, 15: signal processing circuit.

Claims (1)

[Claims] 1. In 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, ( 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 transferred to the sheet. (2) separating the stimulable phosphor sheet, overlaying it on a photographic light-sensitive material, and scanning the stimulable phosphor sheet with excitation light to convert the radiation energy accumulated in the sheet into photostimulated light; The positional information of the radiolabeled substance is obtained as an image on the photosensitive material by emitting the stimulated light and exposing the photographic light-sensitive material to the photostimulated light, and on the other hand, by photoelectrically detecting the stimulated light, the radioactive An autoradiographic measurement method comprising the step of obtaining positional information of a labeling substance as an electrical signal. 2. The autoradiographic measurement method according to claim 1, wherein in the step (2), the excitation light is a laser beam. 3. The autoradiographic measurement method according to claim 2, wherein the laser light is a red laser light. 4. Claim 1, characterized in that in the step (2) above, the positional information of the radiolabeled substance is obtained as an electrical signal by photoelectrically detecting the stimulated light that has passed through the photographic light-sensitive material. Autoradiographic measurement method as described. 5 By converting the electrical signal obtained in step (2) above into a digital signal and then performing signal processing, the position information of the radiolabeled substance can be converted into a symbol and/or
The autoradiographic measurement method according to claim 1, wherein the autoradiographic measurement method is obtained as a numerical value. 6. 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.
Autoradiographic measurement method described in section. 7 Claim 1, characterized in that the support medium for separation and development is a support medium for electrophoresis.
Autoradiographic measurement method described in section. 8. 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. 9. The autoradiographic measurement method according to claim 8, wherein the biopolymer substance is a nucleic acid, a derivative thereof, or a decomposition product thereof. 10 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) (2) absorbing at least a part of the radiation energy emitted from the radiolabeled substance into a stimulable phosphor sheet containing a stimulable phosphor overlaid on the support medium; After stacking the stimulable phosphor sheet on the surface opposite to the support medium side, the stimulable phosphor sheet is inspected with excitation light to release the radiation energy stored in the sheet as photostimulated light, By sensitizing a photographic light-sensitive material with the stimulated light, positional information of the radioactively labeled substance is obtained as an image on the light-sensitive material, and on the other hand, by photoelectrically detecting the stimulated light, the location information of the radioactively labeled substance is obtained. An autoradiographic measurement method comprising the steps of: obtaining position information as an electrical signal. 11 Claim 10, characterized in that in the step (2) above, the excitation light is a laser beam.
Autoradiographic measurement method described in section. 12. The autoradiographic measurement method according to claim 11, wherein the laser light is a red laser light. 13. Claim 10, characterized in that in the step (2) above, the positional information of the radiolabeled substance is obtained as digital data by photoelectrically detecting stimulated light transmitted through the photographic light-sensitive material. Autoradiographic measurement method as described. 14 Claim No. 1, characterized in that the electrical signal obtained in the step (2) 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. The autoradiographic measurement method according to item 10. 15. The autoradiograph according to claim 10, wherein the stimulable phosphor sheet has a support, a phosphor layer made of a stimulable phosphor dispersed in a binder, and a protective film. Measurement method. 16. The autoradiographic measurement method according to claim 10, wherein the support medium for separation and development is a support medium for electrophoresis. 17. The autoradio according to claim 10, wherein the radioactively labeled substance derived from a biological body is a radioactively labeled biopolymer substance, a derivative thereof, or a decomposition product thereof. Graph measurement method. 18. The autoradiographic measurement method according to claim 17, wherein the biopolymer substance is a nucleic acid, a derivative thereof, or a decomposition product thereof.