JP2945547B2 - Autoradiogram measurement method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel autoradiogram measuring method. More specifically, the present invention provides an automatic method comprising obtaining positional information of two or more radiolabeled substances having different average energies distributed in a tissue sample of an organism or a medium sample containing a tissue or substance derived from an organism. It relates to a radiogram measurement method.

[0002]

2. Description of the Related Art After a substance labeled with a radioisotope is administered to a living body, the living body or a part of the tissue of the living body is used as a sample, and this sample and a radiation such as a high-sensitivity X-ray film are used. Autoradiography (radioautography) comprising exposing the film to light (or exposure) by superimposing the film for a certain period of time and obtaining positional information (distribution, etc.) of the radiolabeled substance in the sample from the exposed site. (Also called graphy),
That is, an autoradiogram measuring method is conventionally known. This autoradiography, for example,
It is used to study the metabolism, absorption and excretion pathways and conditions of administered substances in living organisms in detail.

[0003] In recent years, autoradiography involves radioactively labeling a high-molecular substance derived from an organism, such as a protein or nucleic acid, and then subjecting the radio-labeled high-molecular substance, a derivative thereof, or a fragment thereof to gel electrophoresis. It is also effectively used for obtaining positional information of a radiolabeled substance on an electrophoretic medium obtained by separation and development by processing. Further, a method of separating and identifying a polymer substance or evaluating the molecular weight and characteristics of the polymer substance based on the positional information has been developed and actually used.

Further, for elucidation of metabolic pathways in living organisms, for example, autoradiography by a double labeling method (double tracer method) using two types of radioisotopes has been proposed. That is, a radiolabeled substance labeled with two or more different radioisotopes is administered to an organism, and an autoradiogram for each radiolabeled substance (that is, for each radioisotope) is obtained, thereby producing a radioactive substance. Attempts have been made to elucidate metabolism and chemical reactions in the body.

As a radiation film for autoradiography utilizing such a double labeling method, for example, Japanese Patent Publication No. 47-45540 discloses couplers having different hues in an emulsion layer comprising two silver halide layers. was contained, and by adjusting the thickness of the emulsion layer, tritium ⁽³ H) and other color autoradiography for silver halide photographic light-sensitive material for a distribution image fractionating recording radioisotope disclosed Have been.

However, in conventional autoradiography by radiography, it is very difficult to obtain an autoradiographic image (autoradiogram) of a sample that is multiply labeled with different radioisotopes for each isotope. . Even if a radiation film as described above is used, autoradiograms are visualized and recorded on a single radiation film as dye images of different colors. Must be analyzed and analyzed. Further, since different autoradiograms are recorded on one radiographic film, there has been a problem that positional information of a radiolabeled substance in a sample cannot be obtained with high accuracy.

For this reason, recently, double autoradiography (binuclide-labeled autoradiography) utilizing the difference in half-life of radioisotopes has also been used. This method uses two nuclides whose half-lives differ greatly from each other (eg, ¹²³ I: half-life about 13 hours, ¹⁴ C: half-life about 57 hours).
30 years), two types of radiolabeled substances each labeled with the binuclide are administered to a living body, and after a certain period of time, each radiolabeled substance is removed from the living body to examine the distribution in the living body. The sample thus obtained was immediately superimposed on an X-ray film and subjected to autoradiography, images of the two radiolabeled substances labeled with the dinuclear species were obtained on the X-ray film, and then left for a long time. After the radioactivity of the nuclide with a short half-life almost disappeared), the same autoradiography was performed again, and this time,
Only an image of the radiolabeled substance labeled with a long half-life nuclide is obtained on an X-ray film, and then, from the image of the radiolabeled substance obtained in the previous autoradiography, it is obtained by autoradiography after standing. Of the radiolabeled substance labeled with the short-lived nuclide by performing the subtraction process except for the image of the radiolabeled substance (an image showing the distribution of the radiolabeled substance labeled with the long-lived nuclide in the sample). This is a method for obtaining an image showing the distribution in the sample. In this method, each image with high accuracy can be obtained. However, in order to almost completely eliminate the radioactivity of short nuclides, a long time (for example, when the above-described combination of ¹²³ I and ¹⁴ C is used, 2 (About a month), which delays the research. In addition, since there is no linear relationship between the image density of the image (blackened image) generated on the silver halide film and the radiation dose, it is necessary to use a complicated function formula in order to perform the above subtraction. Because
It is easy to be complicated.

Further, there are some problems when autoradiography using an X-ray film (photosensitive silver salt film) is actually used. That is, in order to obtain an autoradiogram of a sample containing a radioactively labeled substance, a sample is superimposed on a radiation film such as a high-sensitivity X-ray film for a certain period of time, and the film is exposed (exposed). This exposure operation requires a long time (tens of hours to several days). This is because the sample to be measured by the autoradiograph is generally not given high radioactivity. And this exposure operation is usually
It must be performed at low temperatures (eg, 0 ° C. to −80 ° C.). The reason is that at a relatively high temperature such as room temperature, the latent image in the silver salt on the film formed by exposure to radiation or fluorescence tends to retreat and become an image that cannot be developed. This is because chemical fog is easily formed due to the migration of harmful components.
When such fog occurs in an image obtained by autoradiography, the accuracy of the positional information of the radioactively labeled substance is significantly reduced. In addition, in order to prevent deterioration in image quality due to chemical fog or the like, a sample containing a radioactively labeled substance must be exposed to a dry film in a state of being superimposed on a radiographic film. For this reason, the sample is usually dried or the sample is wrapped with a synthetic resin film or the like.

The silver salt of the photosensitive component of the radiation film has a disadvantage that it is easily affected not only by chemical stimuli but also by physical stimuli associated with operations such as movement and installation of the film.
This also makes the operation of autoradiography difficult and reduces the accuracy. That is, the radiation film tends to cause a physical fogging phenomenon due to a physical pressure or the like caused by contact with the sample. In order to avoid such physical fogging of the radiation film, the handling operation requires a high degree of skill and care, which further complicates the operation of autoradiography.

Further, in conventional autoradiography, since the exposure operation is performed for a long time as described above, the natural radioactivity contained in the sample other than the radiolabeled substance also contributes to the exposure of the radiographic film, and There is a problem that the accuracy of the positional information of the labeling substance is reduced. In order to eliminate such interference due to natural radioactivity, for example, parallel experiments using a control sample, optimization of exposure time, etc. have been attempted. There is a disadvantage that the whole operation becomes complicated due to the necessity of a preliminary experiment for determining the exposure time and the like.

In order to solve the problems associated with conventional autoradiography using the photosensitive silver halide film photosensitive material as described above, the photosensitive material has a phosphor layer containing a stimulable phosphor. An invention using a stimulable phosphor sheet (radiation image conversion panel) has already been proposed (JP-A-59-83057). In addition, in order to solve the above-mentioned problems accompanying the autoradiography by the double labeling method, in particular, a stimulable phosphor sheet or a stimulable phosphor having a plurality of phosphor layers containing a stimulable phosphor is preferred. An invention using a body sheet laminate has also been proposed (JP-A-62-93679).

The latter invention is effective for solving the above-mentioned problems associated with the conventional autoradiography by the double labeling method using a photosensitive silver salt film. It is necessary to prepare a plurality of stimulable phosphor layers having different thicknesses (laminated body or a plurality of stimulable phosphor sheets), and a radiation image of a plurality of nuclides can be obtained by one reading operation. While having the advantage,
There is a problem that it is not easy to determine the separation conditions of those radiographic images.

[0013]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems associated with the conventional autoradiography by a double labeling method using a photosensitive silver salt film, and to provide a method for producing a plurality of bright silver halide films. There is no need to prepare a stimulable phosphor layer (a laminate or a plurality of stimulable phosphor sheets), and a simple reading operation is repeated, and thereafter, a simple signal processing operation is performed to sufficiently separate the phosphor layers. (That is,
It is an object of the present invention to provide an autoradiogram measurement method capable of obtaining high-precision radiographic images of a plurality of nuclides (the effects of other radiographic images can be ignored).

[0014]

SUMMARY OF THE INVENTION Two or more radiolabeled substances labeled with radioisotopes having different average energies distributed in a tissue sample of an organism or a medium sample containing a tissue or substance derived from an organism. In the autoradiogram measurement method for obtaining the position information of 1), 1) the sample is overlapped on a stimulable phosphor sheet including a stimulable phosphor layer having a protective film on the surface via the protective film for a certain period of time. A step of causing the stimulable phosphor layer to absorb and record the radiation energy emitted from the radioactive labeling substance having a relatively high average energy; 2) stimulable phosphor of the stimulable phosphor sheet; The layer is excited by an electromagnetic wave to emit radiation energy stored and recorded in the stimulable phosphor layer as stimulating light, and the stimulating light is detected. Obtaining an image signal indicating the position information of the radioactive labeling substance having a relatively high average energy; 3) the sample does not have a protective film on its surface, or is less than the protective film used in the above step 1). When the stimulable phosphor sheet including the stimulable phosphor layer having the thin protective film is superimposed on the stimulable phosphor layer directly or through the protective film for a certain time, the average energy is relatively low. A step of absorbing radiation energy emitted from both a high radioactive label substance and a radioactive label substance having a relatively low average energy into the stimulable phosphor layer to record the radiation energy; 4) accumulation through the above step 3) Exciting the stimulable phosphor layer of the stimulable phosphor sheet with electromagnetic waves, emitting radiation energy accumulated and recorded in the stimulable phosphor layer as stimulable light, and detecting the stimulable light. A step of obtaining an image signal indicating both positional information of a radiolabeled substance having a relatively high average energy and a radiolabeled substance having a relatively low average energy; 5) From the image signal obtained in the above step 4) , Above 2)
Obtaining the position information of each of two or more radiolabeled substances in the sample by performing a subtraction process except for between the image signals obtained in the step (a). .

In the present invention, the "positional information" of the radiolabeled substance contained in the sample means various kinds of information centered on the position of the radiolabeled substance or the aggregate thereof in the sample, for example, exists in the sample. It means various types of information obtained as one or any combination of information including the location and shape of the aggregate of radioactive substances, the concentration and distribution of the radioactive substance at the position, and the like. In the present invention, “two or more radioactively labeled substances” also include the same substance having a different radioactively labeled substance.

The stimulable phosphor sheet used in the present invention is also called a radiation image conversion panel, examples of which are described in JP-A-55-12145 and the like. It is known. That is, the stimulable phosphor sheet is made of a stimulable phosphor, and the radiation energy transmitted through the subject or the radiation energy emitted from the subject is absorbed by the stimulable phosphor of the sheet. By exciting the sheet with electromagnetic waves (excitation light) in the visible to infrared region, radiation energy accumulated in the stimulable phosphor of the sheet can be emitted as fluorescence. . Accordingly, the radiation image of the subject or the subject is read out photoelectrically from the fluorescence and converted into an electric signal, and the obtained electric signal is reproduced as a visible image on a recording material such as a photographic film or a display device such as a CRT. Alternatively, it can be represented as numerical information or symbolized position information.

The stimulable phosphor sheet has the following basic structure:
It comprises a support and a phosphor layer provided on one side thereof. However, when the phosphor layer itself is self-supporting, it is not always necessary to provide a support. In general, a transparent protective film is provided on the surface of the phosphor layer (if the support is provided on one side of the phosphor layer, the surface on the side not facing the support), Protects the layer from chemical alteration or physical impact.

The phosphor layer is basically a layer made of a binder which contains and supports a particulate stimulable phosphor in a dispersed state.

As described above, the stimulable phosphor emits stimulable light when irradiated with radiation and then with excitation light, but from a practical point of view, the wavelength is 400 to 900 nm.
It is desirable that the phosphor exhibit a photostimulated emission in the wavelength range of 300 to 500 nm by the excitation light in the range of m.
Examples of the stimulable phosphor used in the stimulable phosphor sheet used in the present invention include the following.

SrS: Ce, Sm, SrS: Eu, Sm, ThO described in US Pat. No. 3,859,527.
₂ : Er, and La ₂ O ₂ S: Eu, Sm;
No. 5-12142, ZnS: Cu, Pb, Ba
O.xAl ₂ O ₃ : Eu (where 0.8 ≦ x ≦ 1
0) and M ^II O.xSiO ₂ : A (where M ^II is M
g, Ca, Sr, Zn, Cd or Ba, and A is C
e, Tb, Eu, Tm, Pb, Tl, Bi, or Mn
And x is 0.5 ≦ x ≦ 2.5),

(Ba _{1 -xy} , Mg _x , Ca _y ) FX: aEu ²⁺ described in JP _-A- 55-12143 (where X is at least one of Cl and Br, x and y is 0 <x + y ≦ 0.6 and xy ≠ 0, and a is 10 ⁻⁶ ≦ a ≦ 5 × 10 ⁻² ),

Ln described in JP-A-55-12144
OX: xA (where Ln is La, Y, Gd, and L
at least one of u, 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),

(Ba _1-x , M ²⁺ _x ) FX: yA (where M ²⁺ is M
g, 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, and x is 0 ≦ x ≦ 0.6, and y is 0 ≦ y ≦ 0.2),

M ^II FX · xA: yLn described in JP-A-55-160078 [where M ^II is Ba, Ca,
At least one of Sr, Mg, Zn, and Cd, A is BeO, MgO, CaO, SrO, BaO, Z
nO, Al ₂ O ₃ , Y ₂ O ₃ , La ₂ O ₃ , In ₂ O
₃ , SiO ₂ , TiO ₂ , ZrO ₂ , GeO ₂ , SnO
₂ , Nb ₂ O ₅ , Ta ₂ O ₅ , and at least one of ThO ₂ , Ln is Eu, Tb, Ce, Tm, D
y, Pr, Ho, Nd, Yb, Er, Sm, and Gd
X is at least one of Cl, Br, and I, and x and y are each 5
× 10 ⁻⁵ ≦ x ≦ 0.5 and 0 <y ≦ 0.2].

(Ba _{1 -x} , M ^II _x ) F ₂ .aBaX ₂ : yEu, zA described in JP-A-56-116777
[Where M ^II is at least one of beryllium, magnesium, calcium, strontium, zinc and cadmium, X is at least one of chlorine, bromine and iodine, A is at least one of zirconium and scandium] , A, x, y, and z
Are respectively 0.5 ≦ a ≦ 1.25, 0 ≦ x ≦ 1, 10 ⁻⁶
≦ y ≦ 2 × 10 ⁻¹ , and 0 <z ≦ 10 ⁻² ].

(B) described in JP-A-57-23673.
^{_{a 1-x, M II x}} ) F 2 · aBaX 2: yEu, zB [ However, M ^II is beryllium, magnesium, calcium,
At least one of strontium, zinc and cadmium, X is at least one of chlorine, bromine and iodine, and a, x, y and z are respectively 0.5 ≦ a ≦ 1.25, 0 ≦ x ≦ 1, 10 ⁻⁶ ≦ y ≦ 2 ×
10 ^-1 and 0 <z ≦ 2 × 10 ^-1 ],

(B) described in JP-A-57-23675
^{_{a 1-x, M II x}} ) F 2 · aBaX 2: yEu, zA [ However, M ^II is beryllium, magnesium, calcium,
X is at least one of chlorine, bromine and iodine, A is at least one of arsenic and silicon, and a, x, y and z are each at least one of strontium, zinc and cadmium. 5 ≦ a
≦ 1.25, 0 ≦ x ≦ 1, 10 ⁻⁶ ≦ y ≦ 2 × 10 ⁻¹ , and 0 <z ≦ 5 × 10 ⁻¹ ].

M described in JP-A-58-69281
^III OX: xCe [where M ^III is Pr, Nd, P
m, Sm, Eu, Tb, Dy, Ho, Er, Tm, Y
b, and at least one trivalent metal selected from the group consisting of Bi, X is one or both of Cl and Br, and x is 0 <x <0.1]. A phosphor represented by the formula:

B described in JP-A-58-206678
a _1-x M _{x / 2} L _{x / 2} FX: yEu ²⁺ [where M is L
L represents at least one alkali metal selected from the group consisting of i, Na, K, Rb and Cs;
Y, La, Ce, Pr, Nd, Pm, Sm, Gd, T
b, Dy, Ho, Er, Tm, Yb, Lu, Al, G
X represents at least one trivalent metal selected from the group consisting of a, In, and Tl; X represents at least one halogen selected from the group consisting of Cl, Br, and I; and x represents 10 ^-2. ≦ x ≦ 0.5, y is 0 <y
≦ 0.1], a phosphor represented by a composition formula:

B described in JP-A-59-27980
aFX · xA: yEu ²⁺ [where X is Cl, Br,
A is at least one halogen selected from the group consisting of and I; A is a calcined product of a tetrafluoroborate compound; and x is 10 ⁻⁶ ≦ x ≦ 0.1, and y is 0 <y
≦ 0.1], a phosphor represented by a composition formula:

B described in JP-A-59-47289
aFX · xA: yEu ²⁺ [where X is Cl, Br,
And A is selected from the group consisting of hexafluorosilicic acid, hexafluorotitanic acid and a salt of a monovalent or divalent metal of hexafluorozirconic acid. X is a calcined product of at least one compound; and x is 10 ⁻⁶ ≦ x ≦ 0.1, and y is 0 <y ≦
0.1], a phosphor represented by a composition formula:

B described in JP-A-59-56479
aFX.xNaX ': aEu ²⁺ [where X and X'
Is at least one of Cl, Br, and I, and x and a are respectively 0 <x ≦ 2 and 0 <a ≦ 0.2].

M described in JP-A-59-56480
^II FX.xNaX ': yEu ²⁺ : zA [where M ^II is
X and X 'are at least one alkaline earth metal selected from the group consisting of Ba, Sr, and Ca;
A is at least one halogen selected from the group consisting of Cl, Br and I; A is V, Cr, M
x is at least one transition metal selected from n, Fe, Co, and Ni; and x is 0 <x ≦ 2, and y is 0
<Y ≦ 0.2 and z is 0 <z ≦ 10 ⁻² ], a phosphor represented by a composition formula:

M ^II described in JP-A-59-75200
FX ・ aM ^I X ′ ・ bM ′ ^II X ″ ₂・ cM ^III X ″ ′ ₃・
xA: yEu ²⁺ [where M ^II is Ba, Sr, and C
is at least one alkaline earth metal selected from the group consisting of a; M ^I is Li, Na, K, Rb, and C
M ′ ^II is at least one divalent metal selected from the group consisting of Be and Mg; M ^III is Al,
X is at least one trivalent metal selected from the group consisting of Ga, In, and Tl; A is a metal oxide;
Is at least one halogen selected from the group consisting of Cl, Br, and I; X ′, X ″ and X ″ ″ are
Is at least one halogen selected from the group consisting of F, Cl, Br, and I; and a is 0 ≦ a ≦
2, b is 0 ≦ b ≦ 10 ⁻² , c is 0 ≦ c ≦ 10 ⁻² , and a
+ B + c ≧ 10 ⁻⁶ ; x is 0 <x ≦ 0.5, y is 0
<Y ≦ 0.2], a phosphor represented by a composition formula:

M ^II described in JP-A-60-84381
X ₂ · aM ^II X ′ ₂ : xEu ²⁺ [where M ^II is Ba,
At least one alkaline earth metal selected from the group consisting of Sr and Ca; X and X 'are Cl, Br
At least one halogen selected from the group consisting of and I, and X ≠ X ′; and a is 0.1 ≦ a ≦
10.0, x is 0 <x ≦ 0.2], a stimulable phosphor represented by a composition formula:

M described in JP-A-60-101173
^{II FX · aM I X ':} xEu 2+ [ However, M ^II is Ba,
M ^I is at least one alkali metal selected from the group consisting of Rb and Cs; M is at least one alkaline earth metal selected from the group consisting of Sr and Ca; X is from the group consisting of Cl, Br and I X 'is at least one halogen selected; X' is F, Cl, Br
And at least one halogen selected from the group consisting of I and I; and a and x are each 0 ≦ a ≦
4.0 and 0 <x ≦ 0.2], a stimulable phosphor represented by a composition formula:

M described in JP-A-62-25189
^I X: xBi [However, M ^I is at least one alkali metal selected from the group consisting of Rb and Cs;
X is at least one halogen selected from the group consisting of Cl, Br and I; and x is 0 <x ≦ 0.2
A stimulable phosphor represented by a composition formula:

L described in JP-A-2-229882
nOX: xCe (where Ln is at least one of La, Y, Gd and Lu, X is Cl, Br and I
At least one of x is 0 <x ≦ 0.2,
The ratio of Ln to X is 0.500 <X / Ln ≦
A cerium-activated rare earth oxyhalide phosphor which has a maximum wavelength λ of 0.998 and a stimulable excitation spectrum having a maximum wavelength λ of 550 nm <λ <700 nm).

The M ^II X ₂ .aM ^II X ' ₂ : xEu ²⁺ stimulable phosphor described in the above-mentioned Japanese Patent Application Laid-Open No. 60-84381 includes the following additives M ^II X ₂・ aM
^II X ′ ₂ may be contained in the following ratio per 1 mol.
BM ^I X described in JP 60-166379 discloses "
(Where M ^I is at least one alkali metal selected from the group consisting of Rb and Cs, and X ″ is F, C
at least one halogen selected from the group consisting of l, Br and I, and b is 0 <b ≦ 10.0); bK described in JP-A-60-221483
_{X "· cMgX"'2 ·} dM III X "" 3 ( where, M ^III
Is at least one trivalent metal selected from the group consisting of Sc, Y, La, Gd and Lu, and X ″, X ″ ′ and X ″ ″ are each selected from the group consisting of F, Cl, Br and I At least one halogen, and b,
c and d are respectively 0 ≦ b ≦ 2.0, 0 ≦ c ≦ 2.
0, 0 ≦ d ≦ 2.0 and 2 × 10 ⁻⁵ ≦ b + c
+ D); yB described in JP-A-60-228592 (provided that y is 2 × 10 ⁻⁴ ≦ y ≦ 2 × 10 ⁻¹ ); and yB described in JP-A-60-228593. bA
(Where A is at least one oxide selected from the group consisting of SiO ₂ and P ₂ O ₅ , and b is 1
0 at ^-4 ≦ b ≦ 2 × 10 ^-1 is); JP 61-1208
No. 83, where b is 0 <b ≦ 3
× 10 ^-2 ); bSnX ″ ₂ described in JP-A-61-120885, wherein X ″ is at least one halogen selected from the group consisting of F, Cl, Br and I; Is 0 <b ≦ 10 ⁻³ ); bCsX ″ · cSn described in JP-A-61-235486.
X "' ₂ (where X" and X "' are each F, Cl,
At least one halogen selected from the group consisting of Br and I, and b and c are each 0 <
b ≦ 10.0 and 10 ⁻⁶ ≦ c ≦ 2 × 10 ⁻² );
And bCsX ".yLn3 ⁺ (where X" is F, Cl, B
rn is at least one halogen selected from the group consisting of r and I, and Ln is Sc, Y, Ce, Pr, Nd, S
m, Gd, Tb, Dy, Ho, Er, Tm, Yb, and at least one rare earth element selected from the group consisting of Lu, and b and y are each 0 <b ≦ 1.
0.0 and 10 ⁻⁶ ≦ y ≦ 1.8 × 10 ⁻¹ ).

Among the above stimulable phosphors, a divalent europium-activated alkaline earth metal halide-based phosphor and a cerium-activated rare earth oxyhalide phosphor are particularly preferable because they exhibit high-luminance stimulable light emission. However, the stimulable phosphor used in the present invention is not limited to the above-described phosphors, and any phosphor can be used as long as it exhibits stimulable emission when irradiated with radiation and then irradiated with excitation light. There may be.

The stimulable phosphor layer of the stimulable phosphor sheet of the present invention is not only composed of a stimulable phosphor and a binder containing and supporting the stimulable phosphor in a dispersed state, but also containing no binder. And a phosphor layer in which a polymer substance is impregnated in the gaps between the stimulable phosphor aggregates.

Next, the method for producing the stimulable phosphor sheet of the present invention will be described, taking as an example the case where the phosphor layer comprises a stimulable phosphor and a binder containing and supporting the stimulable phosphor in a dispersed state.

The phosphor layer can be formed on a support by the following known method. First, a stimulable phosphor and a binder (eg, nitrocellulose, acrylic resin, thermoplastic elastomer) are added to a solvent, mixed well, and the stimulable phosphor is uniformly dispersed in the binder solution. A dispersed coating solution is prepared. The mixing ratio between the binder and the stimulable phosphor in the coating solution is determined by the properties of the target stimulable phosphor sheet,
Generally, the mixing ratio between the binder and the phosphor is from 1: 1 to 1: 100 (weight ratio), although it depends on the kind of the phosphor.
And particularly preferably from the range of 1: 8 to 1:40 (weight ratio).

The coating solution containing the phosphor and the binder prepared as described above is then uniformly applied to the surface of the support to form a coating film. This application operation
It can be performed by using a usual coating means, for example, a doctor blade, a roll coater, a knife coater, or the like.

The support can be arbitrarily selected from materials known as supports for conventional stimulable phosphor sheets. In a known stimulable phosphor sheet, the phosphor layer is used to enhance the bond between the support and the phosphor layer or to improve the sensitivity or image quality (sharpness, granularity) of the stimulable phosphor sheet. A polymer material such as gelatin is applied to the surface of the support on the side to be provided to form an adhesion-imparting layer, or a light-reflective layer made of a light-reflective material such as titanium dioxide, or a light-absorbing material such as carbon black. It is known to provide a light absorbing layer or the like. The support used in the present invention can also be provided with these various layers, and their configuration can be arbitrarily selected according to the desired purpose and use of the stimulable phosphor sheet. Further, JP-A-58-20020
As described in JP-A No. 0, the surface of the support on the side of the phosphor layer (the surface of the support on the side of the phosphor layer, the adhesion-imparting layer, In the case where a layer or a light absorbing layer is provided, the surface thereof means fine irregularities. After forming a coating on the support as described above, the coating is dried to complete the formation of the stimulable phosphor layer on the support.
The thickness of the phosphor layer varies depending on the characteristics of the target stimulable phosphor sheet, the kind of the phosphor, the mixing ratio between the binder and the phosphor, and is usually 20 μm to 1 mm. However, this layer thickness is preferably 50 to 500 μm.

The transparent protective film is made of, for example, cellulose acetate,
Dissolve in a suitable solvent a cellulose derivative such as nitrocellulose; or a transparent polymer such as polymethyl methacrylate, polyvinyl butyral, polyvinyl formal, polycarbonate, polyvinyl acetate, or a synthetic polymer such as vinyl chloride / vinyl acetate copolymer. The solution prepared as described above can be formed by applying the solution to the surface of the phosphor layer. Alternatively, polyethylene terephthalate, polyethylene naphthalate, polyethylene,
A plastic sheet made of polyvinylidene chloride, polyamide, and the like; and a protective film forming sheet such as a transparent glass plate are separately formed and then formed on the surface of the phosphor layer using a suitable adhesive. be able to. Alternatively, the protective film may be formed by a coating film of a fluorine-based resin soluble in an organic solvent, or a perfluoroolefin resin powder or a silicone resin powder may be dispersed and contained in the protective film.

The thickness of the transparent protective film of the stimulable phosphor sheet is suitably selected according to the type and content of the radioisotope contained in the sample and the intensity of the emitted radiation. That is, in order to measure the distribution of a substance labeled with a radioactive label having a relatively high radiation energy in a sample, a stimulable phosphor sheet having a thick protective film is used, and a relatively low radiation energy is used. In order to measure the distribution of the substance labeled with the radioactive label in the sample, a stimulable phosphor sheet having a thin protective film or a stimulable phosphor sheet without a protective film is used.

Examples of radioisotopes used for giving a radioactive label to a sample include ³ H (average energy: 0.0055 MeV) and ¹²³ I (average energy:
0.159 MeV), ¹³¹ I (average energy: 0.0
04MeV), ^14C (average energy: 0.049Me)
V), ³⁵ S (average energy: 0.048 MeV), ³²
P (average energy: 0.0695 MeV), and ¹⁸
F (average energy: 0.64 MeV).

For example, when the sample contains ³ H and ¹⁴ C, the radiation from the radioactive element ( ³ H) that emits radiation having a relatively low average energy can be obtained without interposing a protective film. Is directly superimposed on the stimulable phosphor layer to be absorbed by the stimulable phosphor layer. However, in this case, radiation from a radioactive element ( ¹⁴ C) that emits radiation having a relatively high average energy is also absorbed by the stimulable phosphor layer at the same time. That is, in this operation, both the ³ H-labeled substance image and the ¹⁴ C-labeled substance image are accumulated and recorded in the stimulable phosphor layer on the stimulable phosphor sheet. On the other hand, when the sample is directly superimposed on the stimulable phosphor layer with a protective film (for example, 5 to 50 μm) of an appropriate thickness interposed, radiation from ³ H may pass through the protective film. Although not possible, the radiation from ¹⁴ C has a high penetrating power, passes through the protective film and is absorbed by the stimulable phosphor layer. Therefore, in this case, only the ¹⁴ C-labeled substance image is accumulated and recorded on the stimulable phosphor sheet. Then, by subtracting the radiation image of only the ¹⁴ C-labeled substance image from the radiation image of both the former ³ H-labeled substance image and the ¹⁴ C-labeled substance image, the radiation image of only the ³ H-labeled substance image is obtained. Obtainable. Therefore, both the ³ H-labeled substance image and the ¹⁴ C-labeled substance image can be obtained as independent radiation images. Note that, of course, the order of each exposure operation is not particularly limited, and the exposure operation and the operation of reading the position information of the radioactive marker may be performed continuously,
Alternatively, after all the exposure operations are completed, the operation of reading out the position information of the radioactive label can be performed collectively, and the order of each step is not limited in any case.

The samples were ³ H, ¹⁴ C and ³² C, respectively.
Even if it contains three kinds of radioactive labeling substance labeled with three different radioisotopes P, radiation from radioactive elements ⁽³ H) which emits a relatively low average energy radiation protective film By directly superimposing the sample on the stimulable phosphor layer without intervening, the sample is absorbed by the stimulable phosphor layer. However, in this case, two types of radioactive elements ( ¹⁴ C,
The radiation from ³² P) is simultaneously absorbed by the stimulable phosphor layer. That is, in this operation, the ³ H-labeled substance image, the ¹⁴ C-labeled substance image, and the ³² P-labeled substance image are accumulated and recorded together on the stimulable phosphor layer on the stimulable phosphor sheet. On the other hand, 5-5
When a sample is directly superimposed on the stimulable phosphor layer with a protective film having a thickness of 0 μm interposed, radiation from ³ H cannot pass through the protective film, but from ¹⁴ C and ³² P. Has a high penetrating power, passes through the protective film and is absorbed by the stimulable phosphor layer. Therefore, in this case, the ¹⁴ C-labeled substance image and ³² C
The P-labeled substance image is accumulated and recorded together on the stimulable phosphor sheet. Next, when the sample is directly superimposed on the stimulable phosphor layer with a protective film having a thickness of about 100 μm interposed therebetween,
Radiation from ³ H and ¹⁴ C cannot pass through the protective film, but radiation from ³² P has a very high penetrating power and is absorbed by the stimulable phosphor layer through this thick protective film. You.
Therefore, in this case, only the ³² P-labeled substance image is accumulated and recorded on the stimulable phosphor sheet. Then, the first ³ H-labeled substance image, the ¹⁴ C-labeled substance image, and the ³² P-labeled substance image are all the same radiation image, and the second ¹⁴ C-labeled substance image and the ³² P labeled substance image are both the same radiation image. By subjecting the radiographic image of only the ³² P-labeled substance image to a subtraction process, the ³ H-labeled substance image, the ¹⁴ C-labeled substance image, and
Each radiographic image of the ³² P-labeled substance image can be obtained independently.

That is, the distribution of two or more arbitrary radiolabeled substances is shown by adjusting the kind of radioisotope to be combined as described above and the thickness of the protective film (and / or the presence or absence of the protective film). Radiation images can be obtained independently.

Next, the autoradiogram measuring method (autoradiography) of the present invention will be described. The autoradiogram measurement method of the present invention uses the above-mentioned stimulable phosphor sheet in place of a conventional photosensitive material composed of a radiation film, and the exposure operation is performed by using a sample containing a radioactive labeling substance and a stimulable phosphor sheet. By overlapping the phosphor sheet for a certain period of time, at least a part of the radiation emitted from the radiolabeled substance of the sample is absorbed by the sheet.

The sample to be recorded by the autoradiography of the present invention is a sample selected from the group consisting of a tissue of a living organism and a medium containing a substance derived from the living tissue and / or the living organism. Examples of the medium containing the tissue of the organism and / or the substance derived from the organism include a support medium in which a biopolymer substance such as a protein, a nucleic acid, a derivative thereof, or a degradation product thereof is separated and developed. .

The radiolabeled substance contained in the above sample can be obtained by holding a radioisotope in a sample to be recorded or a specific substance in the sample by a known method.
The radioisotope used in the present invention is radiation (α ray,
Any nuclide that emits β-rays, γ-rays, neutron rays, X-rays, etc. may be used, but typical examples thereof include the aforementioned ³ H, ¹²³ I, ¹³¹ I, and ¹⁴ C. , ³⁵ S,
¹⁸ F, and ³² P. The sample to be recorded in the present invention has a radiolabeled substance radioactively labeled with at least two types of radioisotopes having different radiation energies.

In the exposure operation, the sample is superimposed on the stimulable phosphor sheet as it is or after any treatment such as drying treatment and fixing treatment of the separated and developed product, whereby the autoradiograph of the sample is accumulated. It is stored and recorded on the luminescent phosphor sheet. The state where the sample and the stimulable phosphor sheet are superimposed is usually realized by bringing the sample and the stimulable phosphor sheet into close contact with each other, but it is not always necessary to bring them into close contact with each other, and they are arranged in a state where they are close to each other. It may be. The above-mentioned superposition exposure operation is performed using at least two kinds of stimulable phosphor sheets. For example, the same two stimulable phosphor sheets are prepared except that one is provided with a protective film and the other is not provided with a protective film (the point that the film thicknesses of the protective films are different from each other), A method of performing an overlapping exposure operation on each stimulable phosphor sheet is used. Alternatively, instead of preparing two stimulable phosphor sheets, an overlapping exposure operation is performed once using a stimulable phosphor sheet having a protective film, and then a radiation image reading operation described later is performed. After the radiation remaining on the luminescent phosphor sheet is removed by an erasing operation, the protective film can be removed and the same superposing exposure operation and reading operation can be performed. In addition, it consists of a stimulable phosphor layer and a protective film,
Using a stimulable phosphor sheet without a support, first, a superimposing exposure operation is performed once on the side without a protective film, and then a readout operation of a radiation image is performed, and thereafter, a residual image is left on the stimulable phosphor sheet. After removing the existing radiation by an erasing operation, the same overlapping exposure operation and reading operation can be performed on the side having the protective film. In addition, using a stimulable phosphor sheet composed of a support, a stimulable phosphor layer, and a protective film, first, a superimposing exposure operation is performed once on the protective film side, and then a radiation image reading operation is performed. After the radiation remaining on the stimulable phosphor sheet is removed by the erasing operation, the same superposing exposure operation and readout operation can be performed on the support side. Each of the stimulable phosphor sheets to be used is desirably the same except for the protective film in order to facilitate the subsequent subtraction operation. Body layer material,
It is possible to perform a desired subtraction operation by introducing a variable caused by a difference in thickness or the like.

The exposure time depends on the type of radioisotope contained in the sample, its radiation intensity, the concentration and density of the radiolabel, the sensitivity of the stimulable phosphor sheet, and the positional relationship between the sample and the stimulable phosphor sheet. Although fluctuating, the exposure operation requires a certain time, for example, several seconds or more. However,
When the stimulable phosphor sheet of the present invention is used, the exposure time is significantly reduced as compared with the exposure time required when a conventional radiation film is used.

The temperature at which the exposure operation is carried out is not particularly limited, but autoradiography using the stimulable phosphor sheet of the present invention can be carried out at an environmental temperature of particularly 10 to 35 ° C. However, the low temperature used in conventional autoradiography (for example, 5
The exposure operation may be performed at a temperature of about (° C. or lower).

FIG. 1 shows a method for reading out the position information of the radioactive labeling substance indicated by the autoradiogram stored and recorded on the stimulable phosphor sheet in the present invention.
This will be briefly described with reference to the example of the reading device (or the reading device) shown in FIG. FIG. 1 shows a stimulable phosphor sheet (support,
A layer comprising a stimulable phosphor layer and a protective layer, which may be abbreviated as a sheet in the following.) In order to temporarily read out one-dimensional or two-dimensional positional information of a radiolabeled substance accumulated and recorded in 1) An outline of an example of a reading apparatus including a read-ahead read unit 2 for reading a book and a read-for-main-read unit 3 having a function of reading an autoradiograph stored and recorded on a sheet 1 in order to output positional information of a radioactive labeling substance. FIG.

The read-ahead section 2 performs the following read-ahead operation. The laser light 5 generated from the laser light source 4 passes through the filter 6, so that the stimulable phosphor sheet 1 is excited by the laser light 5.
The part of the wavelength region corresponding to the wavelength region of the stimulated emission generated from is cut off. Next, the laser beam is deflected by an optical deflector 7 such as a galvanometer mirror, and
After that, the light is deflected one-dimensionally and is incident on the sheet 1. The laser light source 4 used here is selected so that the wavelength region of the laser light 5 does not overlap with the main wavelength region of stimulated emission emitted from the sheet 1. The stimulable phosphor sheet 1 is transferred in the direction of arrow 9 with the protective film side up under irradiation of the deflection laser light 5. Therefore, the entire surface of the sheet 1 is irradiated with the deflected laser light. The output of the laser light source 4, the beam diameter of the laser beam 5, the scanning speed of the laser beam 5, and the transport speed of the sheet 1 are set so that the energy of the laser beam 5 in the pre-reading operation is smaller than the energy used in the main reading operation. It is adjusted to. When the stimulable phosphor sheet 1 is irradiated with the above laser light, the stimulable phosphor sheet 1 emits stimulable light in an amount proportional to the radiation energy stored in the stimulable phosphor layer. It is incident on 10. The light guide 10 is disposed close to the stimulable phosphor sheet 1 so that its entrance surface is linear and faces the scanning line on the stimulable phosphor sheet 1, and its exit surface forms an annular shape. It communicates with the light receiving surface of the detector 11. This light guide 10
Is made by processing a transparent thermoplastic resin sheet such as an acrylic synthetic resin, and is configured so that light incident from the incident surface is transmitted to the exit surface while being totally reflected inside the incident surface. I have. Stimulable phosphor sheet 1
The stimulated emission from the light source is guided inside the light guide 10 and reaches the emission surface, is emitted from the emission surface, and is received by the photodetector 11. A filter that transmits only light in the wavelength region of stimulated emission and cuts light in the wavelength region of excitation light (laser light) is attached to the light receiving surface of the photodetector 11, so that only stimulated emission can be detected. It has been like that. The stimulated emission detected by the photodetector 11 is converted into an electric signal, which is further amplified and output by the amplifier 12. The stored record information output from the amplifier 12 is stored in the control circuit 13 of the main readout unit 3.
Is input to The control circuit 13 controls the amplification factor set value a, the recording scale factor b, and the reproduction scale so that an image having the most uniform density and contrast and excellent observation and reading performance can be obtained according to the obtained stored record information. The image processing condition setting value c is output.

The stimulable phosphor sheet 1 for which the pre-reading operation has been completed as described above is transferred to the main-reading reading section 3. In the book reading unit 3, the following book reading operation is performed. The laser beam 15 emitted from the main reading laser light source 14 passes through a filter 16 having the same function as the above-mentioned filter 6, and then the beam expander 17 adjusts the beam diameter strictly. Next, the laser light is deflected by an optical deflector 18 such as a galvanometer mirror, reflected by a plane reflecting mirror 19, and then one-dimensionally deflected onto the stimulable phosphor sheet 1. An fθ lens 20 is disposed between the optical deflector 18 and the plane reflecting mirror 19, so that when the deflected laser beam scans over the sheet 1, a uniform beam speed is always maintained. . The stimulable phosphor sheet 1 is transferred in the direction of the arrow 21 with the protective film side up, as in the case of the pre-reading operation, under the irradiation of the deflected laser light. Therefore, similarly to the pre-reading operation, the entire surface of the sheet 1 is irradiated with the deflected laser light. When the stimulable phosphor sheet 1 is irradiated with the laser beam as described above, as in the pre-reading operation,
It emits stimulated emission of a quantity of light proportional to the stored radiation energy, and this light enters the main reading light guide 22. The book-reading light guide 22 is a pre-reading light guide 10.
It has the same material and structure as those of
The stimulated emission guided while repeating total internal reflection inside 2 is emitted from its emission surface and received by the photodetector 23. A filter that selectively transmits only the wavelength region of stimulated emission is attached to the light receiving surface of the photodetector 23,
3 detects only stimulated emission. The stimulated emission detected by the photodetector 23 is converted into an electric signal, and is amplified to an electric signal of an appropriate level by an amplifier 24 whose sensitivity is set in accordance with the amplification factor setting value a. Is input to A / D converter 25
Is converted into a digital signal with a scale factor suitable for the signal fluctuation range according to the recording scale factor set value b, and is input to the signal processing circuit 26.

The reading operation of the stimulable phosphor sheet as described above is performed twice or more (that is, the stimulable phosphor sheet having an image of a single radiolabeled substance, and the stimulable phosphor having an image of a plurality of radiolabeled substances). After performing each of the body sheets (at least once), the signal processing circuit 26 first stores the position information (image signal) obtained by reading from the plurality of stimulable phosphor sheets in a memory (ie,
After being stored in a buffer memory or a non-volatile memory such as a magnetic disk, a subtraction process is performed on the digital signal. That is, by performing the subtraction between the corresponding positions of the radiographic image to be subtracted, the position information on the radioactive labeling substance can be separated for each radioactive element. The subtraction magnification in the subtraction process is calculated from the thickness of the phosphor layer and the radiation energy,
Alternatively, simultaneously expose a standard (reference),
It can be determined based on that. After multiplying the image signal by the determined magnification, subtraction is performed between the signals. A subtraction processing method for a radiation image is described in detail in, for example, Japanese Patent Application Laid-Open No. 58-163340. Then, based on the reproduced image processing condition set value c for each of the obtained position information, signal processing is performed so that a visible image with proper density and contrast and excellent observation and reading performance is obtained, and then a magnetic tape or the like is used. Is transmitted to a recording device (not shown) via the storage means.

As a recording device, for example, a device for optically recording by scanning a photosensitive material with a laser beam or the like, a CR device,
A recording device based on various principles, such as a device for electronically displaying on a T or the like, a device for recording a radiation image displayed on a CRT or the like on a video printer, a device for recording on a thermosensitive recording material using a heat ray, and the like. Can be used. However, the recording device is not limited to a device that visualizes the autoradiograph obtained as described above, and as described above, one-dimensional or two-dimensional position information of a radiolabeled substance in a sample is used. For example, it can be recorded in a desired form by digitizing or symbolizing.

As for the method for reading out the radiolabeled substance in the sample stored and recorded on the stimulable phosphor sheet, the reading operation including the pre-reading operation and the main reading operation has been described above. The read operations that can be performed are not limited to the above example. For example, if the content of the radioactive substance in the sample, the radiation intensity thereof, and the exposure time of the stimulable phosphor sheet for the sample are known in advance, the pre-reading operation can be omitted in the above example. In addition, as a method for reading out the position information of the radioactive labeling substance in the sample stored and recorded on the stimulable phosphor sheet in the present invention, it is also possible to use an appropriate method other than the above-mentioned examples. .

[0064]

Next, an example in which the autoradiogram measuring method of the present invention is used for simultaneous measurement of cerebral blood flow and cerebral metabolism in rats will be described. (1) Preparation of measurement sample A cerebral infarction model rat and a normal rat 48 hours after arterial occlusion were prepared, and ¹⁴ C was added to each rat according to a conventional method.
2- deoxyglucose (material for brain topical glucose consumption calculation) and N- isopropyl -p- ^[3 H]
Iodoamphetamine (agent for measuring local cerebral blood flow) was sequentially administered from the vein of 40 μCi of the former, and then 2 mμCi of the latter 43 minutes later. Two minutes after the last dose, the rats were decapitated, then the brain was removed and frozen. Serial sections of 20 μm were made from the frozen brain and used as measurement samples.

(2) Preparation of stimulable phosphor sheet A divalent europium-activated barium fluorobromide stimulable phosphor layer is formed on a polyethylene terephthalate sheet support containing titanium dioxide by a conventional method using a polyurethane resin as a binder. Thus, a stimulable phosphor sheet A was prepared. Similarly, on a polyethylene terephthalate sheet support containing titanium dioxide, a divalent europium-activated barium fluorobromide stimulable phosphor layer is formed using a polyurethane resin as a binder, and a transparent layer is formed on the stimulable phosphor layer. A polyethylene terephthalate sheet (protective film, thickness: 20 μm) was stuck to prepare a stimulable phosphor sheet B.

(3) Autoradiography Each of the measurement samples (cerebral infarction model rat and normal rat) was superimposed on the stimulable phosphor sheet A for 12 hours.
An exposure operation was performed. Next, the same measurement sample and the stimulable phosphor sheet B were each superposed and exposed for 12 hours. A stimulable phosphor sheet A, which was exposed to a cerebral infarction model rat and a normal rat sample,
An autoradiogram readout operation was performed for B (total of 4 sheets) using the radiation image reading apparatus shown in FIG. 1, and the stimulable phosphor sheets A and B were read for each sample of the cerebral infarction model rat and the normal rat. And a subtraction process of the position information signal corresponding to the autoradiogram obtained from the above is performed, and ¹⁴ C-2-deoxyglucose and N-isopropyl-p- [ ³ H] of each sample of a cerebral infarction model rat and a normal rat are obtained. When the distribution of iodoamphetamine was examined, a good agreement was observed between the cerebral local glucose consumption and the local cerebral blood flow in normal rats, but the cerebral infarction model rat showed a good correlation between the cerebral local glucose consumption and the local cerebral blood flow. Mismatch was confirmed.

[0067]

The method of the present invention for measuring an autoradiogram solves the above-mentioned problems associated with the conventional autoradiography by the double labeling method using a photosensitive silver salt film, and in particular, provides a method for measuring a plurality of brightnesses. There is no need to prepare a stimulable phosphor layer (laminate or multiple stimulable phosphor sheets),
In addition, by repeating a simple reading operation and then performing a simple signal processing operation, a radiographic image of a plurality of highly separated radioactive labeling substances that are sufficiently separated (that is, the effects of other radiographic images can be ignored) can be obtained. There are advantages that can be obtained.

Further, according to the method of the present invention, not only the exposure time can be significantly reduced, but also the position information obtained even when the exposure is performed at a temperature condition of an ambient temperature or a temperature near the ambient temperature. Does not decrease in accuracy. Therefore, the exposure operation conventionally performed over a long period of time under cooling becomes extremely simple, and the autoradiography operation is simplified.

Further, by utilizing the above-mentioned stimulable phosphor sheet as a photosensitive material used in the autoradiogram measurement method, chemical fogging and physical fogging, which have conventionally been a major problem in the use of radiation films, are substantially reduced. Is also very advantageous in improving the accuracy of the obtained position information and in workability. In addition, the decrease in accuracy due to radioactivity or natural radioactivity of impurities contained in the sample can be easily reduced by electrically processing the position information stored and recorded on the stimulable phosphor sheet. Alternatively, it can be eliminated.

[Brief description of the drawings]

FIG. 1 shows an example of the configuration of a reading (reading) device for reading out positional information of a radioactive label substance in a sample stored and recorded on a stimulable phosphor sheet.

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 stimulable phosphor sheet 2 read-ahead read-out section 3 read-out section for main read-out 4 laser light source 5 laser light 6 filter 7 optical deflector 8 plane reflecting mirror 9 transfer direction 10 light guide for look-ahead 11 photodetector 12 amplifier 13 control circuit 14 Laser light source 15 Laser light 16 Filter 17 Beam expander 18 Optical deflector 19 Planar reflecting mirror 20 fθ lens 21 Transfer direction 22 Book reading light guide 23 Photodetector 24 Amplifier 25 A / D converter 26 Signal processing circuit

──────────────────────────────────────────────────続 き Continuation of front page (72) Inventor Keiji Mori 2-26-30 Nishiazabu, Minato-ku, Tokyo Fuji Photo Film Co., Ltd. (56) References JP-A-62-93679 (JP, A) JP-A Sho 60-233583 (JP, A) Japanese Translation of PCT International Publication No. 3-502729 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) G01N 33/60 G01T 1/29 G21K 4/00

Claims (3)

(57) [Claims] 1. Obtaining positional information of two or more radiolabeled substances labeled with radioisotopes having different average energies distributed in a tissue sample of an organism or a medium sample containing a tissue or substance derived from an organism. 1) superimposing the sample on a stimulable phosphor sheet including a stimulable phosphor layer having a protective film on the surface thereof for a predetermined time via the protective film to obtain an average A step of absorbing radiation energy emitted from a radioactive labeling substance having a relatively high energy into the stimulable phosphor layer and recording the stimulable phosphor layer; and 2) exciting the stimulable phosphor layer of the stimulable phosphor sheet with an electromagnetic wave. The radiation energy stored and recorded in the stimulable phosphor layer is emitted as stimulable light, and the average energy is detected by detecting the stimulable light. A step of obtaining an image signal indicating a relatively high position information of the radiolabeled substance; 3) the sample having no protective film on its surface or a protective film thinner than the protective film used in the above step 1); A stimulable phosphor sheet containing a stimulable phosphor layer having a radioactive label having a relatively high average energy by being superimposed on the stimulable phosphor layer directly or via the protective film for a certain period of time. A step of causing the stimulable phosphor layer to absorb and record radiation energy emitted from both the substance and the radiolabeled substance having a relatively low average energy; and 4) a stimulable phosphor sheet having undergone the above step 3). Is excited by an electromagnetic wave to emit radiation energy stored and recorded in the stimulable phosphor layer as stimulable light, and by detecting this stimulable phosphor, the average energy is reduced. Obtaining an image signal indicating the position information of both the radiolabeled substance having a relatively high energy and the radiolabeled substance having a relatively low average energy; 5) From the image signal obtained in the step 4), )
Obtaining the position information of each of two or more radiolabeled substances in the sample by performing a subtraction process except for between the image signals obtained in the step (a). . 2. The method according to claim 1, wherein each of the radiolabeled substances is ³ H, ¹²³
2. The autoradiogram measurement according to claim 1, wherein the autoradiogram is selected from the group consisting of I, ¹³¹ I, ¹⁴ C, ³⁵ S, ¹⁸ F and ³² P, and has been radiolabeled with different radioisotopes. Method. 3. A radiolabeled substance having a relatively high average energy and a radiolabeled substance having a relatively low average energy are radiolabeled by ¹⁴ C and ³ H, respectively. Autoradiogram measurement method.