JPH0520711B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for displaying autoradiographs in color.

[Technical background of the invention] After administering a radioactively labeled substance to an organism, the organism or a part of the tissue of the organism is used as a sample, and this sample is exposed to radiation such as a highly sensitive X-ray film. Autoradiography is already known, which consists of exposing a film to light by overlapping it with the film for a certain period of time, and obtaining positional information of a radiolabeled substance in the sample from the exposed area.

Autoradiography is also used to obtain positional information of a radiolabeled substance in a tissue of an organism and/or a medium containing an organism-derived substance to which a radiolabel is attached. For example, a radioactive label is attached to a biologically derived polymeric substance such as a protein or a nucleic acid, and the radiolabeled polymeric substance, its derivative, its decomposed product, or its composite is separated using gel electrophoresis or other methods. After separation on a support medium through an operation, the support medium and a radiation film are superimposed and the film is exposed to light. Based on the positional information of the radiolabeled substance on the support medium obtained from the autoradiographic image, the high Methods for separating and identifying molecular substances, and evaluating the molecular weight and characteristics of polymeric substances 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 RNA. There are also southern blotting, northern blotting, and western blotting.
It is also an indispensable tool in genetic screening using hybridization methods such as blotting.

The applicant has already applied for a patent regarding the autoradiograph display method (Patent application 181431/1986).
(No. 61-90603). Special application for the former 1980-
No. 181431 describes an autoradiograph of a sample in which radioactively labeled nucleic acid fragments are separated and developed in a one-dimensional direction on a support medium, based on the position and signal level along the direction of separation and development. A method is disclosed in which a plurality of two-dimensional waveforms are displayed multiplexed at regular intervals in a direction perpendicular to the direction of separation and development (displayed as a so-called bird's-eye view). According to this display method, it is possible to overcome the drawbacks of conventional black and white images, such as difficulty in obtaining quantitative information on radiolabeled substances and difficulty in comparative identification between separated and developed sites (bands). Also, the latter
No. 61-90603 discloses a method of displaying this bird's-eye view in at least two different colors. According to this method, comparative identification between bands can be performed even more accurately and easily.

The applicant has already filed a patent application for a method of using a radiation image conversion method using a stimulable phosphor sheet in place of the conventional radiography method using a radiation film in autoradiography. Publication No. 83057 (1983), Japanese Patent Publication No. 60-
No. 10174, Patent Application No. 173393/1983). The stimulable phosphor sheet is made of a stimulable phosphor, and after radiation energy is absorbed by the stimulable phosphor of the phosphor sheet, it is excited by electromagnetic waves (excitation light) in the visible to infrared region to generate fluorescence. It can be released as According to this method, the exposure time can be significantly shortened, and chemical fog, which has been a problem in the past, does not occur.
Furthermore, since the autoradiograph is stored in a phosphor sheet and then read out in time series as photostimulated light, it can be directly obtained as a digital signal.

[Summary of the Invention] The present invention provides an autoradiograph display method that can easily and appropriately adjust the color level when displaying an autoradiograph having information as a gray scale image in color. .

Further, the present invention provides an autoradiograph display method that can easily display an autoradiograph having information as a grayscale image as a color image that is easy to analyze.

That is, the present invention provides a color autoradiograph having positional information of a sample selected from the group consisting of biological tissue to which a radioactive label has been added and a medium containing biological tissue and/or biological material. In the display method, the autoradiograph is represented by a digital signal having gradation information of n stages (n is a positive integer of 3 or more), and 1. A preset minimum color level l. Based on _p and the maximum color level l _k , each remaining level l _i is calculated as l _i = {(l _k −l _p )/k}·i+l _p (where k is a positive integer smaller than n,
i is a positive integer in the range of 0≦i≦k), and then the gradation in the range of 0 to n steps is determined as (k
+1) dividing the autoradiograph into (k+2) colors by color levels; and 2) dividing the autoradiograph into (k+2) colors by replacing the n levels of gradation with corresponding colors based on the digital signal. The present invention provides an autoradiograph display method characterized by comprising the steps of: displaying in color.

In the present invention, "position information" refers to various information centered on the position of a radiolabeled substance or an aggregate thereof in a sample, such as the location of an aggregate of a radiolabeled substance on a support medium, It means various types of information obtained as one or any combination of information such as concentration, distribution, identification of a radiolabeled substance based on these, identification of a sample, etc.

The method of the present invention, when displaying an autoradiograph as a color image, determines the color level based on a digital signal corresponding to the autoradiograph and having grayscale image information consisting of multiple gradations. This realizes simple and appropriate adjustment of

An autoradiograph of a sample is usually obtained as a grayscale image depending on the intensity of radiation emitted from a radiolabeled substance in the sample. In order to display this autoradiograph in color as a color image on a screen such as a CRT, it is necessary to convert the gradation of the grayscale image into hues classified by color level based on the digital signal corresponding to the autoradiograph. Processing is required. For example, when a digital signal corresponding to an autoradiograph has an information amount of 8 bits per pixel, it includes 256 levels of gradation information. According to the method of the present invention, even if there is a large amount of gradation information in a gray scale image of an autoradiograph, the hue corresponding to the gradation can be easily calculated by inputting the maximum and minimum values of the color level to the signal processing circuit. It is possible to determine the correspondence of
A proper and easy-to-read color image can be obtained.

In addition, when trying to express an autoradiograph by replacing multiple gradations with a relatively small number of colors such as 8 or 16 colors, in order to maintain high analysis accuracy, it is necessary to Depending on the part of the autoradiograph, it is required that the color level (threshold between colors) can be changed freely. According to the present invention, even in such a case, the remaining levels can be automatically set by simply setting two levels, the maximum level and the minimum level, without having to set the color levels one by one according to the number of colors. color can be easily determined and the color change operation is easy. Also, since the total color level is determined by the maximum level and minimum level, it is possible to finely color-code any gradation range (image density range) according to these level settings, and analyze it. You can easily obtain color images.

In particular, it is possible to easily determine and analyze the peak position and magnitude of radiation intensity (image density) at the distribution site of the radiolabeled substance.
The color display method of the present invention can be effectively used in autoradiography for determining base sequences of nucleic acids such as RNA, and genetic screening using Southern blotting methods.

[Structure of the Invention] In the present invention, the sample to be autoradiographed is a tissue of an organism to which a radioactive label has been added, and a medium containing the tissue of an organism and/or a substance derived from an organism. Among these, the method of the present invention is effectively utilized when the sample is a tissue of an organism to which a radioactive label has been added and/or a medium containing an organism-derived substance.

For example, proteins with radioactive labels, nucleic acids,
Examples include macromolecular substances derived from living organisms such as derivatives thereof, decomposition products thereof, and synthetic products. Radioactive labels are applied to these substances in an appropriate manner.
It is given by retaining radioactive isotopes such as ³² P, ¹⁴ C, ³⁵ S, ³ H, ¹²⁵ I, etc. Samples are subjected to various methods such as electrophoresis, thin layer chromatography, column chromatography, paper chromatography, etc. using various known support media such as gel support media to carry out these radioactively labeled substances. It is obtained by separating and developing it on a support medium using a separation and developing method. However, the sample to be subjected to the method of the present invention is not limited to the support medium in which these polymeric substances are separated and developed.

Autoradiographs of samples containing these radiolabeled substances are obtained by radiography using conventional photographic materials or by radiation image conversion using stimulable phosphor sheets, and then appropriate reading is performed. ) system through which a digital signal corresponding to the autoradiograph of the radiolabeled substance is obtained.

When using the former radiographic method, first the sample and a photographic material such as X-ray film are superimposed at low temperature or room temperature for a long time (several hours to several tens of hours), and then the radiographic film is exposed. The autoradiograph of the radiolabeled substance is developed to form a visible image on radiographic film. Next, the autoradiograph visualized on the radiographic film is read using an image reading device. For example, an autoradiograph is obtained as an electrical signal by irradiating a radiation film with a light beam and photoelectrically detecting the transmitted or reflected light. moreover,
By A/D converting this electrical signal, a digital signal corresponding to the autoradiograph can be obtained.

When using the latter radiation image conversion method,
First, the sample and the stimulable phosphor sheet are overlapped at room temperature for a short period of time (several seconds to several tens of minutes), and the phosphor sheet accumulates the radiation energy emitted from the radiolabeled substance, thereby producing an autoradiograph. It is recorded as a kind of latent image on the phosphor sheet. Here, the stimulable phosphor sheet is made of a support made of, for example, a plastic film, divalent europium-activated barium fluoride bromide (BaFBr: Eu ²⁺ )
A phosphor layer made of a stimulable phosphor such as phosphor and a transparent protective film are laminated in this order. The stimulable phosphor contained in the stimulable phosphor sheet absorbs and accumulates radiation energy when it is irradiated with radiation such as X-rays, and then accumulates when excited with light in the visible to infrared region. It has the property of emitting radiation energy as photostimulated light.

Next, the autoradiograph stored and recorded on the stimulable phosphor sheet is read out using a reading device. Specifically, for example, by scanning a phosphor sheet with a laser beam to emit radiation energy as photostimulated light, and detecting this photostimulated light photoelectrically, an autoradiograph of a radioactively labeled substance is converted into a visible image. It can be obtained directly as an electrical signal without any additional processing. Furthermore, by A/D converting this electrical signal, a digital signal corresponding to an autoradiograph can be obtained.

For details on the above-mentioned autoradiograph measurement operation and method for obtaining a digital signal corresponding to the autoradiograph, see the aforementioned Japanese Patent Application Laid-open No. 59-83057;
It is described in various publications such as JP-A-59-126527 and JP-A-59-126278.

In addition, although the above describes a method of using conventional radiography and radiographic image conversion methods as a method of obtaining a digital signal corresponding to an autoradiograph of a radiolabeled substance in a sample, this method is not limited to these methods. The method of the present invention can be applied to digital signals obtained by any other method as long as there is a correspondence with the autoradiograph of the radiolabeled substance.

Furthermore, in any of the above methods, it is not necessary to read (or read out) the autoradiograph over the entire surface of the radiation film (or stimulable phosphor sheet), and it is of course possible to read out the autoradiograph only on the image area. . Furthermore, if the sample is made by separating and developing a radiolabeled substance on a support medium, input information such as the position of each separation and development column and the width of the separation and development site (band) in advance to read out (readout) conditions. is set, and in the readout operation, the light beam is scanned at a scanning line density such that at least one scanning line passes over each band.
You can also save time and get the necessary information efficiently. In the present invention, the digital signal corresponding to an autoradiograph includes the digital signal obtained in this manner.

The resulting digital signal corresponds to each pixel and determines the image density of the autoradiograph (i.e.
Radiation intensity) is the amount of information per pixel (number of bits)
It is included in the digital signal as gradation information consisting of the number of stages corresponding to the number of levels.

The operation of displaying the autoradiograph in color by subjecting the digital signal corresponding to the sample autoradiograph obtained in this manner to signal processing according to the method of the present invention will be described below.

A digital signal corresponding to an autoradiograph is temporarily stored in a memory (buffer memory or nonvolatile memory such as a magnetic disk) in a signal processing circuit.

First, the minimum color level l _p and the maximum color level l _k set within the range of 0 to n levels are input to the signal processing circuit. Here, the color level means a threshold value for classifying each color.

Based on this minimum level l _p and maximum level l _k , each remaining level l _i is calculated as l _i = {(l _k −l _p )/k}・i+l _p (where k is a positive integer smaller than n. ,i
is a positive integer in the range 0≦i≦k). That is, the intermediate levels are determined to equally divide the two maximum and minimum levels.

FIG. 1 shows an example of color levels when the number of colors is eight.

For example, as shown in Figure 1, when displaying in eight colors, the maximum level l 6 is the threshold between the first and second colors, and the maximum level l ₆ is the threshold between the seventh and eighth colors. When the minimum level l _p which is a threshold value is set, the remaining intermediate levels l ₁ to l ₅ are levels obtained by equally dividing the range between l _p and l ₆ into six.

As a result, the gradation in the range of 0 to n steps is (k+
1) The color level is divided into (k+2) colors, and each gradation level is associated with one of the (k+2) colors.

At this time, by setting the maximum and minimum levels, any gradation range can be emphasized by changing a plurality of colors. For example, when trying to emphasize areas with high image density, set the maximum level relatively high to make the range of the first color relatively narrow, as shown in Figure 2, and set the minimum level relatively high. The level can also be set quite high so that the range of the eighth color becomes wider. Note that FIG. 2 shows another example of color levels when the number of colors is eight.

There is no particular limit to the number of gradations of a grayscale image to which the method of the present invention can be applied, but the digital signal corresponding to an autoradiograph is usually 8 to 10 per image.
It has an information amount of bits, and includes gradation information of 256 to 1024 levels. Further, the number of colors to be converted is not particularly limited as long as it is equal to or less than the number of gradation steps, but the method of the present invention can be preferably applied to a relatively small number of colors, for example, 4 to 16 colors.

Next, the n-level gradation in the digital signal is replaced with a corresponding color among (k+2) colors based on the color level. As a result, each pixel of the autoradiograph is converted into a hue corresponding to its gradation.

In this way, the signal having color information is output from the signal processing circuit, and then transmitted to the display/recording device directly or, if necessary, via a recording storage means such as a magnetic disk or magnetic tape, and then transmitted to the autoradio. The graph is displayed in color as a (k+2) color image.

As the display device, for example, an electrical device such as a graphic display or CRT can be used. In addition, recording devices based on various principles include, for example, dot printers for graphics, devices that optically record by scanning photosensitive materials with laser beams, and devices that record on heat-sensitive recording materials using heat rays. A device can be used.

Furthermore, the color distribution of an image once displayed in color can be changed again. In that case, by performing the above-described operation again, the color level can be changed and a desired color image can be displayed and recorded.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing an example of setting color levels for eight colors. FIG. 2 is a diagram showing another example of setting color levels for eight colors.

Claims (1)

[Scope of Claims] 1. An autoradiograph having positional information of a sample selected from the group consisting of biological tissue to which a radioactive label has been added and a medium containing biological tissue and/or biological material. In a color display method, the autoradiograph is represented by a digital signal having gradation information of n stages (n is a positive integer of 3 or more), and 1. A preset minimum color level. Based on l _p and the maximum color level l _k , each remaining level l _i is calculated as l _i = {(l _k −l _p )/k}・i+l _p (where k is a positive integer smaller than n,
i is a positive integer in the range of 0≦i≦k), and then the gradation in the range of 0 to n steps is determined as (k
+1) dividing the autoradiograph into (k+2) colors by color levels; and 2) dividing the autoradiograph into (k+2) colors by replacing the n levels of gradation with corresponding colors based on the digital signal. An autoradiograph display method characterized by comprising the steps of displaying in color. 2 The above autoradiograph is represented by a digital signal having gradation information of 256 or more levels,
Claim 1 characterized in that the autoradiograph is displayed in 4 to 16 colors.
Autoradiograph display method described in section. 3. The autoradiograph display method according to claim 1, wherein the autoradiograph is electrically displayed in color. 4 After the digital signal records the autoradiograph of the sample on the phosphor sheet by overlapping the sample and a stimulable phosphor sheet containing a stimulable phosphor, the phosphor sheet is irradiated with excitation light. 2. The autoradiograph display method according to claim 1, wherein the autoradiograph is obtained by photoelectrically reading out the autoradiograph as photostimulated light. 5. The above-mentioned digital signal is obtained by overlapping the sample and a photographic light-sensitive material, photosensitively recording the autoradiograph of the sample on the photosensitive material, and then photoelectrically reading the autoradiograph visualized on the photosensitive material. An autoradiograph display method according to claim 1, characterized in that: 6. Claim 1, characterized in that the sample is a medium in which a radioactively labeled biopolymer group, a derivative thereof, a decomposition product thereof, or a composite thereof is distributed. How to display an autoradiograph. 7. The autoradiographic display method according to claim 6, wherein the biopolymer substance group is a nucleic acid, a derivative thereof, a decomposition product thereof, or a composite thereof.