Signal detecting method in autoradiography

A signal detecting method in autoradiography, which is applied to an autoradiograph having locational information on radioactively labeled substances such as radioactively labeled biopolymers distributed one-dimensionally, being recorded on a radiosensitive material as a visible image, PA0 which comprises steps of: PA1 (1) preliminarily scanning a part of the radiosensitive material with a light and photoelectrically reading out a portion of the visualized autoradiograph to give an electric signal, to which a signal processing is applied, to determine one-dimensional distribution direction of the radioactively labeled substances; and PA1 (2) finally scanning the radiosensitive material with a light along said one-dimensional distribution direction and photoelectrically reading out the visualized autoradiograph, to obtain the locational information on the radioactively labeled substances as a digital signal.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a signal detecting method in autoradiography. 
2. Description of the Prior Art 
Autoradiography has been known as a method for obtaining locational 
information on radioactively labeled substances distributed in at least 
one-dimensional direction to form a row or rows on a support medium. 
For instance, autoradiography comprises steps of: labeling 
organism-originating biopolymers such as proteins or nucleic acids with a 
radioactive element; resolving the radioactively labeled biopolymers, 
derivatives thereof, or cleavage products thereof (referred to hereafter 
as "radioactively labeled substances") on a gel support (support medium) 
through a resolving process such as electrophoresis to form a resolved 
pattern of the radioactively labeled substances (the resolved pattern is 
not visible); placing said gel support and a high-sensitivity type X-ray 
film together in layers for a certain period of time to expose said film 
and developing said film to give the autoradiograph of the resolved 
pattern as a visible image on the film; and obtaining the locational 
information on the radioactively labeled substances from said visible 
image. Further, the identification of the polymeric substances, 
determination of molecular weight of the polymeric substances and 
isolation of the polymeric substances can be performed based on the 
obtained locational information. The autoradiography has been effectively 
utilized for determining the base sequence of nucleic acids such as DNA. 
In the autoradiography utilizing the above-mentioned radiographic process, 
the autoradiograph is analyzed through visual judgement of investigators. 
Instead of the above analytical method, a digital signal processing method 
for obtaining information on one-dimensional location of radioactively 
labeled substances in the form of symbols and/or numerals, which comprises 
reading out the visualized autoradiograph photoelectrically to give a 
digital signal and applying an appropriate signal processing to the 
digital signal, is described, for instance, in Japanese Patent Application 
No. 58(1983)-1336 (corresponding to U.S. patent application Ser. No. 
06/568875 and European patent application No. 84100147.2). Said signal 
processing method comprises steps of: determining a one-dimensional 
scanning line (namely one-dimensional distribution direction of the 
radioactively labeled substances) for signal processing with respect to 
the obtained digital signal, and detecting sampling points on the scanning 
line. The term "scanning" in this method means the numeral scanning on the 
digital image data. 
According to the above-mentioned signal processing method, the information 
on one-dimensional location of the radioactively labeled substances, which 
has been conventionally obtained through visual judgement by 
investigators, can be obtained automatically and accurately in the desired 
form such as symbols and/or numerals. Consequently, the above-mentioned 
signal processing method improves the accuracy of locational information 
and brings about an increase of the amount of information obtained. 
Another signal processing method in the autoradiography utilizing the 
radiography, which comprises determining the scanning line for detecting 
sampling points with respect to the obtained digital signal, is described 
in Japanese patent application No. 58(1983)-1337 (the content of which is 
disclosed in U.S. patent application Ser. No. 568,873 and European Patent 
Application No. 84100149.8). 
In any signal processing method described above, the one-dimensional 
distribution direction (scanning line on the digital image data for 
detecting sampling points) of the radioactively labeled substances is 
determined, after the digital signal corresponding to the autoradiograph 
having the locational information on the radioactively labeled substances 
is obtained by photoelectrically reading out the autoradiograph visualized 
on the radiosensitive material. Therefore, the obtained digital signal is 
initially stored in a memory of a signal processing circuit and 
subsequently the digital signals is selectively taken out of the memory 
according to the signal processing operation, so as to determine the 
one-dimensional distribution direction of the radioactively labeled 
substances. 
More in detail, the read-out of the radiosensitive material is carried out 
over its entire surface and the digital image data are inevitably obtained 
even on a vacant area of the sheet which does not give any locational 
information on radioactively labeled substances. That is, all of the 
digital signal which is obtained by detecting on the entire surface of the 
radiosensitive material should be temporally stored in the memory of the 
signal processing circuit, and thus the memory requires a great capacity 
for storing it. 
Additionally, the photoelectrically read-out operation on the 
radiosensitive material requires a long period of time, because the 
operation should be carried out over the entire surface thereof. 
SUMMARY OF THE INVENTION 
Accordingly, an object of the present invention is to provide a signal 
detecting method in autoradiography for efficiently detecting a digital 
signal having the locational information on radioactively labeled 
substances. 
The object can be accomplished by preliminarily scanning with a light a 
part of the radiosensitive material on which an autoradiograph having the 
information on one-dimensional location of the radioactively labeled 
substances is recorded, to determine one-dimensional distribution 
direction of the radioactively labeled substances; and subsequently 
scanning with a light the same radiosensitive material along said 
one-dimensional distribution direction to obtain the locational 
information on the radioactively labeled substances as a digital signal. 
That is, the present invention provides a signal detecting method in 
autoradiography, which is applied to an autoradiograph having locational 
information on radioactively labeled substances distributed 
one-dimensionally, being recorded on a radiosensitive material as a 
visible image, 
which comprises steps of: 
(1) scanning the radiosensitive material with a light in at least two 
different positions in such a manner that each scanning traverses the 
one-dimensional distribution of the radioactively labeled substances and 
photoelectrically reading out a portion of the visualized autoradiograph 
to give an electric signal, to which is applied a signal processing 
comprising stages of: 
detecting distribution points of the radioactively labeled substances for 
each scanning, and preparing a continuous line selected from the group 
consisting of a straight line, a polygonal line and a curved line along 
the corresponding distribution points of the radioactively labeled 
substances for each scanning, to assign said continuous line to 
one-dimensional distribution direction of the radioactively labeled 
substances; and 
(2) scanning the radiosensitive material with a light along said 
one-dimensional distribution direction of the radioactively labeled 
substances determined in the step (1) and photoelectrically reading out 
the visualized autoradiograph, to obtain the locational information on the 
radioactively labeled substances indicated by said autoradiograph as a 
digital signal. 
In the present invention, the term "locational information" of the 
radioactively labeled substances means includes a variety of information 
relating to the location of the radioactively labeled substances, or the 
aggregation thereof, being present in the sample, such as the location, 
the shape, the concentration, the distribution and combinations thereof.

DETAILED DESCRIPTION OF THE INVENTION 
According to the present invention, the period of time required for reading 
out the locational information can be shortened, by preliminarily scanning 
a radiosensitive material with a light of low intensity to determine the 
one-dimensional distribution direction of the radioactively labeled 
substances, and subsequently scanning the same radiosensitive material 
along said one-dimensional distribution direction with a light to obtain 
the locational information on the radioactively labeled substances as a 
digital signal. More in detail, the read-out (scanning with a light) of 
the autoradiograph recorded on the radiosensitive material does not have 
to be carried out over the entire surface thereof, but is carried out only 
in a certain area along the one-dimensional distribution direction of the 
radioactively labeled substances, so that the longer period of time which 
has been required in the conventional read-out operation is greatly 
shortened. 
Further, it is not required to store the digital signal corresponding to 
the autoradiograph obtained by scanning the entire surface of the 
radiosensitive material in the memory of the signal processing circuit, 
which is different from the conventional read-out operation, but the 
digital signal only in a certain area along the one-dimensional 
distribution direction of the radioactively labeled substances, namely 
only the digital signal which provides the locational information on the 
radioactively labeled substances, can be stored in the memory, and thereby 
the required memory capacity can be greatly reduced. 
Furthermore, according to the present invention, the one-dimensional 
distribution (resolution) direction of the radioactive labeled substances 
on an autoradiograph can be detected automatically to determine a scanning 
direction for detection of the digital signal, even in the case that there 
occurs overall distortion or dislocation of the autoradiograph recorded on 
the radiosensitive material, which is brought about by locational 
distortion of a resolved row of the radioactively labeled substances on 
the support medium in the resolving procedure or by incorrect arrangement 
between the support medium carrying the resolved row of radioactively 
labeled substances and the radiosensitive material in the exposing 
procedure. Based on the thus determined scanning direction, the 
information on one-dimensional location of the radioactively labeled 
substances can be obtained with a high accuracy. Further, in the case that 
the autoradiograph is composed of a plurality of resolved rows of the 
radioactively labeled substances distributed in one-dimensional 
directions, the distribution direction of every resolved row having the 
distortion can be accurately detected to determine a scanning direction 
for each row. 
In the present invention, the term "one-dimensional distribution" means a 
pattern which comprises the radioactively labeled substances spread in one 
direction in the form of a row of bands or spots, such as an 
electrophoretic row resolved through electrophoresis. 
Examples of the sample employable in the present invention include a 
support medium on which radioactively labeled substances are distributed 
(e.g., resolved) in a one-dimensional direction to form a distributed row 
(e.g., resolved row). Examples of the radioactively labeled substances 
include biopolymers, derivatives thereof, or cleavage products thereof, 
labeled with a radioactive substance. 
For instance, in the case that the radioactively labeled biopolymers are 
polymeric substances such as protein and nucleic acid, derivatives thereof 
and cleavage products thereof, the present invention is useful for 
isolation and identification thereof. Further, the present invention can 
be effectively used to analyze the whole or partial molecular structures 
of these biopolymers and the basic segmental constitutions thereof. In 
particular, the present invention can be effectively used to determine the 
base sequence of nucleic acid such as DNA. 
Representative examples of the method for resolving (or developing) the 
radioactively labeled substances on a support medium include an 
electrophoresis using one of various resolving mediums such as a gel in 
the form of layer, column or the like, a molded polymer film such as a 
cellulose diacetate film, and a filter paper, and a thin layer 
chromatography using a support of material such as silica gel. However, 
the method employable in the present invention is by no means restricted 
by these methods. 
Samples employable in the present invention are by no means restricted to 
the above-mentioned samples, and any other samples can be used, provided 
that the sample is radioactively labeled substances distributed in at 
least one-dimensional direction and is capable of having an autoradiograph 
with the locational information thereof to be recorded on a radiosensitive 
material. 
The radiosensitive material used in the present invention has a basic 
structure comprising a support and a radiographic (photographic) emulsion 
layer. The radiographic emulsion layer comprises a binder such as gelatin 
and silver halide dispersed therein. For instance, the radiosensitive 
material is prepared by providing the above-mentioned emulsion layer onto 
the transparent support such as a polyethylene terephthalate sheet. A 
representative example of the radiosensitive material includes a 
radiographic film such as a high-speed type X-ray film. 
In carrying out the exposing procedure, that is, the procedure of exposing 
the radiosensitive material to the radiation emitted from the support 
medium containing the radioactively labeled substances, at least a portion 
of the emitted radiation is absorbed by the radiosensitive substance of 
the radiosensitive material by placing the support medium and 
radiosensitive material together in layers for a certain period of time. 
The exposure can be accomplished by keeping the radiosensitive material in 
a position adjacent to the support medium, for instance, at a low 
temperature such as a temperature lower than 0.degree. C. for at least 
several days, and then the radiosensitive material is developed. In the 
exposing procedure, it is further possible to enhance the radiographic 
speed of the radiosensitive material by using a radiographic intensifying 
screen or applying thereto a preliminary exposure such as a flash 
exposure. 
The exposing procedure of the radiosensitive material to a sample and the 
developing procedure thereof in the autoradiographic process have been 
well known, and are described, for instance, in the following literature: 
Method in Biochemical Experiment, Volume 6, Method in Tracer Experiment I, 
271-289, "8. Autoradiography" by Toru Sueyoshi and Akiyo Shigematsu (Tokyo 
Kagaku Dozin Ltd., 1977). 
The signal detecting method according to the present invention for reading 
out the information on one-dimensional location of the radioactively 
labeled substances recorded on the radiosensitive material and obtaining 
it in the form of a digital signal will be described in detail 
hereinafter, referring to an embodiment of a read-out system shown in FIG. 
1 of the accompanying drawings and concerning an example of an 
autoradiograph obtained by resolving a mixture of radioactively labeled 
substances on a support medium through a resolving method such as 
electrophoresis. 
FIG. 1 schematically illustrates an embodiment of the read-out system for 
reading out an autoradiograph recorded on a radiosensitive material 1 as a 
visible image, which has the information on the one-dimensional location 
of radioactively labeled substances (an example is shown in FIG. 2). 
FIG. 2 schematically illustrates an example of the autoradiograph of a 
sample recorded on the radiosensitive material, said sample being composed 
of plural kinds of the radioactively labeled substances which are resolved 
in the longitudinal direction on the support medium to form two resolved 
rows. The autoradiograph on the radiosensitive material shown in FIG. 2 is 
an example having a distortion due to the incorrect arrangement between 
the support medium and radiosensitive material in the exposing procedure, 
or the like. 
At first, the preliminary read-out operation is carried out in the 
following manner. 
Laser beams generated by a sensor 2 impinge upon the radiosensitive 
material 1. The sensor 2 comprises a number of optical heads 3 and is 
positioned adjacent to the radiosensitive material 1. Each optical head 3 
comprises a laser diode 3a for generating laser beam and a photosensor 3b 
for detecting the laser beam reflected by the radiosensitive material 1. A 
set of the laser diodes 3a and a set of the photosensors 3b are so 
arranged that the light-radiating faces of the laser diodes 3a and the 
light-receiving faces of the photosensors 3b are located in parallel with 
each other in the sensor 2. The sensor is so arranged downwardly that the 
sensor 2 can be located in parallel with the radiosensitive material 1. 
The number of optical heads 3 is coincident with the number of scans 
needed for determination of the one-dimensional distribution direction of 
the radioactively labeled substances, and the sensor 2 has at least two of 
the optical heads. 
The radiosensitive material 1 is transferred in the direction along the 
arrow 4 under the irradiation of the above-mentioned laser beam, wherein 
the radiosensitive material 1 is so positioned that the transferring 
direction represented by the arrow 4 traverses the one-dimensional 
distribution of the radioactively labeled substances recorded on the 
radiosensitive material 1. Accordingly, when the radiosensitive material 1 
is transferred in the direction along the arrow 4, the laser beams whose 
number coincides with the number of optical heads 3 are simultaneously 
irradiated in parallel with each other on the radiosensitive material 1. 
Each laser beam has a sufficiently large beam diameter which is generated 
by the laser diode 3a to perform the scanning with the laser beam with 
such a width as to catch at least one distributed portion of the 
radioactively labeled substances for each distributed row. 
Each laser beam reflected by the radiosensitive material 1 impinges upon 
the photosensor 3b of the sensor 2. The photosensor 3b, for instance, 
comprises a solidstate imaging device such as CCD, or photomultiplier, 
etc. The reflected light (laser beam) detected by the photosensor 3b is 
converted into an electric signal and then input to a control circuit 5. 
That is, the electric signal with respect to the hatched areas shown in 
FIG. 2 is input to the control circuit 5. 
In the control circuit 5, the obtained electric signal is subjected to the 
signal processing, and thus the one-dimensional distribution direction of 
the radioactively labeled substances is determined. During the signal 
processing, the obtained electric signal is temporally stored in a memory 
(buffer memory) in the control circuit 5. The signal processing for 
determination of the one-dimensional distribution direction of the 
radioactively labeled substances, for instance, is carried out as follows: 
At first, the distribution of intensity of the signal in each scanning 
area is calculated along the scanning direction to obtain such an 
intensity distribution as shown in FIG. 3. Each peak appearing in the 
intensity distribution is then assigned to a distribution point of the 
radioactively labeled substances in each scanning area, and a straight 
line (or polygonal line) is prepared among the corresponding distribution 
points in each scanning area, to be assigned determined aimed 
one-dimensional distribution direction of the radioactively labeled 
substances. Further, the obtained polygonal line may be approximated with 
a suitable curved line so that the one-dimensional distribution direction 
can be determined more accurately. 
In the case of obtaining the electric signal in a great number of scanning 
areas, the distribution points of the radioactively labeled substances may 
be detected for all the scanning areas in the same manner as mentioned 
above to prepare a polygonal line among the corresponding distribution 
points, but it is also possible to selectively find certain preferable 
scanning areas followed by performing the above-mentioned processing only 
with respect to these scanning areas in order to simplify the signal 
processing and shorten the processing time. 
To determine the one-dimensional distribution direction of the 
radioactively labeled substances more accurately, the distance between 
each scanning area is preferably as far as possible. When the 
one-dimensional distribution direction is determined using two scanning 
areas, it is desired to select such scanning areas as to be positioned at 
the top (or vicinity thereof) and the bottom (or vicinity theref) of the 
distribution row of the radioactively labeled substances. 
Thus, the control circuit 5 outputs a scanning condition a of the light 
beam such as a scanning position, direction thereof and width thereof for 
the final read-out operation, according to the determined one-dimensional 
distribution direction of the radioactively labeled substances. 
After finishing the preliminary read-out operation in the above-mentioned 
manner, the final read-out operation is carried out in the following 
manner. 
Laser beam 7 generated by a laser source 6 for the final read-out passes 
through a lens 8 and is subsequently deflected by a movable beam deflector 
9 such as a galvanometer mirror which is set on its deflection according 
to the above-mentioned light beam condition a, and reflected by a movable 
plane reflection mirror 10 which is also adjusted according to the 
scanning condition a. The deflected laser beam impinges one-dimensionally 
upon the radiosensitive material 1. The movable beam deflector 9 and 
movable plane reflection mirror 10 should be continually adjusted 
according to the scanning condition a output from the control circuit 5. 
Consequently, the deflected laser beam is irradiated on the radiosensitive 
material 1 with a certain width along the one-dimensional distribution 
direction of the radioactively labeled substances. 
Then, the deflected laser beam is transmitted by the radiosensitive 
material 1 and enters a movable line-sensor 11. The line-sensor 11 
comprises a solid-state imaging device such as CCD or a combination of a 
light guiding device and a photomultiplier, and can be transferred in the 
direction along the arrow 4 according to the light beam scanning direction 
a. Accordingly, the movable line-sensor 11 is transferred with precise 
relation to the adjustment of both the movable beam deflector 9 and 
movable plane reflection mirror 10, so that the transmitted light (laser 
beam) along the one-dimensional distribution direction of the 
radioactively labeled substance is received by the movable line-sensor 11. 
The transmitted light detected by the movable line-sensor 11 is converted 
to an electric signal, amplified in an amplifier 12 and input to an A/D 
converter 13. The electric signal is then converted to a digital signal in 
the A/D converter 13. 
In the above description on the method according to the present invention 
for reading out the locational information on the radioactively labeled 
substances recorded on the radiosensitive material, the final read-out 
operation wherein the scanning direction of the laser beam is straight has 
been given, but the read-out operation according to the invention is not 
limited to the above-mentioned embodiment. In the case that the 
distribution direction of the radioactively labeled substances is 
approximated with a polygonal line or a curved line, it is also possible 
to perform the scanning of the light beam in such a manner that the 
scanning direction varies coincidently with the distribution direction. 
Further, the above-described method has been given referring to the 
read-out system in which the preliminary read-out operation is performed 
with the different optical equipment from that for the final read-out 
operation, but the read-out system employable in the present invention is 
not limited to this one. For instance, it is possible to perform in the 
same optical equipment both the preliminary scanning with the light beam 
for determining the one-dimensional distribution direction of the 
radioactively labeled substances and the final scanning therewith for 
reading out the locational information along the determined distribution 
direction. 
It is also possible that the spot diameter of light beam in the final 
read-out operation is set according to the light beam scanning condition 
output from the control circuit. By setting the spot diameter of light 
beam to a suitable scanning width, the final read-out operation is more 
simplified. This simplification enables elimination of numeral scanning on 
the digital image data when the obtained digital signal is subjected to a 
signal processing, because only the signal having the locational 
information is obtained, that is, the signal processing for obtaining the 
locational information can be also simplified. 
The thus detected digital signal having the locational information on the 
radioactively labeled substances is input to a signal processing circuit 
14, in which various signal processings are applied thereto to obtain the 
desired information in the form of symbols and/or numerals. 
More in detail, through the processing, sampling points for detecting 
distributed portions of the radioactively labeled substances are 
determined based on the obtained digital signal. In the case that a 
plurality of distributed rows in the one-dimensional direction are 
concerned, the determined sampling points are compared and identified 
between corresponding positions on each distributed row, and thus the 
information on one-dimensional location of the radioactively labeled 
substances can be obtained as symbol and/or numeral. 
Since the digital signal obtained according to the present invention is 
input for individual distributed rows, the determination of scanning 
direction (one-dimensional distribution direction) for detecting the 
sampling points is not required on the digital image data. 
In addition, the amount of digital signal to be stored in the memory 
(buffer memory or non-volatile memory such as magnetic disk) in the signal 
processing circuit 14 can be decreased greatly, because the signal only in 
a certain area along the distribution direction of the radiosensitive 
material is detected. 
In the above-mentioned example shown in FIG. 2 and FIG. 3, the case of two 
distributed rows of the radioactively labeled substances is illustrated, 
but the signal detecting method of the present invention is not limited to 
this case and also applicable to any cases wherein the distributed row 
such as a resolved row is single or plural such as three or more. 
The signal processing to determine the one-dimensional distribution 
direction of the radioactively labeled substances in the control circuit 
is not limited to the above-mentioned one, but for instance, the same 
processing as the digital signal processing described in aforementioned 
Japanese patent application No. 57(1982)-1337 may be employed. 
By employing the signal detecting method of the present invention for 
obtaining the locational information on the radioactively labeled 
substances distributed in one-dimensional direction, the width (spot size) 
of individual distributed portion thereof can be reduced to approximately 
3 mm, because initially the one-dimensional distribution direction thereof 
is determined and the signal is then detected along the distribution 
direction. Consequently, the amount of radioactively labeled substances 
per a resolved row can be decreased and the number of resolvable rows per 
a support medium can be increased. In other words, more information can be 
obtained in a single autographic process than that in the conventional 
autoradiographic process. 
The signal detecting method in autoradiography of the present invention is 
very useful, for instance, for determination of the base sequence of DNA 
or DNA fragment utilizing autoradiography such as Maxam-Gilbert method. In 
Maxam-Gilbert method, DNA or DNA fragment labeled with a radioactive 
element is base-specifically cleaved for each base unit concerning the 
four bases of constitutional units, the mixture of base-specifically 
cleavage products is resolved and developed by electrophoresis to obtain 
an autoradiograph, and then the base sequence of DNA or DNA fragment is 
determined form the obtained autoradiograph. By applying the signal 
detecting method of the present invention to the Maxam-Gilbert method, the 
resolved (developed) direction can be detected based on the electric 
signal and assigned to the scanning direction in the optical operation 
independently of the combination of base-specific cleavage products. Thus, 
the digital signal having the locational information on DNA bases can be 
obtained along the scanning direction.