Autoradiography using radioisotopes is one of the most useful toots for metatturgy ana other material research. Since microautoradiographic techniques, however, have not yet been fully established, a cooperative research on microautoradiography has been carried out by Subcommittee on Autoradio graph, the Physical Sciences and Industry Committee of Japan Radioisotope Association, mainly in order to investigate problems of the resolution, using various methods of sample preparation and experimental operation. Test samples were prepared by alternately laminating radioactive110mAg and nonradioactive Ag layers and rolling them at high or low temperatures. The attained thickness of each layer was between 17 μ and 2.5μ. By using these samples, the resolution of microautoradiography in contact, stripping and dipping methods was studied, and also influences of sample thickness, separating layer thickness, pressure on photographic emulsion and humidity during exposure upon the resolution were examined in detail. Distribution curves of blackening density obtained with several factors affecting autoradiograph of the test sample were theoretically calculated by means of numerical integration. The modulation transfer functions were also obtained from these curves. The results obtained are summarized as follows: 1) The silver sample prepared by the above method is quite suitable for testing the resolution in microautoradiography. 2) Appropriate pressure must be applied on the emulsion layer for obtaining high resolution in the contact method. 3) The limit of resolving power in the stripping method was obtained when the layer thickness was about 5 μ. 4) As the influence of separating layer upon the resolution is remarkable, it is necessary to choose suitable materials and thickness of separating layer. 5) It is desired to keep the humidity between 30% and 40% during the exposure. 6) The modulation transfer function obtained from the experimental results agreed well with that calculated theoretically.
A simple method of correcting autoradiographic background is being proposed to estimate the labelling index of cell nuclei after administration of3H-thymidine. The labelling index (LI) is obtained by the following formula, LI=1-Y0/P0, where Y0is the proportion of cell nuclei without silver grains to the total cell nuclei in a given population, in the autoradio grams of experimental animals which were given3H-thymidine; and P0is that of control animals, or of tissues of the experimental animals in which3H-thymidine was unlikely to have been incorporated. In the present study, the periodontal connective tissue cells of the mandibular incisor of the rat one hour after3H-thymidine injection were observed, in order to estimate the labelling index. The background was also estimated over the same tissue in the control animals; or over the dentine, which does not seem to incorporate the radioisotope, in the experimental animals. There was a reasonable agreement in values of the labelling indices corrected by using the background distributions obtained from two different sources. A previous method of correcting background in which nuclei possessing grains of a certain number or less were neglected by assuming them to be due to background, seems to give an inadequate value of labelling index.
The distribution of 5-131IU and 6-131IP in the bodies of mice in which Ehrlich ascites tumor was implanted was studied by mean of the whole body autoradiography. The results obtained were compared with those previously reported. After intraperitoneal injection of 5-131IU and 6-131IP considerable radioactivities were detected in the tumor tissues, although higher radioactivities were found in the stomach, thyroid and salivary gland. Whole body autoradiography is as useful as scinticamera technique for the investigation of the tumor localizing radioactive agents.
A new autoradio graphic technique of “whole body activation autoradiography” was developed to utillize whole body autoradiographic techniques in studies of non-radioactive materials. This technique is expected to be useful in several area of biological research. However, several difficult problemes associated with interference from induced radioactivities in natural occurring elements in the specimen remain to be solved. Target elements such as Au, Mn, Hg, As, Pr and Eu were given to mice and rats. Freeze-dried whole body sections were irradiated by neutron in the reactor those neutron flux is 4.0×1012n/sec/cm2in pneumatic tube. The activated specimens were placed in contact with X-ray film after adequate cooling. Results showed that the whole body activation autoradiography would be applicable in practical purpose under the limited condition of well selected materials and well designed irradiation and cooling. Several factors which influence minimum detectable amounts were discussed.
The whole body autoradiograms of mice which had received3H- and 14C-labelled compounds injection were obtained by making close contact between the film and the body section or the film and the cellophane tape which covered the section surface. On the film which had been in contact with cellophane tape of 60 μ thick, blackness was found on the spots corresponding to the dorsal subcutaneous tissue which had received the injection of3H-labelled compound. This photographic black image seems to be abnormal considering that β-particles of tritium cannot have the maximum energy which is high enough to penetrate the thickness of the cellophane tape mentioned above. In an effort to find out the cause of these abnormal images, experiments were carried out under various conditions of exposure. Both X-ray and color films were employed. As the result of these experiments, we have concluded that the image obtained is not made by the direct action of β radiation of tritium on the emulsion, but by the interaction between tritium radiation and cellotape, and have assumed that the Bremsstrahlung emission produced by the interaction between tritium radiation and cellophane tape caused the blackness on films. This Bremsstrahlung emission was also observed in other shielding materials, for instance, Al foil, glass plate, carbon paper, scotchtape and lumilar sheet.
Freeze-drying microautoradiograms were made on a few organs of mice for observing3H-methionine transfered into protein synthesizing, degrading and excreting systems in them.3H-methionine was intravenously injected in mice (dd-type) . The mice were sacrificed 0, 30 and 120 minutes after the injection. Liver, spleen, intestine and kidney were cut off and immediately chilled at -70°C. After gently raising of the temperature, the materials were sectioned at -20°C to make 5μ sections. There were many difficulties to keep a good cytological image of cells if the frozen sample sections were dried by a continuously raising of the temperature under vacuum during -20°C to the room temperature. A major part of hydrophylic or lipophylic components was eliminated from some sections by a regular manner for freeze-drying autoradiography. The other sections were covered a stripping type emulsion of Fuji ET-2F by a “Dry-Method”. After elimination of hydrophylic or lipophylic components, the sections were prepared by the same manner as the mentioned above. Autoradio grams showed an exponential decrease of3H-methionine and its water soluble derivatives from cells of every 3 organs following the continuous time after the injection. Similar observation was made on the lipophylic components except very few incorporation of3H compared with the hydrophylic components. This showed no particular manner regarding to the3H distribution of hydrophylic and lipophylic components except the regular excretion of mice. On the other hand, acid-insoluble components containing protein or some polymerized components had an extremely specific manner. The data suggest that some components might be excreted from the kidney after passing through cytoplasm portion of tubules. The other portions were found in the nucleus of liver very rapidly after the injection. As soon as the decrease of its radioactivity was seen, radioactivity in the nucleus of the spleen and intestine tissues were getting increased as if some active cells, fissionable and protein synthetic, in the intestinal basal layer and the splenic parenchyma will receive some efficient precursors which might be produced by a protein synthetic course set up in the liver nucleus.
Adult male mice were administered intraperitoneally with 4μCi of α-, β- and γ-BHC-14C (22, 32 and 21 μg, resp.) per body and the distribution of radioactivity in the body was studied by means of whole body autoradiography. As early as 5 minutes after injection, a remarkable accumulation of α-isomer was observed in the white matter of the central nervous system, which was retained for more than 24 hours. A low but rapid uptake in the brain was also observed for β- and γ-isomers, but the radioactivity showed a rather uniform distribution and disappeared rapidly. An extremely high uptake of radioactivity was observed in the adipose tissues for all the isomers, the concentration being in the order β>>α>γ at 24 hours after administration. After 72 hours, the concentration of both α- and γ-isomers in the body became almost negligible, while β-isomer was found to be still retained in a high concentration in the adipose tissues, the concentration being 40 to 90 times higher than that of γ-isomer. It was also found that the urinary excretion of radioactivity was much lower after administration of β-isomer than γ-isomer, while thin-layer chromatographic separation of the urinary metabolites revealed that β- and γ-isomer appear to have similar ways of metabolism. Possible relations of the toxicities of α- and β-BHC, toxicity of the former being acute and of the latter chronic, to their respective characteristic retentions in the white matter of the CNS and in the adipose tissues were pointed out and discussed.
A technique is described for determining the activity and size distribution of β emitting dust particles using autoradiography. An X-ray film is more sensitive to β rays and more useful than the nuclear emulsion in the β ray autoradiography. Variations of the black spot diameter with the development time and temperature were within a few percent for the deviation of ±0.1 min and ±0.5°C from the standard condition respectively. About fifty micron thick separator of Lyphan did not cause any variation of the spot size for β rays from fission product nuclides. Error in estimating the activity and size distribution of β emitting particles by autoradiography is discussed. The application of the present technique to aerosol analysis is described showing an example.