Improvements in the Coating Technique of Radioautography

The 1958 coating technique of Messier and Leblond for radioautography—the most recent modification of the liquid emulsion method—was investigated in the hope of eliminating artefacts and discovering the optimum conditions for reliable quantitative radioautography. From a review of the properties of the various bulk emulsions available, it is concluded that NTB2 bulk emulsion is the most suitable. The proper handling of the emulsion (transportation, storage and testing operations) is described, as well as the details of an improved coating techniqe. Comparison is made of this method with the other most widely used, the strippingfilm technique. The main features of the improved coating technique are as follows: 1. Various fixatives may be used for radioautography. Zenker and presumably other mercury-containing fixatives are unreliable, but can be used after complete removal of mercury salts. 2. Dipping the sections into celloidin prevents the infiltration of the tissue by emulsion, but is otherwise unnecessary and may be omitted for routine work. 3. Emulsion coating is done by dipping sections in a bubble-free emulsion at 40°C. 4. Drying of the coated preparations is best accomplished by keeping them vertical over moistened tissue paper at 28°C and 80% relative humidity. 5. Exposure is done in a dry atmosphere at 4°C. 6. Processing is carried out at 17-18°C, as even a slight rise in temperature increases the background fog and decreases the efficiency of the emulsion. The optimal duration of development is 2 minutes for D-72 (Dektol), 6 minutes for D-l70 (Dolmi) and 3 minutes for D-178. For fixation, best results are obtained using acid fixer with hardener (or 24% hypo) for 3 minutes. 7. Mounting is done by successive immersions in an alcohol-cedar oil mixture, a xylene-balsam mixture, and balsam. Malinol may replace balsam. The improved coating technique arising from these observations was tested for its quantitative response, measured by grain counts. The suitability of the technique for quantitative radioautography is demonstrated by the fact that the reaction intensity is proportional to exposure up to at least 80 days with hematoxylin-cosin stained sections, and 1 year with unstained sections. The background increases slowly with time, but the increase is more rapid on slides with C 14 -labelled sections than on those labelled with H 3 . In conclusion, the improved coating technique using the NTB2 emulsion is now quite satisfactory. The next major improvement would be for the industry to provide emulsions with a standardized sensitivity and a constant, low level of fog. Nevertheless, in its present state, it is a convenient method permitting the preparation of large numbers of high quality radioautographs and is well suited to the grain counting required for quantitative radioautography.