384 ISOTOPIC TRACERS AND NUCLEAR RADIATIONS [Chap. 14 



The last method (3) to be discussed eliminates the possibility of film dis- 

 tortion due to handling. Here, the cut sections are spread on water and 

 (in the darkroom) are floated onto the emulsion supported on a microscope 

 slide or other film backing cut to convenient size. The preparation is then 

 air dried and stored in a lighttight container until the desired exposure time 

 has elapsed. The emulsion acts as a fixative to hold the section to the slide. 

 The collodion or paraffin is then removed, the picture developed, and the 

 tissue section stained. The resultant preparation consists of the stained 

 section lying on the emulsion. Tissue and radioautograph when examined 

 microscopically under low magnification will appear to lie in the same plane. 

 Under high magnification, they may be examined separately, since the tissue 

 will appear at the uppermost level of focusing while the radioautograph will 

 be brought into focus at a lower level. 



Whatever method is used for assuring contact and alignment, several addi- 

 tional conditions should be examined and regulated to give the most effective 

 results. During preparation of the tissues for sectioning, and through the 

 staining procedure when the radioautograph is to be made after staining, it is 

 important that the radioactive material not be dissolved from the tissue by 

 any of the reagents used. For instance, I 131 in organic molecules may be 

 treated in the usual way without loss of material, whereas P 32 is lost rapidly 

 in acid fixatives. In the case of tissues containing the latter isotope, all 

 solutions used in histological preparation prior to exposure for radioautog- 

 raphy must be neutral. The question of solubility of radioactive material 

 in the histological reagents may be answered only by checking all such 

 reagents used prior to exposure of the film to the section. If the half-life of 

 the radioactive substance under study is short, as with Na 24 , or if the half-life 

 is longer and the amount of material is small, it will be essential to reduce the 

 time of histological preparation. By using small pieces, gentle heat, and 

 agitation during fixing and embedding, the time required for preparing the 

 tissue may be decreased markedly. The time may also be shortened and the 

 problem of solubility eliminated as well by using frozen sections without 

 fixing, although this introduces certain difficulties with regard to both his- 

 tology and radioautography. 



The thickness of the tissue sections used will influence the resolution 

 greatly. Sections of 5 and 10 /x are preferable, since resolution is inversely 

 proportional to section thickness provided that the latter is less than a 

 limiting value, depending on the type of radiation. If the path length of the 

 radiation is short, thickness of section is not critical with regard to resolution 

 since particles from within the tissue will be absorbed. When radiation 

 intensity is low, thicker sections may be necessary to obtain sufficient expo- 

 sure in the requisite length of time. It is difficult to prepare frozen sections 

 as thin as 5 to 10 /x- 



