AUTORADIOGRAPHY 327 



Film should be used as fresh as practicable, since background fog and 

 graininess increase with time and may become a real i)r()l)lem, especially 

 in work with small amounts of radioactivity. Also the film should be 

 stored at refrigerator temperatures in a place where.it is not exposed to 

 extraneous radiation or high humidity. The safelight to be employed 

 during handling should be known to produce no fogging and/or should be 

 tested under actual working conditions. The film should be handled 

 carefully at all times to avoid scratching or fingerprints. Photographic 

 processing is carried out according to the manufacturer's directions with 

 such modifications as are developed by experience for particular circum- 

 stances. A general procedure is as follows: The film is developed in the 

 dark for 2 to 5 min at 18 to 20°C using Eastman D-19 solution. The him 

 is then rinsed for about 15 sec in two changes of water, fixed in the usual 

 hypo for twice the time it takes to clear, and then washed iij running water 

 for 20 to 30 min. All solutions should be maintained at a constant tem- 

 perature to minimize background fog and softening of the emulsion which 

 may occur at higher temperatures. 



Properties of the Radioisotope. Alpha particles allow very high resolu- 

 tion on account of their short range. However, the biological interest in 

 alpha emitters stems primarily from their toxicity in the body. Most of 

 the autoradiograms in biological experimentation are produced from beta 

 particles. Elements with low beta energies such as H'^ (0.0189 Mev), 

 C'^ (0.155 Mev), S^^ (0.16G Mev), and Ca^» (0.254 Mev) give very good 

 resolution. As the beta energy increases, the autogram becomes more 

 and more diffuse. Radioisotopes such as Fe^^ which emit soft K and L 

 X rays give good resolution. Gamma rays, with their great range and 

 low ionization, are relatively inefficient and normally contribute little to 

 the autoradiogram when the isotope also emits beta particles. 



Radioisotopes with half-lives shorter than several hours offer some dis- 

 advantage since, after the experimental period and the time required for 

 histological processing, the tissue sections may be low in activity. In 

 such cases, longer-lived radiocontaminants may present a problem. 



Dosage of Isotope and Exposure Time. There is no simple way to 

 determine the amount of radioactivity that must be put into a biological 

 system in order to ensure an autoradiographic image within a reasonable 

 exposure period. A balance must be struck between high doses, which 

 may produce radiation effects, and low doses, which will require unduly 

 long exposure periods. As a rule of thumb, it has been stated that 

 approximately 5 to 10 million beta particles per sfjuare centimeter must 

 strike an X-ray emulsion diu'ing exposure to produce adecjuate blackening; 

 with alpha particles, I to 2 million particles per square centimeter are 

 required (7). For example, with gross autoradiograms, if a section mon- 

 itored with an end-window counter gives a measurement of 100 counts/- 



