ELECTKON M l( KC )S( < H»Y 



5. Examination of the replicas will show 

 some percentage of particles fractured in a 

 manner that will permit thickness measure- 

 ment of the coating material. 



6. The thin sections are used for optical 

 microscopy to correlate E. M. findings. 



J. H. Bexder and E. H. Kalmus 



Uncurling Carbon Replicas 



The carbon repUca technicjue introduced 

 bj^ Bradley is by now well established among 

 elect ronmicroscopists and modifications are 

 probably as numerous as there are micro- 

 scopists using it. 



A fact familiar to most workers is that 

 sihca and carbon replicas with or without 

 backing have a strong tendency to curl up; 

 straightening out these replicas more often 

 than not becomes extremely difficult and 

 time-consuming, if not impossible. 



During the course of our experiments we 

 noted that carbon replicas are especially 

 prone to curl if primary plastic coatings are 

 used. It was found, however, that such 

 rolled-up squares will unfold immediately 

 and straighten out by transferring them onto 

 the surface of distilled water. 



Replicas made in this manner contain few, 

 if any, holes and/or breaks. 



AV. B. Estill* and E. H. Kalmus 



STAINING, ELECTRON 



Electron staining is a technique for en- 

 hancing contrast in the electron images of 

 biological specimens. The most widely used 

 electron stains are aqueous and/or solvent- 

 soluble compounds containing heavy metal 

 compounds. Examples of the most com- 

 monty used electron stains are phosphotung- 

 stic acid, osmium tetraoxide, and uranyl 

 acetate. Some of these compounds, e.g., os- 

 mium tetraoxide, are also used as fixatives. 



The theoretical basis for the use of heavy 

 metal compounds as electron stains is as fol- 



* Sandia Corporation, Albuquerque, N. M. 



lows. Biological materials for all practical 

 purposes consist of the elements hydrogen, 

 carbon, nitrogen and oxygen. These are low 

 atomic number elements, and therefore have 

 low electron density or electron scattering 

 power. Thus, the difference in electron den- 

 sity of a specimen and its plastic supporting 

 film is very slight. Consequently contrast 

 between the electron image of the specimen 

 and the supporting film is low. However, 

 when one treats the biological specimen with 

 a solution of a compound containing a heavy 

 metal element, the heavy metal, or ion con- 

 taining the heavy metal is (1) absorbed on 

 the surface, (2) absorbed into or (3) chemi- 

 cally combined with specific reactive groups 

 of the specimen. In either event elements of 

 high electron density (or electron scattering 

 power) become part of the specimen, thus 

 enhancing the contrast between the electron 

 image of the biological specimen and its sup- 

 porting film. 



Electron staining reactions may be corre- 

 lated with data obtained by other analytical 

 methods. In the case of collagen an elegant 

 correlation is obtained by comparing electron 

 staining reactions with hydrothermal stabil- 

 ity (shrinkage temperature) data (1). Phos- 

 photungstic acid treatment stains the colla- 

 gen, but has no effect on the hydrothermal 

 stabiUty. From this it may be postulated 

 that the anionic complexes containing tung- 

 sten may combine with certain reactive 

 groups (e.g. — NHs"^ groups of the basic 

 amino acid side chains) but do not introduce 

 cross-links to increase the hydrothermal sta- 

 bility of the collagen. Osmium tetraoxide 

 and uranyl acetate increase the hydrother- 

 mal stability of collagen. Thus it is postu- 

 lated that the collagen reacts with the heavy 

 metal containing ions and in the process 

 cross-links are introduced to increase the 

 hydrothermal stability of the collagen. 



Lead nitrate has a lyotropic effect upon 

 the collagen. The fibrils are markedly swol- 

 len. Hydrothermal stability is decreased 

 substantially yet the fibrils appear to be 



274 



