REPLICA AND SHADOWING TECHNIQUES 



two examples. In biological applications the 

 carbon replica has been used widely in the 

 study of algae, pollen grains, leaf surfaces, 

 viruses, large molecules and bacillus spores. 

 There is no universally correct method for 

 producing replicas, since every type of speci- 

 men may require variations of one or two 

 basic techniques to be developed for it. 



Shadowing 



The technique of shadowcasting (20) was 

 devised for two purposes. First to increase 

 the contrast of a specimen if necessary, and 

 second, to produce a three-dimensional ef- 

 fect in the resulting electron micrograph. 

 These objectives were accomplished by de- 

 positing a thin layer of electron-dense mate- 

 rial by vacuum evaporation at an angle onto 

 the specimen. It can be seen from Figure 6 

 that the "shadow" cast by a protrusion on 

 the surface is transparent, while the sur- 

 rounding area is relatively dense to electrons. 

 Shadows appear bright in the electron micro- 

 scope but dark on the negative of the elec- 

 tron micrograph. Therefore, if the negative 

 of the electron micrograph is viewed, the 

 specimen surface will appear as if it is illu- 

 minated obliquely Avith white light. For this 

 reason shadowed electron micrographs are 

 printed as negatives. It can be seen from the 

 electron micrograph, Figure 4b, that both an 

 improvement of contrast and a three-dimen- 

 sional effect is obtained by the shadowcast- 

 ing technique. 



Materials suitable for preparing replicas 

 are not suitable for shadowcasting. A shad- 

 casting material must be dense to electrons, 

 i.e., it must have a high mean atomic num- 



o 



ber, so that only a thin layer, about 20 A 

 thick, need be deposited on the specimen. If 

 the layer is thick, the material will pile up 

 on the side of the protrusion nearest the 

 source, thus distorting the shape of the 

 protrusion. This is illustrated in Figure 7 

 where spherical particles have been heavily 

 shadowed with a material of low electron 

 density (carbon). 



Shadowing 

 Direction 



Specimen 



Shadowing 

 Metal 



Fig. 6. Diagrammatic representation of the 

 topographical distribution of a layer of shadowing 

 material. 



Fig. 7. Self-shadowed carbon replica of spheri- 

 cal polystyrene latex particles (diameter 2,600 A) 

 showing distortion caused by the piling up of the 

 evaporated carbon on one side. (By courtesy of the 

 British J'ournal of Applied Physics) 



Many materials have been used for shad- 

 owcasting, for example, chromium, an alloy 

 of gold and palladium, platinum, uranium, 

 and tungsten oxide. These can all be evapo- 

 rated in vacuo, either from V-shaped tung- 

 sten filaments, molybdenum foil boats, or 

 tungsten wire baskets. The technique is 



235 



