122 FUNDAMENTALS OF S UB M I C RO S C OP I C MORPHOLOGY 



Film 



to cool, because in vacuo heat cannot be transferred by convection. All that 

 can be done is to withdraw as much of the heat evolved as possible by means 

 of the metallic ring lying on the specimen holder. The best way to do this 

 is to place a fine wire netting over the ring and to irradiate the object through 

 the meshes. 



Only when an organic preparation is thinner than o.i /x, does it become 

 sufficiently transparent to electron rays and can be irradiated for some time 

 without damage. Therefore, the aim is to produce sections lo-ioo times 

 thinner than those used in histology. Various microtomes have been 

 employed for this purpose. Claude and Fullam (1946) produced sections 

 of 0.3-0.6 IX thickness with a special rotating high speed microtome. 

 Bretschneider (1949a) arrived at o.i /z with the rocking microtome. 

 Similar results have been obtained by Danon and Kellenberger (1950). 

 The fine 0.1 ju. movement of the specimen holder of the microtome is handi- 

 capped by the imperfection of the micrometer screw, but it can be achieved 

 by the thermal expansion of a massive metal block which has previously 

 been cooled down by dry ice (Newman and co-workers, 1949). Special 

 devices for the block advance on an inclined plane seem to be coming into 

 general use (Hillier and Gettner, 1950). 



Thin sections of organic materials do not show much contrast in the 

 electron microscope, as their constituents C, N and O produce the same 

 electron scattering as the carrier film. Only cell components which contain 

 phosphorus or which are minerahzed appear to be darker. In certain cases 

 the contrast can be enhanced by osmium fixation of the cells and by staining 

 with phosphotungstic or phosphomolybdic acid. 



The best contrast is obtained by the method oi metal shadowing, developed 

 by Wyckoff (1949). In a vacuum bell jar a small amount of metal is 

 vaporized and deposited obliquely on the preparation (Fig. 81). As a result 



the faces of the specimen 

 turned to the source of metal 

 vapour are coated with metal, 

 whereas the opposite faces are 

 not. Behind the object there is 

 a zone free of metal which is 

 called the shadow of the spe- 

 cimen. From this shadow the 

 height of the object can be cal- 

 culated if the shadowing angle 

 is known (Muller 1942b). 



When a preparation like this 

 is irradiated in the electron 

 microscope, the electrons are 

 greatly scattered at the places where metal has accumulated, passing freely 

 through the zones of shadow. As a result, the picture on the projection 

 screen exhibits an astonishing three-dimensional effect, creating the impres- 



0.00211 

 0.01 u 



O.OOitn 



Specimen 



Fig. 81. Shadowing of a specimen by deposition of 

 metal : s length of the shadow, b height of the spe- 

 cimen, a shadowing angle, h = s tan a. 



