ELECTRON MICROSCOPY 



113 



ELECTRON MICROSCOPY 



field of electron optics. Of most general 

 interest is the electron microscope. 

 With this instrument, using the same 

 equation for resolving power, it should 

 be possible to reach a resolution of at 

 least O.OOl/x- Thus, an improvement of 

 at least 200 times over the limit with 

 visible light might be achieved. The 

 practical limit on magnification has been 

 placed at from 70,000 to 100,000 times. 



Historically the electron microscope 

 is now twenty-four years old. Busch 

 described the first such instrument us- 

 ing magnetic lenses (Busch, H., Arch, 

 f. Elektroteknik, 1927, 18, 583-594). 

 Though many improvements were made 

 in design it was not until 10 years later 

 that the instrument reached the point 

 of being of practical use. Chiefly 

 through the work of Ruska and Borries 

 (numerous papers, 1934-1940) the in- 

 strument was developed to the state 

 that it is in at present. In this country 

 an e.xcellent instrument, capable of 

 giving high resolution has been de- 

 veloped and commercially marketed by 

 the Radio Corporation of America. 

 The apparatus is described by Zwory- 

 kin, V. K. (Science, 1940, 92, 51-53). 

 In this instrument electrons emitted 

 from a hot wire filament are accelerated 

 by a potential of 30,000 or more volts. 

 This beam is condensed and passed 

 through the object which is carried on 

 a wire screen supported nitro-cellulose 

 film. The transmitted electron beam is 

 focussed in a greatly enlarged image by 

 means of two magnetic lenses. The 

 image can be seen on a fluorescent screen 

 or photographed on a sensitive plate. 



The conditions under which good re- 

 sults are obtainable are rather stringent. 

 The ideal object must be very small, or 

 capable of being minutely divided with- 

 out losing its identity; it must maintain 

 its form on drying in vacuum; and it 

 must be rigid enough to resist the heat 

 and disruption caused by exposure to 

 the electronic bombardment. Rela- 

 tively few biological structures fall into 

 this category; hence early studies were 

 confined to bacteria, viruses, connective 

 tissue fibers, blood cells, spermatozoa, 

 etc. 



The great need in histological in- 

 vestigation with the electron micro- 

 scope has been a method for uniformly 

 cutting tissues no thicker than 0.1 ^ 

 and about 1 mm* or larger in area. 

 With these dimensions a section will 

 yield acceptable electron micrographs. 

 Such a method seems to have been 

 found in a modification of the conven- 

 tional microtome whereby the advance 

 of the block is reduced about tenfold, 

 either mechanically (Baker and Pease, 



J. Appl. Phys., 1949, 20, 480) or by 

 means of a thermal expansion device 

 (Newman, Borysko, and Swerdlow, 

 Anat. Rec, 1949, 105, 267). The tissue 

 must be embedded in celloidin or plas- 

 tic, but a special blade is probably 

 necessary for the best results. Since 

 1950 publications have appeared with 

 electron micrographs of nearly all tis- 

 sues of importance. See Excerpta Med- 

 ica, Sect. I, for listings and abstracts. 



Histologists have also borrowed spe- 

 cial methods from metallurgists for 

 investigation of hard, opaque structures 

 like bone, tooth, hair, keratinized epi- 

 thelium, etc. These methods involve 

 the casting of very thin replicas of 

 surface details. Additional contrast is 

 gained by the ingenious process of 

 "shadow casting", either on the original 

 specimen or on its replica. A very thin 

 layer of metal is deposited over the 

 object in a vacuum by evaporation from 

 a hot filament. When done at an acute 

 angle, the elevated portions of the 

 object shadow the depressed areas, thus 

 giving a striking three-dimensional pic- 

 ture (Williams and Backus, J. Appl. 

 Phys., 1949, 20, 98). Methods have 

 also been developed for true stereoscopic 

 images (Heidenreich and Matheson, J. 

 Appl. Phys., 1944, 15, 423). See Shad- 

 dow Casting. 



The old technique of selective stain- 

 ing has been combined with this newest 

 of procedures in histology. It has been 

 shown that the salts of heavy metals, 

 such as osmium and phosphotungstate, 

 are deposited in a highly localized way 

 on certain protein structures, e.g. mus- 

 cle fibrils, thus enabling visualization 

 of practically molecular details (Hall, 

 Jakus, and Schmitt, J. Appl. Phys., 

 1945, 16, 459). 



With the proper selection and com- 

 bination of histological methods now 

 on hand the electron microscopist 

 should have little difficulty in studying 

 any problem in morphology. A practi- 

 cal handbook for the guidance of novice 

 and expert has been prepared by the 

 Royal Microscopical Society: "The 

 Practice of Electron Microscopy", 

 edited by D. G. Drummond and pub- 

 lished as'Part I of vol. 70, 1-141, 1950 of 

 the J. Roy. Micro. Soc. Papers dealing 

 with details of manipulation are to be 

 found regularly in the Journal of Ap- 

 plied Physics. 



2. Emission electron type. The earli- 

 est description of an electron micro- 

 scope in this country was of an entirely 

 different type from the new R.C.A. mi- 

 croscopes that give such prodigious 

 magnifications. McMillan and Scott 

 (J. H. and G. H., R.S.I. , 1937, 8, 288- 



