SHADOW-CASTING 



313 



SHADOW-CASTING 



was first applied successfully to elec- 

 tron microscopy by R. C. Williams and 

 R. W. G. Wyckoff (J. Appl. Phys., 1944, 

 15, 712-716) who used the technique 

 for determining the heights of minute 

 objects by measuring the lengths of 

 their shadows. It was found by them, 

 however, that the process greatly en- 

 hanced the contrast in images of very 

 small objects, or of very small irregular- 

 ities on larger objects, and this advan- 

 tage of the technique has come to out- 

 grow in importance the original purpose 

 of measuring heights. 



Bacteria, viruses and the larger pro- 

 tein molecules have been studied by 

 the use of the shadowing technique 

 (for complete bibliography, see R. W. 

 G. Wyckoff, Electron Microscopy, 1949, 

 Interscience Publishers Inc., New York) 

 as applied to electron microscopy. The 

 advantages of improved contrast are 

 so great that, in the case of the observa- 

 tion of small biological objects and 

 minute chemical aggregates, the tech- 

 nique has improved the practical use- 

 fulness of the electron microscope 

 almost ten-fold. Further applications 

 to both electron and visual microscopy 

 involved a method of studj'ing opaque 

 surfaces by colloidin replicas that are 

 shadowed (Williams, R. C, and Wyck- 

 off, R. W. G., J. Appl. Phys., 1946, 17, 

 23-33). Applications of the method 

 to biological material viewed with the 

 light microscope and an account of the 

 casting apparatus have been presented 

 by W. T. Dempster and R. C. Williams 

 (Anat.Rec, 1946, 96, 27-38). 



For the shadowing of non-opaque 

 objects, materials to be observed with 

 the light microscope, the following 

 procedure is followed: Material is af- 

 fixed to cover slips; smears are thor- 

 oughly dried; paraffin is dissolved from 

 sections with solvents. With no fur- 

 ther preparation, other than thorough 

 drying, the slips are shadowed with a 

 metal deposit in a vacuum chamber. 

 After this, they are mounted face down 

 on slides with clarite or balsam. 



If large, opaque materials are to be 

 examined under the light microscope, 

 a surface replica can be taken by flow- 

 ing a dilute solution of celloidin over 

 the surface, allowing it to harden, and 

 stripping the celloidin film off after 

 drying. The film is then shadowed with 

 its replicating surface uppermost. 



For electron microscopy, regular 

 screen grids with a thin colloidin film 

 over the mesh are used as substrates 

 for suspensions; replicas are placed 

 directly on the mesh. A droplet of 

 suspension containing small biological 

 objects is placed on the substrate and 



allowed to dry. Salts are then rinsed 

 off with distilled water and the speci- 

 men is ready for shadowing; then the 

 specimen may be studied with the elec- 

 tron microscope. 



A metal to be used in shadowing for 

 light microscopy should be readily 

 evaporated and it should be relatively 

 opaque when jjresent in a very thin 

 film. Metallic chromium appears to 

 be the best metal to use for light micros- 

 copy since it transmits less than 50% 

 of the incident light in a thickness less 

 than 0.03 micra. For electron micros- 

 copy chromium is generally satisfac- 

 tory for objects as large as bacteria, 

 but, for finer detail, the metals palla- 

 dium and uranium are superior, as they 

 can be applied to yield satisfactory 

 contrast in thicknesses of only 0.001 

 micra. 



The casting technique is similar for 

 the different metals. Shadow-casting 

 produces a visually structureless de- 

 posit which sticks to all surfaces save 

 those directed away from the hot fila- 

 ment and shadow areas due to obstruc- 

 tions. Surfaces perpendicular to 

 straight line paths from the filament 

 get the heaviest deposit; oblique sur- 

 faces get less and shadows none. Metal 

 deposited at a rather oblique angle has 

 a distribution much like light from a 

 point source shining obliquely on three- 

 dimensional objects. Highlights and 

 shadows are produced. Through the 

 microscope, shadows in the prepara- 

 tions transmit light and appear bright; 

 highlights are dark. The eye, however, 

 readily adapts to this reversal of tone. 

 Photographic negatives or prints made 

 from glass positives reverse the micro- 

 scope appearance; highlights then are 

 bright, shadows are dark, and varia- 

 tions of surface texture are shown by 

 gradations of tone. 



Electron-microscopic negatives show 

 the same natural appearance of light 

 and dark. Although the electron mi- 

 crographs are taken by transmitted 

 electrons, in complete analogy with 

 photomicrographs, the negative prints 

 give one the impression that one is 

 looking down on the surface of the ma- 

 terial being examined. 



The apparatus for shadowing con- 

 sists of a bell jar and a base plate with 

 vacuum tight electrical connections. 

 Electrodes raised above the level of 

 the base plate carry a tungsten filament 

 on which the metal is placed for vapor- 

 izing. Cover slips with affixed ma- 

 terial (or the grid screens) are arranged 

 in a semicircle at a predetermined 

 distance from the filament and the 

 metal thereon to be vaporized. The 



