Metal shadowing for electron microscopy

Abstract

Metal shadowing of bacteria, viruses, isolated molecules, and macromolecular assemblies is another high-resolution method for observing the ultrastructure of biological specimens. The actual procedure for producing a metal shadow is relatively simple; a heavy metal is evaporated from a source at an oblique angle to the specimen. The metal atoms pile up on the surfaces that face the source, but the surfaces away from the source are shielded and receive little metal deposit, creating a "shadow." However, the process of producing biological specimens that are suitable for metal shadowing can be very complex. There are a whole host of specimen preparation techniques that can precede metal shadowing, and all provide superior preservation in comparison to air drying, a required step in negative staining procedures. The physical forces present during air drying (i.e., surface tension of the water-air interface) will literally crush most biological specimens as they dry. In this chapter I explain the development of and procedures for the production of biological specimens from macromolecular assemblies (e.g., DNA and RNA), purified isolated molecules (e.g., proteins), and isolated viruses and bacteria preparations suitable for metal shadowing. A variation on this basic technique is to rotate the specimen during the metal deposition to produce a high-resolution three-dimensional rendering of the specimen.

Fig. 1

One of the first images of metal shadowed influenza virus ever recorded. This slightly fuzzy image was published as a photographic negative of the original image. The authors had to produce a photographic positive on film and then print that back as the negative image (a second generation print) to produce the “realistic shadow effect”. Each individual virus particle is approximately 100 nm in diameter. Reproduced from Sharp et al. [3], with the permission of the Journal of Biological Chemistry

Fig. 2

Metal shadowed isolated chicken gizzard myosin molecules, sprayed onto a freshly cleaved mica sheet and rotary shadowed with platinum–carbon at 6°. The image was recorded at 60 KX. Courtesy of Dr. Roger Craig, University of Massachusetts Medical School

Fig. 3

Myosin V, Glycerol spraying/low-angle rotary shadowing (6°) with platinum–carbon on carbon stabilized Formvar support films. Reproduced from Krementsoy et al. [15], with the permission of Rockfeller University Press

Fig. 4

Metal shadowed bacteria, Proteus mirabilis, rotary shadowed with platinum–palladium at 30°. The image was recorded at 20 KX

Fig. 5

Loading spraying apparatus

Fig. 6

Spraying samples onto prepared grids

Fig. 7

Single strand DNA with PC4 protein prepared as described above in the Droplet method with 1 % formaldehyde, rotary shadowed at 6° with Platinum–Palladium 80:20. When the single strand DNA is spread in the presence of the protein PC4, the strands form loops, without the protein all you see are linear strands. Images courtesy of Dr. Lijain Yu at the University of Massachusetts Medical School

Fig. 8

Adeno-Associated Virus (AAV), first negative stained with 1 % uranyl acetate to stabilize the virions and then rotary shadowed with platinum–palladium (80:20) at 30°. The image was recorded at 46 KX, the stage tilted to 45°. Courtesy of Dr. Guangping Gao, University of Massachusetts Medical School

Fig. 9

Denton 502B Vacuum Evaporator

Fig. 10

The inside view and setup of the vacuum evaporator for metal shadowing

Fig. 11

Floating carbon–metal film off of the sheet of mica. In this case there are two sheets of mica (opposing sides of the mica sandwich). Both sides will be metal shadowed and carbon coated to produce the metal rendering of the molecular sample

Fig. 12

Picking up the metal renderings on bare copper grids. Simply pick up pieces of the film as it floats on the water surface. Many grids are collected from each side of the mica sandwich