High resolution biological scanning electron microscopy: A comparative study of low temperature metal coating techniques

Structural information on the surface of biological specimens can be resolved within molecular dimensions by “in-lens” field emission scanning electron microscopes when cryo-methods are used to adequately preserve the native state of the specimen. The visual definition of molecular surface structures depends largely on the metal coating. The thickness of the coating, as well as the temperature at which it is deposited, are among the most important parameters affecting visual definition. These were evaluated on T4 polyheads and T4D phages using chromium double-axis rotary shadowing (DARS). Micrographs of optimally DARS coated T4 polyheads and T4D phages were compared with chromium planar-magnetron sputtering (PMS) and unidirectional shadowing with platinum/carbon. Metal deposition was carried out at low temperatures during all three procedures. Optimal visual definition of structural details on the surface of DARS coated T4 polyheads and T4D phages (capsomeres of T4 polyheads and their subunits with diameters of 8 and 3 nm; T4D phage tail fibres with a thickness of 3 nm) is achieved at a thickness of the chromium film greater than the minimum required for metal film coalescence. Chromium DARS coating at room temperature resulted in poor structural definition, whereas DARS at specimen temperatures of ?85°C and ?150°C, with the chromium thickness optimized for each temperature, yielded good visual detail of polyhead substructures. The visual definition was slightly reduced when DARS coating was carried out at a specimen temperature of ?250°C. Adequate structural visibility of T4D phage and T4 polyhead surface structures was achieved with the three coating techniques tested. Smaller filamentous structures, however (e.g., phage tail fibres), were more clearly identified after chromium DARS coating or unidirectional platinum/carbon shadowing than after optimized PMS with chromium. Each method has its own merits.