Scanning electron microscopy at macromolecular resolution in low energy mode on biological specimens coated with ultra thin metal films.

In this report, conditions for attaining high resolution in scanning electron microscopy with soft biological specimens are described using the currently available high resolution scanning electron microscopes in emission mode of low energy electrons (secondary and charging electrons). Retinal rod outer segments, red blood cells, intestinal mucosa, and ferritin molecules were all used as biological test specimens. From uncoated specimens a new source of signal, referred to as a discharge signal, can provide a high yield of low energy electrons from an excitation area approximately the size of the beam's cross section. Additionally, under these conditions sufficient topographic contrast can be achieved by applying ultra thin metal coatins. A 0.5 nm thick gold film is found sufficient for generating the total signal, whereas increased coating thickness causes additional topographic background signal. However, a 2.0 nm film is needed for imaging surface details with the present instrument. Ultra thin, even, and grainless tantalum films have been found effective in eliminating the charging artifacts caused by external fields, and the decoration artifacts caused by crystal growth as seen in gold films. To improve, in high magnification work on ultra thin coated specimen, signal-to-noise ratio, methods for obtaining saturation of the signal with discharge electrons are shown. The necessity of confirming the information obtained in SEM by independent techniques (TEM of stereo-replicas or ultra thin sections) is discussed.