A new generation of high performance field emission scanning electron microscopes (FSEM) is now commercially available (JEOL 890, Hitachi S 900, ISI OS 130-F) characterized by an "in lens" position of the specimen where probe diameters are reduced and signal collection improved. Additionally, low voltage operation is extended to 1 kV. Compared to the first generation of FSEM (JE0L JSM 30, Hitachi S 800), which utilized a specimen position below the final lens, specimen size had to be reduced but useful magnification could be impressively increased in both low (1-4 kV) and high (5-40 kV) voltage operation, i.e. from 50,000 to 200,000 and 250,000 to 1,000,000 x respectively.At high accelerating voltage and magnification, contrasts on biological specimens are well characterized1 and are produced by the entering probe electrons in the outmost surface layer within -vl nm depth. Backscattered electrons produce only a background signal. Under these conditions (FIG. 1) image quality is similar to conventional TEM (FIG. 2) and only limited at magnifications >1,000,000 x by probe size (0.5 nm) or non-localization effects (%0.5 nm).
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