Field emission scanning electron microscopy (FESEM) entering the 21st century: Nanometer resolution and molecular topography of cell structure

The field of scanning electron microscopy (SEM) has undergone a revolution in terms of resolution obtainable on biological samples in the last 4 decades. In the late 1960s, the introduction of electron guns using tungsten filaments for SEM provided resolution on the order of 10-20 nm and valuable information on cell shape and morphology was obtained that had hitherto been inaccessible. In the late 1980s, the development of stable cold field emission SEM (FESEM) with in-lens specimen positioning and above the lens collection for secondary electron (Se) imaging provided a tremendous advance with resolution in the nm range being possible. Accompanying these changes in resolution have been improved procedures in thin film technology with metal film thickness being reduced from 10-20 nm to less than 1 nm for FESEM. Developments in cryoSEM have led to interpretable structure on biological samples of about 1-2 nm. Improvements in instrumentation, refinements in ultrathin film technology, and specimen preparation may lead to atomic level resolution exceeding 1 nm in the next decade.