Dual-stage choreography of bulk biological specimens by Schottky field-emission SEM: Quality imaging at all magnifications

Abstract

The utility of a dual stage scanning electron microscope boasting a Schottky field emission (FE) source, for imaging bulk biological samples is its versatility to select the quality of the secondary electron signal. The detector placement and the specimen staging, either in or below the final probe forming lens, results in two distinct secondary electron images dominated by either the backscatter electron (BSE) component or by specimen specific secondary electrons (SE) that generate a high resolution image. It is only the voltage range to operate the FESEM and whether or not to enhance the SE-I contrast from biological specimens, with the coating of an ultrathin continuous fine grain metal film, which need to be determined for successful image recordings. Our facility has employed 1 nm chromium films on bulk biological specimens in order to restrict the electron beam interaction volume and generate a more intense SE-I signal contrast. Due to the extreme stability and minimal 1 nm beam diameter of the Schottky FESEM operated at high voltage (20-30 keV), very high resolution images of membrane ectodomains composed of hydrocarbons <10 nm in diameter (not colloidal gold markers) were recorded when the specimen was coated with Cr.