On the molecular profiling of cell surfaces by SEM

Abstract

Cell surfaces interface with a variety of environments and, as a consequence, cell surface properties are of considerable functional importance to the biological organism. SEM immunocytochemistry (SEM-IC) is one of a range of techniques used to analyse cell surface properties. A major goal of SEM-IC centres on extended survey or high-magnification morphological analysis of cell and tissue surfaces combined with molecular profiles of these surfaces as established by gold-labelling. The properties of colloidal gold make it the marker of choice for SEM-IC and a representative gold-labelling protocol is outlined. The SEM-IC gold-labelling technique has been applied advantageously to the analysis both of cell surfaces and cytoskeletal and extracellular matrix elements: a tabulation of the main SEM-IC biomedical applications is given. Illustrated examples demonstrate how SEM-IC provides a highly effective approach for analysis both of cell and tissue differentiation-maturation sequences, and of pathological change involving not only the entire tissue or cell surface but also minute changes in microdomain characteristics of the individual cell surface. Steps in exploiting the technique of colloidal gold SEM-IC have been several-fold and include: use of backscattered electron imaging; accurate localization of gold particles by superimposition on topographical maps of the cell surface; and use of small (1-10 nm) gold probes followed by silver enhancement in order to minimize steric hindrance. Factors under assessment include: use of low voltage SEM; BE imaging of samples coated with ultrathin metal films; and use of gold-labelled SEM-IC for direct quantification of the numbers of target molecules exposed on cell surfaces by automated image analysis of the digitized BE image.