HIGH-RESOLUTION BACKSCATTER ELECTRON DETECTION OF CELL-SURFACE MOLECULES ON HUMAN PLATELETS USING THE DOUBLE-LAYER COATING METHOD AND CRYO FIELD-EMISSION SCANNING ELECTRON-MICROSCOPY

A model system utilizing cryo scanning electron microscopy (SEM) for t he detection of putative cell adhesion molecule(s) on the surface of h uman platelets is described. Plunge freezing was used for cryoimmobili zation of unactivated and activated platelets after prefixation. Extra cellular ice was removed by sublimation to expose the surface of the p latelet membrane. Cryosamples were coated by the double-layer method, in which unidirectional shadowing is performed at an angle of 45 degre es with 2 nm of platinum by thermal evaporation, followed by evaporati on of 5 nm of carbon at an angle of 90 degrees for stabilization of th e platinum film. The topography of the extracellular surface of the un stimulated platelet membrane was dominated by small spherical protrusi ons, while that of the activated platelet had not only similar spheric al projections, but also possessed numerous rod-like protrusions, pres umably representing the upregulation of the cell adhesion molecule, P- selectin, from intracellular a granules. These results clearly demonst rate that cryo field-emission SEM can detect molecular topography on t he extracellular surface of cells consistent with the dimensions and s hape of membrane cell adhesion molecules.