High-resolution CryoFESEM of individual cell adhesion molecules (CAMs) in the glycocalyx of human platelets: Detection of P-selectin (CD62P), GPI-IX complex (CD42a/CD42b alpha,b beta), and integrin GPIIbIIIa (CD41/CD61) by immunogold labeling and stereo imaging

The aim of this study was to develop a model for the detection of individua l cell adhesion molecules (CAMs) in the glycocalyx of spread human platelet s using high-resolution cryo-field emission scanning electron microscopy (c ryoFESEM). Three surface glycoprotein CAMs, P-selectin (CD62P), GPlba in th e GPI-IX complex (CD42a/CD42b alpha ,b beta), and the integrin GPIIbIIIa (C D41/CD61) in the human platelet were selected on the basis of their unique topographic shape. Spread human platelets were indirectly immunolabeled wit h IO-nm colloidal gold and then cryoimmobilized. After sublimation of water from the cryoimmobilized sample, partially freeze-dried platelets were coa ted unidirectionally with Pt, stabilized with carbon, and examined in an in -lens cryoFESEM using high-resolution backscattered electron imaging. CAMs were detected by indirect immunogold labeling and the length of each type o f CAM was determined using analysis of differences in parallax as measured in the software program Sterecon. Our results demonstrate the efficacy of u sing high-resolution cryoFESEM to recognize and detect individual CAMs in t he glycocalyx. Further advances in production of metal coatings with finer granularity, together with improvements in imaging (tilting and angle of st ereo images), may provide better definition of the topography associated wi th glycosylation and formation of multimeric CAM complexes.