Erlandsen S L, Bittermann A, Krumweide M, Nelson R D, Chen Y, White J G
Department of Cell Biology and Neuroanatomy, University of Minnesota School of Medicine, Minneapolis 55455, USA.
Scanning. 1997 Aug;19(5):356-60. doi: 10.1002/sca.4950190502.
A model system utilizing cryo scanning electron microscopy (SEM) for the detection of putative cell adhesion molecule(s) on the surface of human platelets is described. Plunge freezing was used for cryoimmobilization of unactivated and activated platelets after prefixation. Extracellular ice was removed by sublimation to expose the surface of the platelet membrane. Cryosamples were coated by the double-layer method, in which undirectional shadowing is performed at an angle of 45 degrees with 2 nm of platinum by thermal evaporation, followed by evaporation of 5 nm of carbon at an angle of 90 degrees for stabilization of the platinum film. The topography of the extracellular surface of the unstimulated platelet membrane was dominated by small spherical protrusions, while that of the activated platelet had not only similar spherical projections, but also possessed numerous rod-like protrusions, presumably representing the upregulation of the cell adhesion molecule, P-selectin, from intracellular a granules. These results clearly demonstrate that cryo field-emission SEM can detect molecular topography on the extracellular surface of cells consistent with the dimensions and shape of membrane cell adhesion molecules.
描述了一种利用低温扫描电子显微镜（SEM）检测人血小板表面假定细胞粘附分子的模型系统。在预固定后，采用骤冷冷冻法对未活化和活化的血小板进行冷冻固定。通过升华去除细胞外冰，以暴露血小板膜表面。冷冻样品采用双层镀膜法，其中通过热蒸发以45度角进行2nm铂的无定向阴影投射，随后以90度角蒸发5nm碳以稳定铂膜。未刺激的血小板膜细胞外表面的形貌以小的球形突起为主，而活化血小板的形貌不仅有类似的球形突起，还具有许多棒状突起，推测这代表了细胞粘附分子P-选择素从细胞内α颗粒上调。这些结果清楚地表明，低温场发射扫描电子显微镜可以检测与膜细胞粘附分子的尺寸和形状一致的细胞外表面分子形貌。
