SURFACE-TOPOGRAPHY AND INTRACELLULAR ORGANIZATION OF HUMAN-CELLS IN SUSPENSION AS REVEALED BY SCANNING ELECTRON-MICROSCOPY

This report presents a new procedure to study the ultrastructure of human cells in suspension by means of scanning electron microscopy. Living cells were maintained in suspension within cell culture flasks located on a rotating tilting table within an incubator. These cells were injected into warm glutaraldehyde/formaldehyde fixative. After washing in buffer, fixed cells were attached to propylamine-derived glass carriers. However, saturating free aldehyde groups of fixed cells and blocking amine groups of the derived glass carriers prevented cells from attachment to these carriers. Thus, we postulated that bonds between the fixed cell-free aldehyde groups and the carrier amine groups were responsible for cell-to-carrier attachment. Fixed cells attached to the carriers were subsequently dehydrated, dried, and coated for surface topography studies. For studies of internal cell organization, these attached cells were immersed in agar or gelatin as extracellular embedments and infused with sucrose or polyvinyl-pyrrolidone as cryoprotectants. Cells then were frozen and fractured. Fractured cells were either thawed, dehydrated, critical point dried, and ion beam sputter-coated, or freeze-substituted, dried, and planar magnetron sputter-coated. Finally, cell preparations were observed in the scanning electron microscope. Due to high cell attachment yield, both approaches samples observed in the electron microscope were representative of the entire cell population.