High voltage cryomicroscopy of human blood platelets

We have analyzed the ultrastructure of human blood platelets using ultrarapid freezing techniques followed by either freeze-substitution fixation or direct imaging of frozen hydrated specimens in a high voltage electron microscope. Freeze substitution preserved a regularity in the arrangement of microtubules when compared with chemically fixed specimens, as identified by a preferred center-to-center distance of 30 nm. Platelets imaged in the frozen hydrated state during different stages of adhesion and spreading were rich in cytoplasmic detail and revealed novel features not previously reported. Different granule populations were distinguished in the unstained samples, and the morphology of the cells was dramatically different from that of dried cells. Membrane channels formed as the cells spread, increasing in size and number as the number of granules in the cell decreased, suggesting a fusion of granule membranes with one another or an additional membrane system to release their contents. The data support the idea that direct cryoimaging is an optimum method for characterizing rapid structural changes of labile organelles in whole cells.