Morphology of hyaluronidase-sensitive cell coats as seen in the SEM after freeze-drying.

Many adherent cells in vitro are surrounded by a transparent exclusion zone or halo, several micrometers thick, which red blood cells, bacteria and carbon particles cannot penetrate. This halo is rapidly and specifically removed by hyaluronidase and its high degree of hydration is demonstrated by the fact that, although fixation does not eliminate the halo, solvent dehydration does. This latter observation means that the halo cannot be visualized by conventional electron microscopic techniques. We report here that the exclusion-zone material can, however, be seen in the scanning electron microscope if cells are fixed and frozen rapidly and then freeze-dried. Many cells in cultures from a murine fibrosarcoma or from human embryonic lung treated in this way appear to be covered by a matrix that obscures the microvilli that are visible on critical-point-dried or hyaluronidase-treated, freeze-dried cells. Only where the coat is, for some reason, missing can microvilli be seen on freeze-dried cells. The coat structure varies from amorphous to an assembly of fine fibres approximately 100 nm in diameter and its appearance is very similar to that of small drops of hyaluronic acid (10(-5) micrograms ml-1) treated in the same way. Halo material is fragile and detaches itself from the cell surface within an hour of fixation. These observations suggest that the halo phenomenon reflects only the production of extracellular matrix and its turnover. The fragility of the haloes implies that, if they do exist in vivo, they are unlikely to play any structural role. The results suggest that the technique will yield information on other highly hydrated, unstable structures.