Scanning Microscopy


The standard preparation of cell suspensions (e.g., blood cells, cell suspensions derived from monolayer cultures or from various tissues, etc.) for scanning electron microscopy (SEM) includes fixation of the cells in suspension and subsequent dehydration and critical point drying (CPD) of the cells after their preliminary attachment to the special substrata. In the course of the SEM examination of cell suspensions of various origins, unusual morphological cell surface structures, filopodia-like protrusions (FLP), were consistently detected in 3-20% of the cells in the populations. FLP can be effectively observed only by using a stage tilt angle of no less than 30°. FLP were single or multiple thin threads extending from basal parts of a spherical cell and attaching to the substratum surface used. FLP strikingly resembled substratum-attached filopodia formed by a viable cell at its earliest stages of spreading. The percentages of the cells with FLP were not significantly affected by the character of cell fixation (primary aldehyde fixation alone or primary aldehyde with subsequent OsO4 post-fixation) or the raising of the temperature and pressure inside the CPD bomb. It seems that the protrusions imitating the natural cell surface structures can probably be formed at later stages of the preparation of cell suspensions for SEM, namely during dehydration and (or) CPD when the cells undergo substantial shrinkage. One of the possible mechanisms by which the cell shrinkage could induce FLP formation follows. A prefixed spherical cell which settles down to the substratum sticks to it at some discrete points on the cell surface. As a result of the subsequent cell shrinkage, FLP could be formed and then stretched, connecting the same points on the cell surface with the points of the initial cell-substratum adhesion.

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