The effective osmotic pressure of the fixative for transmission and scanning electron microscopy

In previous studies concerning the morphogenetic movements in the invaginating lens placode of the chick eye, a discrepancy between the TEM and SEM images was observed. With a fixation procedure giving good results in the TEM, certain changes seemed to have occurred in the SEM specimens (opening of apical cell blebs, bubble-like swelling of microvilli). These changes were suspected of being artefacts arisen during processing. Comparing SEM and TEM images of the invaginating lens placode and the surrounding surface applying two different osmolalities of the glutaraldehyde/formaldehyde fixative vehicle, and comparing SEM as well as TEM images with LM images of living embryos using differential interference contrast, yielded to following information. Recent reports in literature that ideal fixation techniques for SEM, certainly those preserving delicate surface structures as apical blebs and microvilli, can differ from those suitable for TEM, were confirmed. Apparently, the cell blebs which can be demonstrated in vivo to be closed, burst during SEM processing as a consequence of a relative hypo-osmolality of the fixative vehicle (a too low effective osmotic pressure). The TEM images appeared to suffer much less from changes in the osmolality of the fixative vehicle. It is suggested, therefore, that the artefacts are brought about by some step specific to the processing for the SEM (E.g. critical point drying).