First disclosure of lipid droplet substructure and myelin translucency in embedment-free section electron microscopy.

Abstract

In conventional transmission electron microscopy (EM), thinly sectioned specimens embedded in epoxy resin are observed. However, because of a substantial level of electron density of epoxy resin, the possibility cannot be ruled out that bio-structures having electron density similar to that of epoxy resin are not clearly recognized and thus are neglected or misinterpreted in conventional EM. This was the reason to require for embedment-free EM. Embedment-free sections have already been made available reliably by transient embedding in polyethylene glycol (PEG) and subsequent de-embedding through immersion in water, and further by critical-point drying, and this embedment-free EM is thus termed PEG-EM. However, this PEG-EM has not been successful to attract reasonable attention from electron microscopists and instead been misunderstood as a non-reliable method. In this paper, the remarkably enhanced contrast and electron translucency of any observation targets in PEG-EM are clearly demonstrated by comparing with images in conventional EM of adipocytes and neural myelin as examples. These features of PEG-EM, together with faithful correspondence in EM images of any individual substructures between the two methods, confirm the reliability of PEG-EM. Furthermore, the much higher thickness of embedment-free sections together with these features makes the PEG-EM more advantageous than the conventional EM for three-dimensional appreciation of structural elements, which is made by stereo-viewing of sections or by EM tomography. Therefore, the PEG-EM is regarded as an important adjunct to the conventional EM for histological studies and wide application of this method may unravel a new level of histology.