Specimen Preparation For Scanning Electron Microscopy Research Articles

Backscattered electron image of osmium-impregnated/macerated tissues as a novel technique for identifying the cis-face of the Golgi apparatus by high-resolution scanning electron microscopy.

The osmium maceration method with scanning electron microscopy (SEM) enabled to demonstrate directly the three-dimensional (3D) structure of membranous cell organelles. However, the polarity of the Golgi apparatus (that is, the cis-trans axis) can hardly be determined by SEM alone, because there is no appropriate immunocytochemical method for specific labelling of its cis- or trans-faces. In the present study, we used the osmium impregnation method, which forms deposits of reduced osmium exclusively in the cis-Golgi elements, for preparation of specimens for SEM. The newly developed procedure combining osmium impregnation with subsequent osmium maceration specifically visualised the cis-elements of the Golgi apparatus, with osmium deposits that were clearly detected by backscattered electron-mode SEM. Prolonged osmication by osmium impregnation (2% OsO4 solution at 40°C for 40 h) and osmium maceration (0.1% OsO4 solution at 20°C for 24 h) did not significantly impair the 3D ultrastructure of the membranous cell organelles, including the Golgi apparatus. This novel preparation method enabled us to determine the polarity of the Golgi apparatus with enough information about the surrounding 3D ultrastructure by SEM, and will contribute to our understanding of the global organisation of the entire Golgi apparatus in various differentiated cells.

Quantitation of platelet deposition on human arteries: Assessment of the disparity between results obtained with 111indium ( 111in)-labelling versus scanning electron microscopy

Quantitation techniques for measuring platelet deposition (PD) to vessel surfaces are important to an understanding of thrombogenesis. In previous studies, scanning electron microscopy (SEM) has been shown to indicate a lower extent of PD than platelet 111In-scintigraphy. Part of this disparity may be explained by nonspecific binding of 111In to the vessel surface during perfusion, or loss of adherent 111In-labelled platelets by lysis or dissociation from the surface during specimen preparation for SEM. To assess whether these independent processes occur, we used a previously described human placental artery (HPA) perfusion model to quantify vessel 111In retention. Of the total 111In that bound to the vessel surface during perfusion, 77 ± 42% (N=9) was platelet associated 111In ( 111In-labelled platelets) and 23 ± 19% (N=9) was non-platelet associated 111In (nonspecific binding). After specimen fixation, 67 ± 32% (N=9) of the initial total surface 111In remained. This decrease is due to dissociation of both adherent 111In-labelled platelets, and non-platelet associated 111In. After fixation, 57 ± 34% (N=9) of the initial total surface 111In remained as 111In-labelled platelets and 10 ± 13% (N=9) remained as non-platelet associated 111In. Fixation caused no measurable lysis of platelets. These data suggest that PD may be overestimated by 111In-scintigraphy because of nonspecific binding of 111In and underestimated by SEM because of dissociation of adherent platelets during specimen preparation for SEM.

A Nuclepore Filter Bag for Preparation of Small Biological Specimens for Scanning Electron Microscopy.

A method for the preparation of minute, delicate and dilute suspended organisms for scanning electron microscopy (SEM) by using of Nuclepore filter bags (NFB) is described. NFBs are easily made by welding with a heat sealer and are inert with solvents used in the preparation of specimens for SEM. The use of NFB avoids the risk of unintentional air-drying, physical damage, contamination, and loss of specimens during preparation. Specimens prepared by NFB show fine details of three-dimensional surface structure.