Specimen preparation for "block face" scanning electron microscopy (BFSEM). An energy-dispersive X-ray spectroscopy study

ABSTRACT

In this paper we describe a SEM block-face technique in which block faces of large dimensions can be examined in a high-resolution SEM under high vacuum. The results of different tissue contrast methods have been studied and, in addition to osmium, potassium permanganate has been used as a staining medium for the first time in BFSEM. The study also examined the effects of uranyl acetate and phosphotungstic acid. The following organs of adult albino rats were examined colonic mucosa, spinal ganglion, anterior pituitary gland and exocrine pancreas.Six preparation protocols, referred to here as treatments, were applied and evaluated according to three criteria 1st the visual quality of the digital images, 2nd the measurements of the signal-noise ratio (SNR) of the digital images with and without beam deceleration (BD), and 3rd the X-ray microanalysis of samples, treated according to the 6 proposed protocols, demonstrating the presence and relative quantity of the elements used to stain the cellular structures, enabling visualisation with the electron microscope. In conclusion, it can be said that treatments with osmium produced better results than those containing potassium permanganate. Treatments with the addition of thiocarbohydrazide (TCH) considerably increased the osmium deposits (ligand effect) and proved highly effective. Finally, it should be noted that the method proposed, called here 2D BFSEM, can be very useful not only in histology but also in histo-pathology, for example in the study of biopsies and - last but not least - in embryology all these are situations in which it is important to avoid a loss of material due to preparation exigencies

RESUMEN

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