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

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

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The isolated colonic mucosa: a new substrate for tissue reconstruction. Isolation, decellularization and characterization by immunohistochemistry and electron microscopy

This study proposes the use of isolated colonic mucosa as a "scaffold" for cell cultures and potentially for tissue reconstruction. The main goal of this study was to obtain complete decellularization of the specimens while preserving the superficial basement membrane (BM) as a place for cell attachment and growth. This decellularization technique uses a chelating agent in combination with mechanical vibration, followed by detergents and DNase. The grade of decellularization achieved is assessed by counting the number of cell nuclei stained with propidium iodide (PI). BM marker proteins such as collagen IV, laminin and perlecan were detected by immunohistochemistry (ICC). Transmission (TEM) and scanning electron microscopy (SEM) were used to examine the BM ultrastructure and surface topography. The results show that the protocol used is suitable for rat colonic mucosa. During the process, material of the lamina lucida (LL) was partly removed from the BM, whereas the lamina densa (LD) seems to have remained unchanged. The BM had become thinner than the control specimens. The nanotopography of the BM surface is characterized by globules of 34-60 nm in diameter. Human fetal fibroblasts were successfully cultured on this substrate confirming that cells can adhere to and grow on this substrate, at least for the particular cell line used. It can be said that the colonic mucosa is an interesting substrate for in vitro stud-ies with cells and presumably also for tissue reconstruction

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