Significance of preparation of tissue samples for electron microscopy for observation and diagnosis

Transmission Electron Microscopy (TEM) is used to provide descriptive, morphological information and plays important role in cell biology for the visualization of intracellular organelles. It uses a beam of electrons which passes through the specimen and provides an image of objects 1 nm in size. The aim of this study is to compare the varyes of techniques that have been developed to prepare TEM specimen ready for observation and diagnosis. Significance of preparation of the samples for EM requires precise execution of each step in the entire procedure. The standard procedure for sample preparation is described in the following steps: extraction of tissue, fixation and dehydration with different percentages of acetones, resin infiltration, followed by embedding and polymerization and ultramicrotomy. Following steps are crucial in sample preparations. Generally, formaldehyde as a fixative penetrates better into the tissue, but glutaraldehyde better preserves the structure. After initial fixation, we used osmium tetroxide as a second fixative for lipid preservation. During the dehydration step, water from the samples is removed by solvents such as ethanol or acetone, this prevents holes-artifacts in the sections. Infiltration and embedding in resin provides stability of the tissue sample and formation of indestructible and insoluble block, suitable for section on ultramicrotome .Finally ultramicrotomy produce semi-thin and ultrathin sections which contrast can be introduced using heavy metals such as uranyl acetate and lead citrate. The alternative procedure for sample preparation is from an already paraffin-embedded tissue previously treated by the method of histopathological analysis. The procedure is very similar and differs only initial but contributes to significant differences in the final quality of the specimen. Tissue must be first deparaffinized in xylol, processed in serial diluents of ethanol in order ,and then washed in PBS to remove residue xylol, continues with following steps of the standard procedure, starting with the glutaraldehyde fixation shortly, then with osmium tetroxide, dehydration with acetone, resin infiltration, embedding and polymerization. Modification in procedure is mostly emphasized during the ultramicrotomy. Every step of the sample preparation is important for having a valid electron-microscopic image, and every modification can result in an inadequate results. The aim of this study was to compare alternative procedures, and we found that the method based on paraffin embedded samples has several disadvantages, including disruption of continuity of the tissue and low quality of the electron micrographs. But tissue already embedded in 89 paraffin still can be used for TEM although initially it was not intended to be analyzed by a transmission electron microscope. However, this method can be useful for diagnostic purposes