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

Jovanka Laskova; Darko Bosnakovski; Gordana Petrusevska

Jovanka Laskova Institute of Pathology,Medical Faculty,University “Ss Cyril and Methodius”-Skopje R.Macedonia
Darko Bosnakovski Faculty of Medical Sciences, University Goce Delcev-Stip
Gordana Petrusevska Medical Faculty, University “Ss Cyril and Methodius”-Skopje

Abstract

Electron microscopy (EM) is used to provide descriptive, morphological information and plays important role in cell biology for the visualization of intracellular organelles. Electron microscope uses a beam of electrons to provide an image of the specimen. Two types of electron microscope are available; Transmission Electron Microscope (TEM) where electron beam passes through the specimen and objects of 1nm in size can be observed, and second, the Scanning Electron Microscope (SEM) where topographical information of the surface can be provided.

Significance of preparation of the samples for EM requires precise execution of each step in the entire procedure. This presentation gives an overview of the variety of techniques that have been developed to prepare TEM specimen ready for observation and diagnosis. Following steps are crucial in sample preparations: extraction of tissue, fixation, dehydration, resin infiltration, embedding, ultramicrotomy and staining. If the tissue block is too large or if the sample is kept for a short period in fixative and resin, they will not penetrate to the middle of the specimen, and the tissue in the middle will be soft. This sample is not suitable for section on ultramicrotome. Generally, formaldehyde better penetrates into the tissue and glutaraldehyde better preserves the structure. After initial fixation, for the second fixative osmium tetroxide is used to preserve lipids. During dehydration step, water from the samples is removed by solvents such as ethanol or acetone. This prevents creations of holes in the sections. Infiltration and embedding in resin provides stability of the tissue sample and formation of indestructible and insoluble block, suitable for ultramicrotomy.

Inadequate fixation, dehydration or infiltration of specimen leads to further tissue destruction, making it inappropriate for diagnosis. The aim of this study was to compare alternative procedures, for preparation biological sample for TEM. We found that method based on paraffin embedding of the samples has several disadvantages including disruption of continuity of the tissue. However, this method still can be useful for diagnostic purposes.

Keywords:

Sample preparation, TEM, paraffin embedding