Introduction
Electron microscopy has become established as a useful diagnostic tool in pathology. Diagnostic electron microscopy entails the utilization of electron microscopy and its associated methods in all of their ramifications for the study of human disease as well as animal disease (Zond & Cosmi 2001). Modern advances in imaging and immunolabeling techniques for electron microscopy (EM) have contributed to resolution enhancement and improved diagnostic accuracy. Many scientists have described EM as a valuable tool in both research and teaching areas. Pathologists rely increasingly on the electron microscope as an aid in the morphologic diagnosis of disease. Its diagnosis is however limited because of either strategic reasons or budgetary constraints. A recent review (Tucker, 2000) discusses modes of clinical diagnoses in which a small but significant proportion of cases (3–8%), especially of cancer and nonneoplastic renal diseases, can be identified solely by EM. These numbers probably underestimate the potential contribution of EM, since its usage is limited not only by lack of utility in many clinical situations, but also by cost, time required to produce results, and relatively low output compared to that of histological methods. In the last decade, EM has been considered as an important tool in virology for the basic understanding of viruses and more important for the rapid and unequivocal recognition of their presence (Zond & Cosmi 2001) An important concept in general pathology is the correlation between structure and function at the cellular level which has been made observable through the integration of methods in the fields of microscopy, immunology, biochemistry and physiology. EM is a fundamental tool in those investigations since it is at the level of resolution provided by this instrument those more structural correlations with function and metabolism are visible. Diagnostic electron microscopy in kidney is one of the oldest and well known developed applications. A number of renal diseases cannot be distinguished clinically. They however, differ in prognosis and response to therapy. When a light microscope is used, the lesions are very similar and almost identical in appearance. Fortunately, they are readily differentiated by electron microscopy. In addition, EM has been found to be extremely useful in diagnosis of the etiology of infectious disease in both surgical and autopsy pathology. This is more important since some causative organisms cannot be cultured or in other culture results are questionable or cannot be interpreted. For this aspect, in addition to the examination of standard stained ultrathin sections, negative staining of the specimens is also employed (Zond & Cosmi 2001). Accurate diagnosis and classification of carcinomas and sarcomas are serious and critical problem among pathologists. The applications of EM coupled with cytochemistry give a new classification and histogenesis of lung carcinomas. Moreover, the diagnosis of soft tissue tumors which has for a long time been impossible has been improved by electron microscopy. Combination of scanning and transmission electron microscopy greatly augments the knowledge of classification, typing and estimation of prognosis in prostatic carcinoma.
Specimens
In diagnostic pathology, good laboratory practice must be observed at all times, especially when handling unfixed human tissues. However, practical and ethical considerations limits clinicians who are responsible for the care of the patient with regard to the nature and extent of human tissue specimens made available by pathologist. Tissue biopsies and resections must be restricted to the minimum necessary for diagnosis or treatment. There is one method of preparing specimens for electron microscopy that is suitable for rapid diagnosis and that is the technique of negative staining. Negative staining is useful for electron microscopic examination of small biological objects such as viruses and bacteria particularly for specimen taken from cutaneous vesicular lesions, serum or stool filtrates (Howell et al. 1998). The virus is surrounded by heavy metal atoms which act as electron stain, the electron beam can pass through the low electron density of the virus but not through the metallic background hence the term negative stain- a light virus against a dark background. In the past decades, only EM examination was available for the identification of certain types of specimens but novel techniques have been developed which are better suited for dealing with large number of specimens. For instance, the diagnosis of hepatitis B antigen carrier was always confirmed, if not established, by electron microscopy. Now, radioimmunoassay and passive haemagglutination are the techniques of choice in reaching a diagnosis. Almost any biological material will yield a specimen that can be examined by EM. However, the limitations of EM are set by two main factors: the absolute amount of virus present in a specimen and the ratio of this virus to the background material. A concentration of 105 virus particles in urine will yield a good specimen as there is little contaminating material present; however, the same concentration of virus in sputum would almost certainly not be suitable because of background material. The absolute amount of virus required for success also varies with the virus type. For instance, 105 particles of a small cubic virus would not be visualized unless a more sensitive technique of immune electron microscopy (IEM) is used. This involves adding specific antiserum to the virus containing specimen so that antigen-antibody aggregates are formed (Howell et al. 1998). The small cubic virus present in the form immune complexes is much more readily visualized than its unaggregated counterpart. Fortunately, both viable and non-viable particles are visualised in the EM and a specimen containing 106/ ml physical particles may contain as few as 103 infectious virions per ml.
Specimen preparation
Three basic rules entails the preparation of all specimens for negative staining. The diluent employed is distilled water which has an advantage of lysing cellular structures but leaves viruses unharmed. Secondly, the concentration by centrifugation should always be at the lowest possible speed. Virus is often entangled with fragments of cell debris and will be deposited at surprisingly low speeds. The large pieces of cellular material offer no problem in the EM and low molecular weight material will not be deposited. The duration and speed of centrifugation will vary somewhat with different types of specimen but many can be handled by centrifuging for one hour at 15,000 g. When the final pellet has been obtained, it is of the utmost importance that the last drops of fluid in the tube be removed. If fluid is present when the pellet is re-suspended, low molecular weight material contaminates the specimen (Sobrinho-Simões, Nesland & Johannessen 1981).
Usefulness of EM
The X-ray microprobe analysis is growing rapidly and has become so important that every institution with pathology department should at least have access to this technology. For example, granulomas was described by light microscopy as being caused by “particles of plastic” which were in fact caused by talc as confirmed by X-ray microanalysis (Howell et al. 1998). The application of electron microscopy requires simplified methods of the tissue preparation. One key strategy is the introduction of fixatives which are possible to fix tissues for both light and electron microscopy and moreover to permit prolonged storage in the fixative. This enables later retrieval of specimens for EM when indicated by either diagnostic application or the interest and education of the pathologists. All materials fixed in this manner should be available for subsequent ultra-structural study when required. In recent years, much progress has shifted in the training department where both technical and medical personnel are educated to achieve greater skills needed in application of electron microscopy.
References
- Howell, D. N., Payne, C. M., Miller, S. E., et al. (1998). Special techniques in diagnostic electron microscopy. Human Pathology, 29(12), 1339-46
- Sobrinho-Simões, M., Nesland, M., & Johannessen J. V. (1981). Diagnostic ultrastructural pathology–sub-speciality or special stain? Diagnostic Histopathology, 4(3), 223-36
- Tucker, J. A. (2000). The continuing value of electron microscopy in surgical pathology. Ultrastructural Pathology, 24(6), 383-9
- Zond, J. R., & Cosmi, S. A. (2001). Electron microscopy as a diagnostic tool in pathology. The Journal of the American Osteopathic Association, 92(1), 102- 4.