Electron Microscope Definition and Working Principle PDF

**Electron microscope definition** An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. It is a special type of microscope having a high resolution of images, able to magnify objects in nanometres, which are formed by controlled use of elect...

**Electron microscope definition** An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. It is a special type of microscope having a high resolution of images, able to magnify objects in nanometres, which are formed by controlled use of electrons in vacuum captured on a phosphorescent screen. Ernst Ruska (1906-1988), a German engineer and academic professor, built the first Electron Microscope in 1931, and the same principles behind his prototype still govern modern EMs.  **Working Principle of Electron microscope** Electron microscopes use signals arising from the interaction of an electron beam with the sample to obtain information about structure, morphology, and composition. The electron gun generates electrons. Two sets of condenser lenses focus the electron beam on the specimen and then into a thin tight beam. To move electrons down the column, an accelerating voltage (mostly between 100 kV-1000 kV) is applied between tungsten filament and anode. The specimen to be examined is made extremely thin, at least 200 times thinner than those used in the optical microscope. Ultra-thin sections of 20-100 nm are cut which is already placed on the specimen holder. The electronic beam passes through the specimen and electrons are scattered depending upon the thickness or refractive index of different parts of the specimen. The denser regions in the specimen scatter more electrons and therefore appear darker in the image since fewer electrons strike that area of the screen. In contrast, transparent regions are brighter. The electron beam coming out of the specimen passes to the objective lens, which has high power and forms the intermediate magnified image. The ocular lenses then produce the final further magnified image. **Types of Electron microscope** There are two types of electron microscopes, with different operating styles: **The transmission electron microscope (TEM)** The transmission electron microscope is used to view thin specimens through which electrons can pass generating a projection image.   The TEM is analogous in many ways to the conventional (compound) light microscope.  TEM is used, among other things, to image the interior of cells (in thin sections), the structure of protein molecules (contrasted by metal shadowing), the organization of molecules in viruses and cytoskeletal filaments (prepared by the negative staining technique), and the arrangement of protein molecules in cell membranes (by freeze-fracture). **The scanning electron microscope (SEM)** Conventional scanning electron microscopy depends on the emission of secondary electrons from the surface of a specimen.  Because of its great depth of focus, a scanning electron microscope is the EM analog of a stereo light microscope. It provides detailed images of the surfaces of cells and whole organisms that are not possible by TEM. It can also be used for particle counting and size determination, and for process control.  It is termed a scanning electron microscope because the image is formed by scanning a focused electron beam onto the surface of the specimen in a raster pattern.  **Parts of Electron microscope** EM is in the form of a tall vacuum column which is vertically mounted. It has the following components: **Electron gun** The electron gun is a heated tungsten filament, which generates electrons. **Electromagnetic lenses** Condenser lens focuses the electron beam on the specimen. A second condenser lens forms the electrons into a thin tight beam. The electron beam coming out of the specimen passes down the second of magnetic coils called the objective lens, which has high power and forms the intermediate magnified image. The third set of magnetic lenses called projector (ocular) lenses produce the final further magnified image. Each of these lenses acts as an image magnifier all the while maintaining an incredible level of detail and resolution. **Specimen Holder** The specimen holder is an extremely thin film of carbon or collodion held by a metal grid. Image viewing and Recording. **Review** **Application of transmission electron microscopy to the clinical study of viral and bacterial infections: present and future** Alan Curry et al. Micron. 2006. Free PMC article Show details Abstract    PubMed    PMID  Full text linksCite **Abstract** Transmission electron microscopy has had a profound impact on our knowledge and understanding of viruses and bacteria. The 1000-fold improvement in resolution provided by electron microscopy (EM) has allowed visualization of viruses, the existence of which had previously only been suspected as the causative agents of transmissible infectious disease. Viruses are grouped into families based on their morphology. Viruses from different families look different and these morphological variances are the basis for identification of viruses by EM. Electron microscopy initially came to prominence in diagnostic microbiology in the late 1960s when it was used in the rapid diagnosis of smallpox, by differentiating, on a morphological basis, poxviruses from the less problematic herpesviruses in skin lesions. Subsequently, the technique was employed in the diagnosis of other viral infections, such as hepatitis B and parvovirus B19. Electron microscopy has led to the discovery of many new viruses, most notably the various viruses associated with gastroenteritis, for which it remained the principal diagnostic method until fairly recent times. Development of molecular techniques, which offer greater sensitivity and often the capacity to easily process large numbers of samples, has replaced EM in many areas of diagnostic virology. Hence the role of EM in clinical virology is evolving with less emphasis on diagnosis and more on research, although this is likely only to be undertaken in specialist centres. However, EM still offers tremendous advantages to the microbiologist, both in the speed of diagnosis and the potential for detecting, by a single test, any viral pathogen or even multiple pathogens present within a sample. There is continuing use of EM for the investigation of new and emerging agents, such as SARS and human monkeypox virus. Furthermore, EM forms a vital part of the national emergency response programme of many countries and will provide a frontline diagnostic service in the event of a bioterrorism incident, particularly in the scenario of a deliberate release of smallpox virus. In the field of bacteriology, EM is of little use diagnostically, although some bacterial pathogens can be identified in biopsy material processed for EM examination. Electron microscopy has been used, however, to elucidate the structure and function of many bacterial features, such as flagellae, fimbriae and spores and in the study of bacteriophages. The combined use of EM and gold-labelled antibodies provides a powerful tool for the ultrastructural localisation of bacterial and viral antigens. **EM Methods Currently Used in Medical Diagnosis** Generally it can be stated that EM is of high value in the investigation of clinical specimens related to renal diseases (see above), tumor processes (especially for questions concerning the grade of differentiation of tumor cells), storage disorders and the identification of infectious agents many experts recommend the inclusion of EM in kidney biopsy protocols, or at least to take some biopsy tissue in reserve for additional EM studies, should they become necessary. This is surely one if not the standard paradigmatic case in which it can be said that EM is necessary as a routine method in biopsy diagnosis. In some countries, these circumstances alone have moved those responsible for education to include EM. **asma al ameer:** An example would be a group of patients with a clinical situation characterized by repeated infections of the upper and lower respiratory tracts. These patients moreover have a reduced mucociliary clearance that can be well measured with colored indicators at the nasal fossae. The reason for these deficits is the reduced or altered motility of the kinocilia (for instance, Kartagener syndrome). Both TEM and scanning electron microscopy (SEM) contribute significantly to the clarification of the structure of the kinocilia in biopsies obtained \>from the wall of the respiratory tract, which are of course indicated in these patients. Depending on the type of disorder, the external shape (\"hockey-stick\") or the exonemal microtubular apparatus of the kinocilia can be altered. EM is indicated in cases in which suspicion of a primary ciliary diskynesia (PCD) exists \[3\]. In the case of other diseases it is necessary to identify and locate specific molecules (markers), for which immuno-cytochemistry can be used (in pre- and post-embedding). For this purpose, numerous variations in the chemical composition of fixatives, temperature conditions during preparation and embedding (PLT, freeze-substitution) as well as the most useful type of resin are available. For instance, in studies on sperm fertility the criteria used to evaluate the state of the cells include both structural, immuno-cytochemical and microanalytical aspects. In such investigations cryo-techniques are used increasingly in TEM (cryo-fixation and cryo-ultramicrotomy to observe native vitrified sections). In pathological processes like storage disorders and those related to occupational medicine, heavy metals and crystals can accumulate in cells and organs. In such cases microanalytical investigations (EDX, EELS) have an important place in the diagnostic strategy. The specimens can be prepared directly from fresh tissue, but material already embedded in paraffin for light microscopy can also be used for correlative LM/TEM studies. In this last case the preservation of ultrastructure is rather poor, which can mean a significant handicap for evaluation. Depending on which elements are to be micro-analyzed, appropriate fixation and embedding protocols are necessary. For easily diffusible elements cryo-preparation and cryo-sectioning are required (see Jonas et al. in this meeting). In recent years the application of EM techniques in pathology has been aided by continuous progress in the technical development of specimen preparation. An important disadvantage of standard TEM protocols is the duration of processing, which can be as long as 3 to 5 or more days. Relevant modifications in fixation, dehydration and embedding management allow shortening processing times of small tissue blocs considerably. Today rapid processing for TEM can be performed in 2-3 hours, which is very attractive for diagnosis The term \"bio-adhesion\" applies primarily to interactions between proteins and surfaces.  The formation of proteinaceous biofilms can be studied with TEM but also with SEM In relation to the adhesion of bacteria to surfaces, the study of the ultrastructure of flagella is currently a much-discussed topic. The structure of flagella can be clarified down to the macromolecular level using EM methods such as negative staining and electron tomography \[7\]. The attachment of cells to a surface is a complex phenomenon in which the properties of the surface (topography, roughness) and the cells themselves are very important. During attachment and depending on the affinity to the surface they become flattened, displaying characteristic morphologies. During the cell cycle, the cell shape changes in correlation with variations in the cell adhesion. The density and distribution patterns of surface profiles (microvilli, kinocilia, small blebs) in adherent cells depend on the grade of attachment to the substrate. With SEM and also with ESEM (wet mode as well as low pressure and large field detector, LFD) the changes of the cell surface associated with these processes can easily be investigated.  The cell can be observed in the scanning electron microscope and a very precise selection of the point or area that should be cut is possible. **صقرIn medical research the situation is completely different from the one in clinical laboratory medicine, and the newest EM methods can be applied, the only limitation being the financial support. The topic of neurosciences is a good example, ** Electron microscopy, considered by some to be an old technique, is still on the forefront of both clinical viral diagnoses and viral ultrastructure and pathogenesis studies. In the diagnostic setting, it is particularly valuable in the surveillance of emerging diseases and potential bioterrorism viruses. In the research arena, modalities such as immunoelectron microscopy, cryo-electron microscopy, and electron tomography have demonstrated how viral structural components fit together, attach to cells, assimilate during replication, and associate with the cellular machinery during replication and egression.  Early virus classification depended heavily on morphology as shown by EM , and many of the intestinal viruses were discovered by EM examination of feces after negative staining. Cossart et al., in noticing an anomalous reaction while testing normal blood for hepatitis B virus, excised a precipitation band from a gel and, using EM, demonstrated that it contained a very small virus (parvovirus B19) (16). That virus was later determined to be the cause of transient aplastic crisis in patients with sickle cell disease and of "fifth disease," a childhood exanthem. Even today, taxonomy books include electron micrographs of viruses in their descriptions . Even today, in the age of molecular diagnostics, EM is a mainstay in detecting new and unusual outbreaks. EM continues to serve to confirm infection in quality control of molecular techniques. EM was instrumental in elucidating the viral agent of the first outbreak of Ebola virus in Zaire in 1976 and in identifying the Ebola Reston infection of a monkey colony in Reston, VA, in 1989 as being caused by a filovirus. In 1999, the causative agent of a strange skin infection in an immunosuppressed patient, coined trichodysplasia spinulosa, was identified by EM as a polyomavirus ; since then, eight additional cases have been described and confirmed by EM of thin sections of skin biopsy specimens. Further, the Henipavirus (Hendra and Nipah) outbreaks in Australia and Asia were first described by use of EM. in 2003, EM recognized lymphocytic choriomeningitis virus as the cause of fatalities of recipients of organs transplanted from a single donor. Much work was done trying to identify the severe acute respiratory syndrome (SARS) agent before it was classified by EM as a coronavirus , and the monkeypox outbreaks in the United States in 2003 were discovered by EM to be caused by a poxvirus. Viruses stored in various solutions for extended periods are not viable for culture detection and may be unsuitable for molecular testing. However, EM does not require live or intact virus; it has been used to identify variola virus in infected tissue preserved for decades, in many cases, in unknown solutions . Besides its use in diagnostic virology, EM has been and continues to be valuable in elucidating mechanisms of virus attachment and replication. This information can be useful in the discovery and design of antiviral agents and vaccines. Another exciting area in this arena is the ultrastructural examination of virus-like particles (VLPs), which are viral cClinical Electron Microscopy (EM) is a powerful diagnostic tool used to assist in the diagnosis of Kidney Disease, Muscle Disorders, Neurological Disorders, Ciliary Dysfunction, Viral Gastroenteritis, Viral Infections or any disorder that may benefit from the analysis of the fine structures of a biopsy.apsids formed by using viral proteins but not genetic material .  It has also undergone a resurgence in recent years and can now be combined with fluorescence techniques in correlative methods giving unprecedented levels of structural and functional detail.   EM the preferred technique to: obtain information on the morphology and the chemical-physical composition of the sample; analyze the bioaccumulation of contaminants in the tissues, and evaluate the protein expression at subcellular level  Several cellular events may be missed if conventional ultrastructural studies are not complemented with details concerning the subcellular localization of a wide range of specific proteins. Thus, immunoelectron microscopy emerges as a technique that links the information gap between biochemistry, molecular biology, and ultrastructural studies, by placing macromolecular functions within a cellular context. IMMUNOELECTRON MICROSCOPY Immunoelectron microscopy is one of the best methods for detecting and localizing proteins in cells and tissues. This procedure can be used on practically every unicellular and multicellular organism, and often provides unexpected insights into the structure-function associations. It uses transmission electron microscope for visualisation. These days scanning electron microscope is also use. This technique uses antibodies to detect the intracellular location of structures of particular proteins.Ultra thin sections are labeled with antibodies against the required antigen and then labeled with gold particles. Gold particles of different diameters enable two or more proteins to be studied simultaneously. Immunogold labelling - colloidal gold particles are most often attached to secondary antibodies which are in turn attached to primary antibodies designed to bind a specific antigen or other cell component. Gold is used for its high electron density which increases electron scatter to give high contrast \'dark spots\'. Immunostaining (immunohistochemistry)- staining technique of tissue section so that the cells can be visualized under the microscope. The structure and location of antigen can be easily detected. Immunofixation -- used for identification of antibodies for specific antigens. IMMUNOGOLD LABELING Used for the identification, localization, and distribution of proteins, antigens, and other macromolecules of interest, at an ultrastructural level. Powerful technique for identifying active sites and the presence of biomarkers in the cells. A primary antibody is designed to bind onto a specific antigen in the cells. Gold conjugated secondary antibody designed to bind to primary antibody. Gold probe with its excellent electron scattering property is an important element for immunohistochemistry in the electron microscope. ANTIGEN-ANTIBODY REACTIONS Immunogold labeling is focused more on indirect patterns (gold conjugated secondary antibodies bind with specific primary antibodies in a microenvironment) Indirect pattern is more favorable than the direct pattern for two reasons: (a) higher density of secondary antibody and (b) increased sensitivity, since the secondary antibody is able to bind with multiple sites on primary antibody. Success of immunogold labeling technique depends on -- Quality of protein antigen preservation Antigen-primary antibody specificity Antibody's ability to infiltrate cells and tissues. GOLD PARTICLES AS A PROBE Gold became the most reliable choice for immunogold labeling due to Large specific surface area Good biocompatibility and High electron density, which offers easy detection The size of gold particles used for immunogold labeling varies from 1 to 40µm, chosen according to the type of labeling techniques employed Detecting multiple antigens within a cell may require the selective use of different sizes of gold particles. Smaller gold particles (2 nm or 5 nm) produce a higher labeling intensity and lower steric hindrance. Larger particle sizes (10 nm or more) reduce the potential labeling intensity due to their sheer size but are more easily seen at lower magnifications.. Pre-Embedding Immunogold Labeling is used primarily for the detection of proteins, antigens, and other macromolecules of interest, that are located on the surface or the exterior of cells, virus particles, and other extracellular biological specimens. In this technique specimens used are to be ultra-thin sectioned, for the examination of both the interior and exterior of cells, tissues, and other biological specimens. The different techniques normally used for immunogold labeling, dependent on the type of sample submitted and the location of the protein or macromolecule of interest. Post-Embedding Labeling techniques are used exclusively for the detection of proteins, antigens, and other macromolecules of interest that are located in the interior or intracellular regions of cells, virus particles, and other extracellular biological specimens. Immunoelectron Microscopy of Parasites  Immunoelectron Microscopy is a powerful tool for studying host--parasite interactions, and it is playing an important role in identifying specific immune targets and characterizing the precise subcellular localization, transport, and expression of parasite antigens.  This technique helps to clarify specific functions of subcellular organelles, which may not otherwise be detected by standard electron microscopy or biochemical techniques. So, Immunoelectron Microscopy contributes to a better understanding of the relationship between structure and function in parasites.  In studies of Plasmodium, Immunoelectron Microscopy has been, especially valuable in characterizing the antigenic composition of intracellular compartments, for example, parasitophorous vacuole and cytoplasmic clefts, that cannot be isolated, purified, and studied by current biochemical procedures. المجهر الإلكتروني والمجهر الضوئي إنّ المجهر الضوئي هو النوع المعتاد من المجاهر والذي قد تجده في غرفة الصف أو مختبر العلوم. يستخدم المجهر الضوئي الضوء لتكبير الصورة حتى 2000 x ، وعادة ما يكون أقل بكثير، كما ويمتلك دقة تبلغ حوالي 200 نانومتر.  يستخدم المجهر الإلكتروني حزمة من الإلكترونات بدلاً من الضوء لتشكيل الصورة. قد تصل نسبة تكبير المجهر الإلكتروني إلى 10000000 x ، مع دقة تصل إلى 50 بيكومتر (0.05 نانومتر). الإيجابيّات والسلبيّات من مزايا استخدام المجهر الإلكتروني بدلًا من المجهر البصري هو قدرته على التكبير والدقة. من سيّئات هذا المجهر التكلفة وحجم المعدات والحاجة لتدريب خاص لإعداد العينات للفحص المجهري واستخدام المجهر والحاجة إلى عرض العينات في فراغ، على الرغم من امكانيّة استخدام بعض العينات المائية. كيف يعمل المجهر الإلكتروني؟ إنّ أسهل طريقة لفهم كيفية عمل المجهر الإلكتروني هو مقارنته بمجهر الضوء العادي. تنظر من خلال العدسة لرؤية صورة مكبرة للعينة في المجهر الضوئي. تتكون إعدادات المجهر الضوئي من عينة وعدسات ومصدر ضوء وصورة يمكنك رؤيتها. تأخذ حزمة الإلكترونات مكان حزمة الضوء في المجهر الإلكتروني. يجب تحضير العينة بعناية حتى تتفاعل الإلكترونات معها. يتم ضخ الهواء خارج غرفة العينة لتشكيل فراغ لأن الإلكترونات لا تسافر لمسافة بعيدة بوجود الغاز. تركز اللفائف الكهرومغناطيسية حزمة الإلكترون بدلاً من العدسات. تحني المغناطيسات الكهربائية شعاع الإلكترون بنفس الطريقة التي تحني بها العدسات الضوء. يتم إنتاج الصورة بواسطة الإلكترونات، لذلك يتم مشاهدتها إما عن طريق التقاط صورة (صورة مجهرية إلكترونية) أو من خلال عرض العينة من خلال جهاز العرض. هناك ثلاثة أنواع رئيسية من المجهر الإلكتروني والتي تختلف وفقا لكيفية تشكيل الصورة وكيفية إعداد العينة ودقة الصورة. هذه الأنواع هي المجهر الإلكتروني النافذ (TEM) والمجهر الإلكتروني الماسح (SEM) ومجهر المسح النفقي (STM).