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2. Electron Microscopy.pdf

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Anatomy & Cell Biology
ANAT 262
John Presley

Electron Microscopy: To get a sense of scaling for electron microscopy, lets start by looking at someones finger, about 20mm across, and then zoom into a spot by 10x every time 1X the whole finger is visible 20mm 10X the skin can be seen close up as ridges 2mm 100X the skin layers can roughly be seen (low mag. LM) 0.2mm 1000X start seeing distinct cells and some organelles (very high power LM) 20 microns 10KX details of organelles 2 microns 100KX large complexes become visible from a low mag. 0.2 microns 1MX Individuals ribosomes can be seen (and other macromolecular complexes) 20nm 10MMX atoms becomes visible (atomic resolution) 2nm 100MX individual atoms can be seen 0.2nm LM, as we saw before, has a resolving power of about 200nm, at which point cells like bacteria (1 micron) begins to look like dots Techniques like X-Ray NMR are used for atomic resolution to give detailed structural biology at the level of Angstroms (1A is 0.1nm) o NMR becomes impractiral, though, for looking at larger molecules and complexes Therefore, to fill the gap between LM and NRM, theres electron microscopy, EM, for which there are different types o While EM is not good for atomic resolution, it can be used to look at things smaller than 200nm The limit of resolution, d, is the smaller distance between two objects with which they can still be distinguished It can be calculates using the formula to the right, and is dependent on three things: o The angle of the rays collected by the objective lens; this value can maximize at a value of 1 o n is the refractive index of the medium between the specimen and, usually o The wavelength of light is directly proportional to d, and is often the factor we play around with to try and change the resolving power Typically in LM, we use the visible spectrum of light as a lambda value, from about 400- 700 nm o For EM, much smaller values are used In EM, the wavelength of electrons is dependent on voltage; we typically use a voltage of 100kV, which gives a lambda of 3.7 x 10 nm o This would produce, in theory, a resolution limit of about 0.002nm, which is 100,000 time that of LM =12 o However, a modern EM at about 300kV with lambda approx. equal to 2.2 x 10 m will only have a resolution of about 1A, 0.1nm, because only the very center of the lens can be used as the aperture The first electron microscope was built in 1931by Ernst Ruska and Max Knollat the Berlin Technische Hochschule Although this crude initial instrument was capable of magnifying objects by 400X, it demonstrated the principles of an electron microscope o This was more a proof of concept than as an effective machine Two years later, Ruska constructed an electron microscope that exceeded the resolution possible with an optical microscope It was greatly developed through the 1950sand has allowed great advances in the natural sciences and physics The advantage of an electron beam is that it has a much smaller wavelength, which allows a higher resolution-the measure of how close together two things can be before they are seen as one o Light microscopes allow a resolution of about 200nm, whereas electron microscopes can have resolutions as low as 0.1 nm (1A) EM can commonly be used to try and answer the following things: Morphology and changes in morphology caused by certain diseases (i.e. whats going wrong in the cell) Protein localization and trafficking, and thus the mistargeting and mislocating caused by disease Understanding protein function which can allow us to, for example, develop drug which prevent bacterial ribosome function (disease prevention) There are two main types of EM: transmission EM, or TEM, and scanning EM, or SEM TEM is used for infrastructural components and has more versatile applications, and has a high resolution than SEM SEM is used mainly for topographical information, and not for detailed work on cell infrastructure LM, SEM, and TM do have a similar design and serve similar purposes; to make small things bigger for us to see them In LM, the light source comes from a light, but in EM, electrons are produced from a heated filament Instead of glass lenses, EM techniques used magnetic coils to condense and magnify the electron beam o In TEM, the sample is placed right after the condenser, like an LM, and the electrons pass through it (everything is analogous to LM)o In SEM, the sample is located at the bottom after a series of lenses, and electrons are scattered off of it A beam deflector allows you to move the electron beam and literally scan along the sample surface The bouncing pattern of the electrons is recorded on a viewing screen, now usually a computer In all EM, electrons can collide with air particles, so the process must take place inside a vacuum o Presence of air will cause the image to blur There are different types of interactions between the electrons and the sample depending on the type of EM used The source of light comes from the incident high kV electron beam which comes from the heated filament All EMs experience some background noise which can come from backscattered electrons (BSE), Characteristic X-Rays, visible light, inelelastically scattered electrons and Bremsstrahlung X-rays TEM uses elastically scattered electrons in comparison with direct beams, which both go through the sample SEM uses secondary
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