Electron Microscope 'Filters' Energy

Over the last several decades, a detailed description of the, fine struc " ture of cells and tissues has emerged, due in a large part to transmission electron microscopy (TEM). With improvements in microscope design, sectioning techniques, and fixation and staining methodology, scientists can now examine biological structures with nanometer resolution. In addition, regularly spaced structures, such as cytoskeletal polymers, can be described in molecular detail via electron diffraction. Despite the power of conventional TEM, however, a number of technical limitations exist, which, to date, have placed restrictions on TEM as an analytical tool. Now it appears that some of these restrictions have been overcome by a new product from Carl Zeiss Inc., Thornwood, N.Y. The company has developed the EM 902, a unique energy-filtering electron, microscope that broadens the image analysis capabilities of transmission electron microscopy in the biological and materials sciences. Electron microscopic images are generated by electron scatter of the illuminating beam by atoms in the specimen. Electrons are scattered either elastically by the nucleus of the atom or inelastically by shell electrons. Elastic scatter results in no loss of electron energy, while inelastic scatter results in an element-specific loss of electron energy. The difference in energy between the elastically and inelastically scattered electrons results in a chromatic aberration of the objective lens, which in - turn compromises the quality of the resultant image. This effect is noticeable particularly as the thickness of the specimen increases. Until the development of the EM 902, the only way to reduce chromatic aberration was to increase the accelerating voltage of the electron beam. Thus, the high-resolution imaging of thick sections required the use of costly high-voltage (1 MeV) electron microscopes. The EM 902 is the first series-manufactured TEM to feature an integrated imaging electron spectrometer, affording high-resolution images with energy-selected (monochromatic) electrons. Electron spectroscopic imaging (ESI) utilizes an electron optical prism/mirror/prism combination in the projector lens system of the TEM, which, in combination with an energy selective slit, permits only elastically scattered electrons to reach the image plane. Thus, ESI (also termed elastic brightfield imaging) permits distortion-free, high-resolution imaging of specimens up to 1 mm in thickness at relatively low (80 KV) accelerating voltages. A second advantage of ESI is the improvement of darkfield imaging, a technique commonly used in the examination of unstained specimens or ultrathin sections. Darkfield imaging (as with brightfield imaging of thick sections) is particularly impaired by the presence of inelastically scattered electrons in the image plane. With ESI, the inelastically scattered electrons are selectively filtered out resulting in a superior image. A third image enhancement capability of ESI is due to the fact that the energy loss associated with inelastic scatter is element-and-shell specific. In contrast to elastic brightfield imaging—which selectively filters out inelastically scattered electrons— contrast enhancement of unstained specimens may be obtained by selectively imaging inelastically scattered electrons. The energy-loss spectrum shows sharp absorption edges for the ionization of electrons from the inner shells of a particular element. In order to image with electrons that have been inelastically scattered with a specific energy loss, the accelerating voltage is increased selectively (via a microprocessor) so that only the specific energy-loss electrons remain in the final image plane. This imaging technique is particularly useful for unstained or weakly stained specimens. Conventional TEM imaging of unstained biological specimens results in very low contrast images. With ESI however, high contrast images are generated by element specificities within the specimen. Selective electron energy-filtering offers numerous applications by virtue of the ability to selectively image specific elements in a specimen. High-resolution images of specific elements within a specimen can be obtained by subtraction of an element-specific image from a reference image (obtained at a different accelerating voltage). Sequential element-specific imaging of a specimen followed by subsequent image analysis permits the localization of several elements within the same specimen. Applications include specific imaging of ions, metabolites, and immunolabeled structures within a cell. In addition, trace components in a metal alloy can be localized, providing applications in the materials sciences. Other material analysis capabilities of the EM 902 are realized via electron spectroscopic diffraction (ESD) and electron energy loss spectra (EELS). With. ESD, diffraction patterns obtained are significantly crisper due to the absence of inelastically scattered electrons. EELS permits chemical analysis of a specimen area (10 nm). In addition to providing a technical advance in TEM, Zeiss’s new EM 902, priced at $240,000, has many of the features seen in high-quality microscopes. The specimen positioning is microprocessor-controlled and can “mark and find” up to 100 positions. Magnification is from 150x to 400,OOOx, and focus is retained while changing magnification. The accelerating voltage of the EM 902 is 50 or 80 KV, and energy loss values can be selected from 0-2000 eV in 0.5 eV steps. A microprocessor-controlled sheet film camera is utilized for obtaining hard copy. An optional image analysis computer is available from Zeiss for use with the new energy filtering electron microscope. Wendy Wilson Sheridan is associate director of foundation and coporate relations, and V. Richard Sheridan is a post doctoral associate pharmacology both at Fox Chase Cancer Center, Philadelphia.

Electron Microscope 'Filters' Energy

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