The goal of the proposed research is to determine the effectiveness of using charging-free versions of two fundamentally different types of microfabricated phase-contrast devices, designed for use in the transmission electron microscope. The advantages of using in-focus phase contrast for applications in biomedical research are expected to be: Images of protein complexes as small as 200 kDa should be more easily identified and selected for computer processing, due to the improved contrast transfer function (CTF) at low spatial frequencies that is provided by in-focus phase contrast. Structural information at high resolution will no longer be corrupted or lost due to use of large values of defocus to achieve the needed amount of phase contrast. The improved image quality may result in improvement in the ability to detect structural (compositional) and conformational differences amongst particles in a structurally non-homogeneous sample. One type of device that will be evaluated is a microfabricated aperture that blocks a half-plane of the electron wave that is scattered at small angle, while at the same time allowing the rest of the scattered wave to contribute in the usual way to image formation. The second type of device is a novel, 2-electrode """"""""electrostatic drift tube"""""""" design that applies a 90 degree phase shift to the unscattered electrons relative to the scattered electrons. Our fabrication of charging-free devices will employ high-temperature annealing and refinement of materials and devices, chemical and physical cleaning of surfaces, and the use of sensitive tools for detecting surface contamination. Fabrication methods will include traditional lithographic wet-fabrication as well as laser micromachining and focused-ion beam milling. The effectiveness of these devices will be determined from cryo-EM images of biological test specimens such as monolayer crystals of streptavidin and tobacco mosaic virus. Applications to current research will include studies on structural plasticity of microtubules and the interactions of various regulatory proteins with microtubules.
It is expected that our research will greatly improve the usefulness of electron microscopy of unstained, ice-embedded specimens (cryo-EM) for basic bio-medical research in the fields of biochemistry and cell biology. The improvement in image contrast that will be achieved will greatly increase the information that one can derive from images of complex protein assemblies and macromolecular machines. It may also make it possible to identify a much greater number of such molecular complexes in three-dimensional electron tomograms of whole cells.
|Glaeser, Robert M; Han, Bong-Gyoon; Csencsits, Roseann et al. (2016) Factors that Influence the Formation and Stability of Thin, Cryo-EM Specimens. Biophys J 110:749-55|
|Han, Bong-Gyoon; Watson, Zoe; Kang, Hannah et al. (2016) Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules. J Struct Biol 195:238-244|
|Glaeser, Robert M; Müller, Holger (2014) Generalization of the Matsumoto-Tonomura approximation for the phase shift within an open aperture. Ultramicroscopy 138:1-3|
|Grob, Patricia; Bean, Derek; Typke, Dieter et al. (2013) Ranking TEM cameras by their response to electron shot noise. Ultramicroscopy 133:1-7|
|Glaeser, Robert M; Sassolini, Simone; Cambie, Rossana et al. (2013) Minimizing electrostatic charging of an aperture used to produce in-focus phase contrast in the TEM. Ultramicroscopy 135:6-15|
|Glaeser, Robert M (2013) Invited review article: Methods for imaging weak-phase objects in electron microscopy. Rev Sci Instrum 84:111101|
|Glaeser, Robert M; Typke, Dieter; Tiemeijer, Peter C et al. (2011) Precise beam-tilt alignment and collimation are required to minimize the phase error associated with coma in high-resolution cryo-EM. J Struct Biol 174:1-10|
|Buijsse, Bart; van Laarhoven, Frank M H M; Schmid, Andreas K et al. (2011) Design of a hybrid double-sideband/single-sideband (schlieren) objective aperture suitable for electron microscopy. Ultramicroscopy 111:1688-95|
|Hall, R J; Nogales, E; Glaeser, R M (2011) Accurate modeling of single-particle cryo-EM images quantitates the benefits expected from using Zernike phase contrast. J Struct Biol 174:468-75|
|Müller, H; Jin, Jian; Danev, R et al. (2010) Design of an electron microscope phase plate using a focused continuous-wave laser. New J Phys 12:|
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