We are applying for funds to purchase an Integrated Cryo-Processing System for High-Resolution Transmission Electron Microscopy. This new integrated system includes a high-pressure freezer, an automatic freeze substitution system, and an ultramicrotome that will be incorporated into the existing Molecular Microbiology Imaging Facility at Washington University School of Medicine. The primary need for this instrumentation is to elevate our electron microscopy capabilities with the goal of providing investigators access to state-of-the-art fixation techniques for optimal preservation of cellular ultrastructure. The Molecular Microbiology Imaging Facility processes biological specimens for morphological and immunocytochemical high-resolution electron microscopy analyses. Despite optimization, our techniques relying on chemical fixation remain inferior to those attainable by high-pressure freezing, the most current standard fixation method for immobilizing complex macromolecular events in near native state. Cryofixation by high-pressure freezing, in combination with subsequent freeze substitution, would provide superior preservation of fine ultrastructure and antigenicity circumventing many of the artifacts and deficiencies imposed by our standard processing techniques. One of the most powerful techniques that our investigators rely on in the analysis of subcellular details is in situ localization of target molecules by means of specific antibodies and analyses of these interactions at the ultrastructural level. High-pressure freezing is one of the most powerful sample preparation methods for antigenic and architectural retention, bridging the gap between high labeling efficiency and excellent ultrastructure. Incorporation of the proposed integrated cryo-processing system into our existing Biosafety Level 2 (BSL2)- approved Imaging Facility would provide users working with live BSL2-level pathogens, unique access to cryofixation by high-pressure freezing. The user groups include fifteen NIH-funded laboratories all working in areas of research related to human diseases, with ten users affiliated with the Program in Molecular Microbiology and Microbial Pathogenesis and working in the areas of host-pathogen interactions. Acquiring this integrated cryo-processing system would greatly strengthen the biomedical research programs of these highly productive groups by providing access to cutting-edge instrumentation for superior preservation of sub-cellular detail in combination with reliable ultrastructural immunolocalization.
Elucidating the subcellular details and events in host-pathogen interactions and model systems for human disease is a requisite component of biomedical research. Acquisition of the most innovative instrumentation for electron microscopy sample preparation will strongly benefit ongoing health-related research, greatly advancing our current antiquated techniques to state-of-the-art technology.
| Fischer, Kerstin; Beatty, Wandy L; Weil, Gary J et al. (2014) High pressure freezing/freeze substitution fixation improves the ultrastructural assessment of Wolbachia endosymbiont-filarial nematode host interaction. PLoS One 9:e86383 |
