The technological novelty is three-fold, i) Establishing a correlative multi-mode imaging system using live cell single particle imaging, super-resolution single molecule imaging, and cryoET imaging advances our previously established correlative live-cell and cryoET methodology to determine HIV-i protein interactions with host cell factors. At one end of the imaging spectrum, the dynamic behavior of a specific target in the context of a living biological system can be characterized with compromised resolution, while at the other end, high resolution structural snapshots of regions of interest can be obtained. Super-resolution molecular imaging bridges these methods and allows identification and localization of specific target proteins associated with cryoET structures. 2) Creating cell lamella in vitreously frozen cells, similar to yo-ultramicrotomy, but without sectioning artifacts, is a novel approach to generate suitably thin vitreous biological specimens and enhances the applicability of cryoET for high resolution 3D structural analysis of native cells and tissues. 3) Using a short fusion peptide with high affinity to a gold surface constitutes a novel approach to overcome some of the difficulties in developing a GFP-equivalent taggable EM label. Ultimately the innovation of the technology program relies on the integration of several approaches developed by us (correlative light microscopy/cryoEM imaging) and by others (super resolution imaging). Our proposed studies build on our prior work and develop new capabilities.
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