Ultra-stable, photon-efficient cryogenic super-resolution fluorescence imaging for visualizing vitrified biological samples with molecular-scale resolution

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ABSTRACT Cryogenic electron microscopy (CryoEM) and cryo-electron tomography (CryoET) are powerful techniques for visualizing macromolecular assemblies at a near-native state and in their functional context inside cells; however the undiscriminating/non-specific contrast of such techniques often complicates identifying regions of interest and specific molecular targets in high-resolution cryoEM/cryoET datasets. Super-resolution (SR) fluorescence methods have exquisite molecular specificity, but SR techniques have only achieved limited spatial resolutions (~20-60 nm FWHM) when imaging vitrified biological samples at cryogenic temperatures. The proposed project will create new ultra-stable, photon efficient cryogenic SR methods for imaging vitrified biological specimens with true molecular-scale resolution (<2-3 nm FWHM), closing the gap towards the (sub-)nanometer resolution of cryoEM/cryoET. The increased spatial resolution will be leveraged for further development of powerful correlative light-electron microscopy (CLEM) approaches that will ultimately enable elucidating the structural basis for many critical macromolecular processes inside cells.