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.

Cryogenic electron microscopy (CryoEM) and electron tomography (CryoET) can visualize macromolecules in their native state inside cells, however the undiscriminating contrast of such techniques complicates identifying regions of interest and specific molecular targets in high-resolution cryoEM/cryoET data. Super-resolution (SR) fluorescence methods have exquisite molecular specificity, but SR techniques that image vitrified biological samples at cryogenic temperatures have only achieved limited spatial resolutions (~20-60 nm). 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 and paving the way for powerful correlative light-electron microscopy, with transformative potential of elucidating the organization of macromolecular assemblies in cells.