tauSTED: Fluorescent Lifetime Upgrade for Stimulated Emission Depletion Super-Resolution Microscope

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Fluorescence microscopy has been extensively used to study subcellular structures and organelles and, uniquely, molecular dynamics in living cells. However, its application to structural analysis of macromolecular complexes is restricted by the diffraction limit of visible light (~200 nm). Super-resolution microscopy has broken the diffraction barrier, and various super-resolution approaches have been developed. Stimulated emission depletion (STED) microscopy applies a donut-shaped depletion laser to confocal excitation, achieving lateral resolutions of ~50 nm. However, STED resolution is directly proportional to the power of the depletion laser, resulting in substantial photobleaching and phototoxicity for live cell imaging. Here we apply for funding to upgrade our Leica SP8 STED microscope to tauSTED, in which fluorescence lifetime information is used to increase resolution, reduce phototoxicity and photobleaching and eliminate uncorrelated background. Using NSERC RTI funding, we upgraded our STED microscope with a 775 nm depletion laser and 86X STED white objective, enhancing multichannel imaging and improving resolution ~65 nm for live and ~50nm for fixed samples. However, live imaging is limited to short time lapse series (max ~100 frames) and 3D imaging limited to fixed samples due to photobleaching. tauSTED reduces the laser intensity required, enhancing resolution, enabling gentler live-cell STED imaging for longer time-lapse experiments and increasing multicolor applications. As new tauSTED super-resolution microscopes cost ~$1.5-2 million, tauSTED upgrade of our STED microscope for $150,000 represents a highly cost-effective approach to enhance our super-resolution capabilities to best-of-class. STED microscopy has contributed enormously to the research programs of the applicants, enabling the novel characterization of endoplasmic reticulum ER nanodomains (Nabi) and ER reorganization upon Zika virus infection, now being extended to ER reorganization upon SARS-CoV-2 infection (Nabi, Jean, Hamarneh). STED data has enabled machine learning analysis of ER organization, ER-mitochondria contact sites and ER nanodomains (Hamarneh, Nabi). Weidberg will use these approaches to develop novel assays for mitochondrial protein import and Rideout to study lipid droplet dynamics and autophagy in during the lipometabolic switch associated with testes differentiation and spermatogenesis in Drosophila. Vogl uses STED to study disruption of cortactin or oxysterol binding protein-related protein 9 at membrane contact sites at tubulobulbar complex (TBC) in testes. Gold will use tauSTED for prolonged time-lapse analysis of B cell receptor (BCR) organization during immune synapse formation. tauSTED upgrade will substantially enhance the live cell capabilities of our STED microscope and the research output of the applicants, as well as other users of the microscope.