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

荧光显微镜已广泛用于研究亚细胞结构和细胞器，以及活细胞中的分子动力学。然而，它在大分子配合物结构分析中的应用受到可见光衍射极限（~200 nm）的限制。超分辨显微技术打破了衍射障碍，各种超分辨方法得到了发展。受激发射耗尽（STED）显微镜应用环形耗尽激光共聚焦激发，达到~50 nm的横向分辨率。然而，STED分辨率与耗尽激光的功率成正比，导致活细胞成像的大量光漂白和光毒性。我们申请资助将我们的Leica SP8 STED显微镜升级为tauSTED显微镜，利用荧光寿命信息来提高分辨率，减少光毒性和光漂白，消除不相关背景。利用NSERC RTI资助，我们升级了STED显微镜，使用775 nm耗尽激光器和86X STED白色物镜，增强了多通道成像，并提高了活体样品的分辨率~65 nm和固定样品的分辨率~50nm。然而，实时成像仅限于短时间推移系列（最大~100帧），而由于光漂白，3D成像仅限于固定样品。tasted降低了所需的激光强度，提高了分辨率，使更温和的活细胞STED成像更长的延时实验和增加多色应用。由于新的超分辨率显微镜的成本约为150万至200万美元，我们的STED显微镜的升级成本为15万美元，这是一种极具成本效益的方法，可以将我们的超分辨率能力提升到一流水平。STED显微镜对申请人的研究项目做出了巨大贡献，使内质网内质网纳米结构域（Nabi）和寨卡病毒感染时内质网重组的新特征成为可能，现在已扩展到SARS-CoV-2感染时的内质网重组（Nabi, Jean, Hamarneh）。STED数据使机器学习分析内质网组织、内质网线粒体接触位点和内质网纳米结构域成为可能（Hamarneh, Nabi）。Weidberg将利用这些方法开发线粒体蛋白输入和Rideout的新检测方法，以研究与果蝇睾丸分化和精子发生相关的脂质代谢开关过程中的脂滴动力学和自噬。Vogl利用STED研究了睾丸小管球复合体（TBC）膜接触部位的接触蛋白或氧甾醇结合蛋白相关蛋白9的破坏。Gold将使用tauSTED进行免疫突触形成过程中B细胞受体（BCR）组织的长时间延时分析。升级将大大提高我们的STED显微镜的活细胞能力和研究成果的申请人，以及显微镜的其他用户。

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.