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

荧光显微镜已被广泛用于研究亚细胞结构和细胞器，特别是活细胞中的分子动力学。然而，它在大分子络合物结构分析中的应用受到可见光(~200 nm)衍射极限的限制。超分辨显微镜突破了衍射的障碍，各种超分辨方法相继问世。受激射电子耗尽(STED)显微镜采用环状耗尽激光共聚焦激发，横向分辨率可达~50 nm。然而，STED的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。在这里，我们申请资金将徕卡SP8 STED显微镜升级到tauSTED，其中使用荧光寿命信息来提高分辨率，减少光毒性和光漂白，并消除不相关的背景。利用NSERC RTI资金，我们将STED显微镜升级为775 nm耗尽激光和86倍STED白色物镜，增强了多通道成像，并将活样品的分辨率提高到约65 nm，固定样品的分辨率提高到~50 nm。然而，由于光漂白，实时成像仅限于短时间序列(最大~100帧)，而3D成像仅限于固定样本。TauSTED降低了所需的激光强度，提高了分辨率，实现了更温和的活细胞STED成像，适用于更长时间的延时实验，并增加了多色应用。由于新的tauSTED超分辨率显微镜的成本约为150,000美元，升级我们的STED显微镜的成本为150,000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同类最佳。STED显微镜为申请者的研究计划做出了巨大贡献，使内质网ER纳米结构域(NABI)和寨卡病毒感染后ER重组的新表征成为可能，现在已扩展到SARS-CoV-2感染后ER重组(Nabi，Jean，Hamarneh)。STED数据使机器学习能够分析内质网组织、内质网线粒体接触部位和内质网纳米结构域(Hamarneh，NaBi)。Weidberg将利用这些方法开发线粒体蛋白输入和输出的新分析方法，以研究果蝇在与睾丸分化和精子发生相关的脂代谢开关过程中的脂滴动力学和自噬。Vogl使用STED研究睾丸小管球复合体(TBC)膜接触部位皮质蛋白或氧固醇结合蛋白相关蛋白9的破坏。Gold将使用tauSTED对免疫突触形成期间的B细胞受体(BCR)组织进行长时间推移分析。升级tauSTED将大大提高我们的STED显微镜的活细胞能力，以及申请者和其他显微镜用户的研究成果。

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.