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，以及独特的活细胞中的分子动力学。然而，其在大分子复合物结构分析中的应用受到可见光（~200 nm）衍射极限的限制。超分辨率显微镜已经打破了衍射障碍，并且已经开发出各种超分辨率方法。受激发射损耗 (STED) 显微镜采用环形损耗激光器进行共焦激发，实现​​约 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，导致活细胞成像出现严重的光漂白和光毒性。在这里，我们申请资金将 Leica SP8 STED 显微镜升级为 tauSTED，其中荧光寿命信息用于提高分辨率、减少光毒性和光漂白并消除不相关的背景。利用 NSERC RTI 资金，我们用 775 nm 耗尽激光器和 86X STED 白色物镜升级了 STED 显微镜，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，由于光漂白，实时成像仅限于短延时系列（最多约 100 帧），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，实现更温和的活细胞 STED 成像，以进行更长的延时实验并增加多色应用。由于新型 tauSTED 超分辨率显微镜的成本约为 150,000 美元，因此花费 150,000 美元对我们的 STED 显微镜进行 tauSTED 升级代表了一种极具成本效益的方法，可将我们的超分辨率能力增强至一流水平。 STED 显微镜为申请人的研究项目做出了巨大贡献，实现了内质网 ER 纳米结构域 (Nabi) 的新表征和寨卡病毒感染时的 ER 重组，现在已扩展到 SARS-CoV-2 感染时的 ER 重组 (Nabi、Jean、Hamarneh)。 STED 数据支持对 ER 组织、ER 线粒体接触位点和 ER 纳米域（Hamarneh、Nabi）进行机器学习分析。 Weidberg 将利用这些方法开发线粒体蛋白导入和 Rideout 的新测定方法，以研究与果蝇睾丸分化和精子发生相关的脂质代谢转换过程中的脂滴动力学和自噬。 Vogl 使用 STED 研究睾丸管球复合体 (TBC) 膜接触位点皮质素或氧甾醇结合蛋白相关蛋白 9 的破坏。 Gold 将使用 tauSTED 对免疫突触形成过程中的 B 细胞受体 (BCR) 组织进行长时间延时分析。 tauSTED 升级将大幅增强我们 STED 显微镜的活细胞功能以及申请人以及显微镜其他用户的研究成果。

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.