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

tauSTED：受激发射损耗超分辨率显微镜的荧光寿命升级

• 批准号: RTI-2022-00465
• 负责人: Nabi, Ivan
• 金额: $ 10.93万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=743072
• 关键词: tauSTED; Fluorescent; Lifetime; Upgrade; Stimulated

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.

荧光显微镜已被广泛用于研究活细胞中的亚细胞结构和细胞器以及独特的分子动力学。但是，它在大分子复合物的结构分析中的应用受到可见光的衍射极限（〜200 nm）的限制。超分辨率显微镜打破了衍射屏障，并开发了各种超分辨率方法。刺激的发射耗竭（STED）显微镜将甜甜圈形耗尽激光施加到共聚焦激发，达到〜50 nm的横向分辨率。但是，Sted分辨率与耗尽激光器的功率成正比，从而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Leica SP8 Sted显微镜升级到TAUSTED，其中荧光寿命信息用于增加分辨率，减少光毒性和光漂白并消除无关的背景。使用NSERC RTI资金，我们用775 nm的耗尽激光器和86倍sted White物镜升级了Sted显微镜，增强了多通道成像，并改善了现场直播的分辨率〜65 nm，固定样品的〜50nm。但是，由于光漂白，实时成像仅限于短时间段系列（最大〜100帧）和3D成像限于固定样品。 Tausted降低了所需的激光强度，增强了分辨率，从而为更长时间的延时实验和增加多色应用提供了更柔和的活细胞sted成像。随着新的Taust超分辨率显微镜的成本约为15-200万美元，以150,000美元的价格升级了我们的Sted显微镜，这是一种高度成本有效的方法，可以增强我们对最佳级别的超级分辨率能力。 STED显微镜为申请人的研究计划做出了巨大贡献，从而使内质网的新表征在Zika病毒感染后的内质性网状纳米结构群（NABI）和ER重组，现在延伸到SARS-COV-2感染后ER重组（Nabi，Jabi，Jayean，Hamarneh）。 STED数据已使ER组织，ER-Mochondria接触站点和ER纳米域（Hamarneh，Nabi）的机器学习分析。 Weidberg将使用这些方法来开发用于线粒体蛋白导入和骑行的新颖测定法，以研究与果蝇中睾丸分化和精子发生相关的LIPOMET代谢开关中的脂质液滴动力学和自噬。 VOGL使用St Sted研究睾丸中膜的膜接触部位的皮质素或氧蛋白酶结合蛋白相关的蛋白9的破坏。在免疫突触形成期间，黄金将使用TAUSTED进行B细胞受体（BCR）组织的延时分析。 Tausted升级将大大增强我们Sted显微镜和申请人的研究输出以及显微镜的其他用户的活细胞功能。