Ring TIRF microscope with FRAP and ablation

带 FRAP 和消融功能的环形 TIRF 显微镜

• 批准号: 497257693
• 金额: --
• 依托单位: Universität des Saarlandes
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2022
• 资助国家: 德国
• 起止时间: 2021-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/497257693?language=en
• 关键词: Ring; TIRF; microscope; FRAP; ablation

We apply for a total internal reflection fluorescence (TIRF) microscope with fluorescence recovery after photobleaching (FRAP) and laser ablation. The microscope allows low-background, high-contrast imaging of near-surface events in diverse in vitro and in cellulo systems. By eliminating out-of-focus illumination, TIRF microscopy enhances the signal-to-noise ratio and allows single molecule imaging. While conventional point TIRF systems suffer from non-uniform illumination and interference patterns, the ring TIRF technology ensures even illumination by rapidly spinning the excitation light on a circle, thus averaging out any interference patterns, producing higher quality images and permitting quantitative fluorescence measurements as required by the applicants. The only available TIRF microscope at Saarbrücken campus is already used to capacity by six research groups, and it does not include the ring TIRF technology necessary for high-quality TIRF imaging. In contrast, the setup presented here will allow quantitative fluorescence measurements, thus presenting an important technical advancement over the available system. The ring TIRF setup provides unique, powerful characteristics that enable quantitative, near-surface fluorescence microscopy, even on the level of single molecules. In the past, we used ring TIRF to uncover new aspects of cytoskeletal dynamics and function: We showed that – contrary to common belief – microtubules are not only dynamic at their tips, but also along their shaft far from the tips, leading to major advances in the microtubule cytoskeleton field and demonstrating the potential of ring TIRF microscopy. In the future, we intend to employ ring TIRF microscopy to continue investigating cytoskeletal processes as well as other, near-surface events on the cellular and subcellular scale. In addition to TIRF imaging, in order to achieve a sound understanding of in vitro and subcellular processes, the applicants need to determine the mobility of molecules in vitro and in cells. The FRAP function allows flexible, controlled photobleaching in user-defined regions, thus meeting this demand. In addition, the ablation function permits moving beyond mere observational experiments through active and precise manipulation of the sample: by damaging or severing in vitro and subcellular elements such as cytoskeletal filaments, we will be able to directly probe the structural and functional aspects of these elements. The microscope we apply for will not only satisfy the increased demand for near-surface fluorescence microscopy at the Saarbrücken campus, but also enable advanced TIRF microscopy suitable for quantitative imaging, as well as provide state-of-the-art photomanipulation tools. The microscope will thus contribute to the infrastructure to promote scientific advances. It will provide a pillar for the Saarland University NanoBioMed strategy for excellence, as evidenced by the Strategy Grants awarded to two of the applicants.

我们申请了具有光漂白（FRAP）和激光烧蚀后荧光恢复功能的全内反射荧光（TIRF）显微镜，该显微镜可以对各种体外和细胞系统中的近表面事件进行低背景、高对比度成像。 TIRF 显微镜消除了离焦照明，从而提高了信噪比并允许单分子成像。传统的点 TIRF 系统存在照明不均匀和干涉图案的问题。环形 TIRF 技术通过在圆周上快速旋转激发光来确保均匀照明，从而平均任何干涉图案，产生更高定量质量的图像并允许按照申请人的要求进行荧光测量萨尔布吕肯园区唯一可用的 TIRF 显微镜已经用于测量。六个研究小组的能力，并且它不包括高质量 TIRF 成像所需的环形 TIRF 技术，相比之下，这里介绍的设置将允许定量荧光测量，从而呈现出比现有系统重要的技术进步。 TIRF 装置提供了独特、强大的特性，可以实现定量、近表面荧光显微镜检查，甚至在单分子水平上。过去，我们使用环形 TIRF 来揭示细胞骨架动力学和功能的新方面：我们表明，与常见的相反。我们相信，微管不仅在其尖端处是动态的，而且沿远离尖端的轴方向也是动态的，这导致了微管细胞骨架领域的重大进展，并展示了环形 TIRF 显微镜的潜力。使用环形 TIRF 显微镜继续研究细胞骨架过程以及细胞和亚细胞尺度上的其他近表面事件 除了 TIRF 成像之外，为了充分了解体外和亚细胞过程，申请人还需要： FRAP 功能允许在用户定义的区域进行灵活、受控的光漂白，从而满足这一需求。此外，消融功能可以通过主动和精确的操作超越单纯的观察实验。样品：通过破坏或切断体外和亚细胞元件（例如细胞骨架丝），我们将能够直接探测这些元件的结构和功能方面，我们所使用的显微镜​​不仅可以满足对近表面荧光日益增长的需求。萨尔布吕肯校区的显微镜，还支持适用于定量成像的先进 TIRF 显微镜，并提供最先进的照片处理工具，因此该显微镜将为促进科学发展的基础设施做出贡献。它将为萨尔大学纳米生物医学卓越战略提供支柱，授予两名申请人的战略补助金就证明了这一点。