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.

我们申请了全内反射荧光(TIRF)显微镜，具有光漂白(FRAP)和激光消融后的荧光恢复。该显微镜允许在不同的体外和细胞系统中对近表面事件进行低背景、高对比度成像。通过消除焦外照明，TIRF显微镜提高了信噪比，并允许进行单分子成像。虽然传统的点式TIRF系统存在照明不均匀和干涉图案的问题，但环形TIRF技术通过在圆形上快速旋转激发光来确保照明均匀，从而平均消除任何干涉图案，产生更高质量的图像，并允许根据应用程序的要求进行定量荧光测量。萨尔布吕肯校区唯一可用的TIRF显微镜已经被六个研究小组满负荷使用，它不包括高质量TIRF成像所需的环形TIRF技术。相反，这里提供的设置将允许定量荧光测量，从而提供了比现有系统重要的技术进步。环形TIRF设置提供了独特而强大的特性，使定量、近表面荧光显微镜成为可能，甚至在单分子水平上也是如此。在过去，我们使用环TIRF来揭示细胞骨架动力学和功能的新方面：我们表明-与通常的信念相反-微管不仅在其顶端是动态的，而且沿着远离顶端的轴是动态的，这导致了微管细胞骨架领域的重大进展，并展示了环TIRF显微镜的潜力。在未来，我们打算使用环形TIRF显微镜来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，为了更好地了解体外和亚细胞过程，申请者还需要确定分子在体外和细胞内的流动性。FRAP功能允许在用户定义的区域中灵活、受控地进行光漂白，从而满足这一需求。此外，消融功能允许通过主动和精确地操作样本而不仅仅是观察实验：通过破坏或切断体外和亚细胞元素，如细胞骨架细丝，我们将能够直接探索这些元素的结构和功能方面。我们申请的显微镜不仅将满足萨尔布吕肯园区对近表面荧光显微镜日益增长的需求，还将使先进的TIRF显微镜能够适用于定量成像，并提供最先进的光操纵工具。因此，显微镜将为促进科学进步的基础设施做出贡献。它将为萨尔兰大学纳米生物医学卓越战略提供支柱，其中两名申请者获得的战略补助金就是明证。