Ring TIRF microscope with FRAP and ablation

我们申请了一个全内反射荧光（TIRF）显微镜与荧光恢复后光漂白（FRAP）和激光烧蚀。该显微镜允许低背景，高对比度成像的近表面事件在不同的体外和cellulo系统。通过消除离焦照明，TIRF显微镜提高了信噪比，并允许单分子成像。虽然传统的点TIRF系统受到不均匀照明和干涉图案的影响，但是环形TIRF技术通过使激发光在圆上快速旋转来确保均匀照明，从而使任何干涉图案平均化，产生更高质量的图像并且允许如申请人所要求的定量荧光测量。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使用，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。环TIRF设置提供了独特的，强大的特性，使定量，近表面荧光显微镜，即使在单分子的水平。在过去，我们使用环TIRF揭示细胞骨架动力学和功能的新方面：我们表明-与普遍的看法相反-微管不仅在其尖端是动态的，而且沿着远离尖端的轴，导致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，我们打算采用环TIRF显微镜继续调查细胞骨架的过程，以及其他，近表面的细胞和亚细胞尺度上的事件。除了TIRF成像，为了实现对体外和亚细胞过程的充分理解，申请人需要确定体外和细胞中分子的迁移率。FRAP功能允许在用户定义的区域进行灵活、可控的光漂白，从而满足这一需求。此外，消融功能允许通过对样品的主动和精确操作超越单纯的观察实验：通过破坏或切断体外和亚细胞元件，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。我们申请的显微镜不仅可以满足萨尔布吕肯校区对近表面荧光显微镜日益增长的需求，还可以实现适用于定量成像的先进TIRF显微镜，并提供最先进的光操纵工具。因此，显微镜将有助于促进科学进步的基础设施。它将为萨尔兰大学纳米生物医学卓越战略提供支柱，正如授予两名申请人的战略赠款所证明的那样。

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.