Ring TIRF microscope with FRAP and ablation

我们申请了一种全内反射荧光（TIRF）显微镜，在光漂白（FRAP）和激光消融后荧光恢复。显微镜允许低背景，高对比度成像近表面事件在不同的体外和纤维素系统。通过消除失焦照明，TIRF显微镜提高了信噪比，并允许单分子成像。传统的点TIRF系统存在不均匀的照明和干扰模式，而环形TIRF技术通过在圆上快速旋转激发光来确保均匀的照明，从而平均任何干扰模式，产生更高质量的图像，并允许申请人要求的定量荧光测量。saarbr<e:1> cken校区唯一可用的TIRF显微镜已经被六个研究小组使用，而且它不包括高质量TIRF成像所必需的环形TIRF技术。相比之下，这里介绍的设置将允许定量荧光测量，从而在现有系统上呈现重要的技术进步。环形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，甚至在单分子水平。在过去，我们使用环状TIRF来揭示细胞骨架动力学和功能的新方面：我们表明-与普遍看法相反-微管不仅在其尖端动态，而且沿着远离尖端的轴也是动态的，导致微管细胞骨架领域的重大进展，并展示了环状TIRF显微镜的潜力。在未来，我们打算使用环形TIRF显微镜继续研究细胞骨架过程以及其他细胞和亚细胞尺度上的近表面事件。除了TIRF成像，为了更好地了解体外和亚细胞过程，申请人需要确定体外和细胞内分子的流动性。FRAP功能允许在用户定义的区域灵活、可控的光漂白，从而满足这一需求。此外，消融功能允许通过主动和精确的样品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，如细胞骨架细丝，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saarbr<e:1> cken校区对近表面荧光显微镜日益增长的需求，还可以使先进的TIRF显微镜适用于定量成像，并提供最先进的图像操作工具。因此，显微镜将为促进科学进步的基础设施做出贡献。它将为萨尔大学的卓越纳米生物医学战略提供一个支柱，正如向两名申请人颁发的战略补助金所证明的那样。

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.