Ring TIRF microscope with FRAP and ablation

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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.