Single Molecule sensitive CLSM for FLIM FRET

单分子荧光显微镜已成为一种成熟的技术，申请人帮助塑造了许多方法学的发展。特别是，荧光共振能量转移实验和荧光共振能量转移实验允许生物分子复合物和光子结构的复杂相互作用的可视化。在这种情况下，荧光寿命起着至关重要的作用。它是一个正交参数，与强度无关，这意味着作为一个强度变量，它可以提供更精确和更强大的见解。所要求的仪器是关于单分子共聚焦技术的瑞士军刀。它将允许申请人的实验室和整个慕尼黑社区以高通量进行复杂的单分子研究。申请人在他的博士论文中建立了荧光寿命成像，并在许多应用中使用了这种方法。在过去的五年中，在20多个同行评审的出版物中使用了侧重于荧光寿命的单分子测量。到目前为止，申请人的实验室已经建立了自己的共聚焦荧光显微镜设备，并在方法开发中发挥了形成性作用。与此同时，动态开发已经完成，商业供应商的产品在功能和灵活性方面已经赶上。此外，商业仪器在工程性能、自动化和系统集成方面具有优势。在这里，我们申请了一个共聚焦，激光扫描显微镜优化的单分子检测和众多的单分子光谱应用，如单分子测量的解决方案或表面上，荧光共振能量转移（FRET）显微镜，荧光相关光谱（FCS），特别是荧光寿命成像（FLIM）的基础上的时间相关的单光子计数。此外，单分子测量应该具有高通量和高自动化，以便单分子测量可以常规进行，而不仅仅是由专家和方法开发人员进行。为了实现与宽视场显微镜相同的吞吐量，将需要用于固定化单分子的自动聚焦和自主检测例程。

Single-molecule fluorescence microscopy has become an established technique with the applicant helping to shape numerous methodological developments. In particular, multicolor experiments and experiments with fluorescence resonance energy transfer allow the visualization of complex interactions of biomolecular complexes and photonic structures. In this context, fluorescence lifetime plays a crucial role. It is an orthogonal parameter and intensity-independent, which means that as an intensive variable it can provide more precise and robust insights. The requested instrument is a Swiss army knife regarding single-molecule confocal techniques. It will allow the applicant’s laboratory and the entire community in Munich to perform sophisticated single-molecule investigations at high throughput. The applicant established fluorescence lifetime imaging in his PhD thesis and has used this method in numerous applications. In the last five years, single molecule measurements focusing on fluorescence lifetime were used in over 20 peer-reviewed publications. So far, the applicant’s laboratory has built its own confocal fluorescence microscopy equipment and has played a formative role in method development. Meanwhile, the dynamic development has been completed and products from commercial suppliers have caught up in functionality and flexibility. Further, commercial instruments have advantages in terms of engineering performance, automation, and system integration. Here, we apply for a confocal, laser-scanning microscope optimized for single molecule detection and for numerous single-molecule spectroscopic applications such as single molecule measurements in solutions or on surfaces, fluorescence resonance energy transfer (FRET) microscopy, fluorescence correlation spectroscopy (FCS), and especially fluorescence lifetime imaging (FLIM) based on time-correlated single photon counting. Furthermore, multicolor measurements should be possible with high throughput and high automation, so that single-molecule measurements can be performed routinely, not only by specialists and method developers. To achieve the same throughput as a widefield microscope, autofocus and autonomous detection routines for immobilized single molecules will be required.