Single Molecule sensitive CLSM for FLIM FRET

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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.