Advanced Confocal Laser-Scanning-Microscope

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In this proposal we apply for funding of a state-of-the-art, fast and highly sensitive confocal laser scanning microscope (CLSM), with spatial (super)resolution beyond the diffraction limit and spectral detection. Both research units at the faculty Biology, Chemistry and Pharmacy and the integrated core unit BioSupraMol Optical Microscopy of Freie Universität Berlin will highly benefit from installation of this instrument as it will permit fast, but highly sensitive image acquisition relying on a high pixel dwell time for quantitative densitometric measurements in 3D and for high-throughput screening of biological samples and tissues, without affecting the signal-to-noise ratio. The novel detection technology will improve the lateral, and even more the axial resolution, permitting the detection of objects beyond the diffraction limit of classical confocal microscopes. The versatility of the instrument is reflected by a high scanning speed in combination with an improved signal-to-noise ratio and enhanced spatial resolution, so that fast in situ screenings of subcellular nano-structures (below 200 nm) even in large volumes are possible. The highly sensitive detector elements permit simultaneous spectral detection of more than ten fluorophores in the visible spectrum. The system relies on direct de-mixing of the different fluorophores in situ and therefore offers not only efficient multichannel imaging with a single scan, but also elimination of (chlorophyll-) autofluorescences. In addition, the novel system will provide the possibility to detect molecule interactions via Förster Resonance Energy Transfer (FRET), either by spectral FRET or by Flurorescence Lifetime Imaging (FLIM). Importantly, the system will bridge a substantial gap at the optical microscopy units at Freie Universität Berlin, as no other system currently combines high speed and superresolution with spectral demixing. The new microscope will be incorporated in the DFG funded Core Facility BioSupraMol and be located in the laboratories of the research building SupraFAB which is optimized for majors instrumentations.