Confocal Laser Scanning Microscope (CLSM) with Fluorescence Lifetime Imaging Microscopy (FLIM)

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Protein/protein or protein/nucleic acid (DNA or RNA) interaction play a fundamental role in the understanding of complex cell- and molecular biological networks. Colocalization studies between two or more fluorescently tagged interaction partners could indicate potential molecular interaction, but are limited by the resolution of light (250 nm lateral, 500 nm axial or deconvolved 125 nm lateral, 200 nm axial). In order to discriminate between local colocalization and molecular interaction (distances of less than 10 nm), we would like to apply for equipment to establish the FLIM-FRET technique (Fluorescence Lifetime Imaging Microscopy - Förster Resonanz Energie Transfer) at IMB’s microscopy core facilities. FLIM has emerged as a powerful tool for the characterization of molecular interaction and the sensing of local environmental conditions. FLIM directly measures the lifetime of the excited state of fluorophores (fluorescence lifetime). Fluorescence lifetime is very sensitive not only to the local microenvironment of the fluorophore (pH, temperature), but also to energy transfer processes such as FRET. Combined with the recent technological advances in FLIM, this allows for much higher imaging speeds, making FLIM the most suitable technique for probing molecular interaction quantitatively via FLIM-FRET in high-spatial and temporal resolution. The Institute of Molecular Biology (IMB) provides excellent equipped Core Facilities and offers service to IMB scientists as well as the scientific community in Mainz. The Microscopy and Histology Core Facility (MHCF) offers access to different high-tech microscopes. We would like to provide the scientists at IMB, as well as researchers from the university medical center and the Johannes Gutenberg University, access to the latest technique in the field of confocal microscopy for their research and teaching. There are quite a few exciting innovations in the field, e.g. graphic card (GPU) supported deconvolution algorithms that provide deconvolution on the fly or improved laser techniques and detector sensitivity. FLIM offers additional functions like improved image quality by removing autofluorescence, multiplexing of fluorophores with overlapping spectra, measuring molecular interaction or assessing cellular metabolism with FRET sensors. The requested confocal laser scanning microscope with fluorescence lifetime imaging microscopy (FLIM) should replace two confocal microscopes that are already 10 years old. With a usage time of more than 2000 annual hours, the current confocal systems are our working horses. We would like to keep one of the current confocal systems to reduce the workload.