High resolution Microscope for Multi-parameter Fluorescence Image Spectroscopy

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Fluorescence spectroscopy and imaging are important biophysical techniques to study the structure and dynamics of fluorescently labeled biomolecules and bridge these with their function under in vitro conditions or in living cells. To this end, we rely on the unique capabilities of recently developed fluorescence-based spectroscopy and imaging techniques in combination with multi-parameter detection that give maximum information and selectivity together with spatial resolution. The proposed microscope consists of two closely connected modules: (1) a confocal laser scanning unit with an ultimate dynamic range in time (picoseconds to minutes), and (2) a total internal reflection unit for super-resolution microscopy. Distances between two dyes on the molecular scale between 2 and 15 nm can be resolved by Förster Resonance Energy Transfer (FRET) measurements. If the inter-dye distances exceed 15 nm, super-resolution microscopy with single-molecule localization and colocalization analysis is most appropriate. For closing the gaps in time and space in fluorescence spectroscopy and imaging, we want to perform performing multi-modal microscopy on the same sample. We will realize a seamless implementation of a combination of FRET spectroscopy and super-resolution microscopy. In this way, we achieve molecular resolution and map structural and dynamic features of the biomolecular assemblies over a wide range of length scales. For gaining the maximum of information from the fluorescence signal, we want to probe more degrees of freedom in biomolecular dynamics by FRET. While the same distance distributions are obtainable from three two-color FRET experiments, information about the correlation of distance changes and thus the coordination of molecular movements is only obtained using three-color FRET. This is possible since three-color FRET experiments contain information about the co-occurrence of distances for the individual FRET pairs. In the proposed setup, we want to study single immobilized biomolecules under in vitro conditions and to perform seamless super-resolution FRET microscopy of biomolecules in a cellular context. To characterize single-molecule reactions, we want to employ microfluidics to shift reversible equilibria and to trigger reactions of immobilized molecules by adding ligands or binding partner or by varying reaction conditions (buffer, ionic strength, pH). The confocal module will be used to register single-molecule fluorescence intensity traces of single immobilized molecules for several colors with an ultimate time resolution. In addition, we will employ super-resolution FRET microscopy to study processes in biomolecular systems such as (1) Monitoring the membrane translocation and chaperone-dependent folding of a lipase, (2) Mapping functional molecules of innate immune defence on different length scales and (3) Deciphering the cellular signal initiation determinants of the apoptosis signaling complex.