Single-molecule spectrometer with multi-parameter fluorescence detection

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The Ebbinghaus research group investigates how proteins fold in the cell and how they retain their functionality in space (i.e. spatially inside mammalian cells) and time (i.e. age-dependent changes). The group further investigates disease states in which proteins aggregate and phase separate. Therefore, novel techniques are developed and utilized with which kinetic and thermodynamic properties of biomolecules can be assessed directly in vivo. The requested instrument will allow to measure protein folding on nanosecond to millisecond timescales in the test tube and the cell. It will further allow to access the full stability curve of a protein in its native cellular environment. Such measurements will allow to analyze how different physicochemical factors (such as crowding, confinement, hydration) or biological factors (such as chaperones, cell stresses) modulate folding kinetics and thermodynamics (including the enthalpic and entropic contributions to the standard free energy of folding) in cellular health and disease conditions. This information cannot be obtained with any other available technique and will be crucial to the success of the current and planned research projects in the group. Specifically, the planned instrument encompasses a single molecule spectrometer with multi-parameter fluorescence detection (e.g. FCS, FCCS, FLCS, nsFCS, smFRET, anisotropy, FLIM) and fast infrared laser heating. A multi-laser setup for pulsed interleaved excitation as well as a multi-detector setup for simultaneous recording of multiple fluorophores in different polarizations is required. The Walla research group will specifically use this configuration for pairwise cross-correlational analysis of multi-protein complexes and develop a general scheme for higher-order multi-correlation fluorescence microscopy. To perform live cell experiments at different temperatures a stable incubation system is required.