TIRF microscope for single-molecule FRET applications

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Observing the action of biomolecular machines at the single-molecule level has revolutionized the understanding of countless biological processes. Prominent examples are the hand-over-hand walking mechanism of kinesin motors along microtubules or the rotation of the ATP synthase. Whereas conventional fluorescence microscopy techniques have the potential to reveal large-scale movements of biomolecules along linear polymers such as cytoskeletal components or DNA, the application of single-molecule fluorescence resonance energy transfer (smFRET) has furthermore enabled the detection of dynamic movements within individual macromolecules at the low-nanometer scale.Here, we request funds for the acquisition of a total internal reflection (TIRF) microscope to study the dynamics of molecular assemblies that control the three-dimensional organization of eukaryotic genomes by single-molecule microscopy methods. The laboratories that contribute to this proposal share a central interest in protein machines that dynamically bind chromatin to alter its topology and, concomitantly, its function during cell proliferation; including transcription factor assemblies that account for long-range promoter-enhancer DNA contacts or motor complexes that enzymatically drive the extrusion of DNA loops. Dissecting the intricate mechanisms of these molecular machines requires a methodology not only with the temporal and spatial resolution to follow individual events over time, but also with the capability to simultaneously detect conformational changes.The requested instrument will be able to resolve single molecules labelled with different fluorophores at millisecond timescale and enable time-resolved, dynamic measurements of molecular conformations by smFRET. Sensitive detection of emission for smFRET experiments and biologically delicate samples with an EMCCD camera is complemented by an sCMOS camera that allows the acquisition of a fourfold larger area at the same resolution whenever higher photon yields (>20 photons/pixel) are achieved. A motorized TIRF module allows for switching between TIRF and highly inclined and laminated optical sheet (HILO) illumination of individual laser lines to fine-tune the depth of imaging. A temperature-controlled box enables imaging at temperatures relevant to diverse model systems, including human cell lines.