14 November 2014 STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA (presentation video)
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Abstract
Dense coverage of DNA by proteins is a ubiquitous feature of cellular processes such as DNA organization, replication and repair. We present a single-molecule approach capable of visualizing individual DNA-binding proteins on densely covered DNA and in the presence of high protein concentrations. Our approach combines optical tweezers with multicolor confocal and stimulated emission depletion (STED) fluorescence microscopy. Proteins on DNA are visualized at a resolution of 50 nm, a sixfold resolution improvement over that of confocal microscopy. High temporal resolution (<50 ms) is ensured by fast one-dimensional beam scanning. Individual trajectories of proteins translocating on DNA can thus be distinguished and tracked with high precision. We demonstrate our multimodal approach by visualizing the assembly of dense nucleoprotein filaments with unprecedented spatial resolution in real time. Experimental access to the force-dependent kinetics and motility of DNA-associating proteins at biologically relevant protein densities is essential for linking idealized in vitro experiments with the in vivo situation.
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Iddo Heller, Iddo Heller, Gerrit Sitters, Gerrit Sitters, Onno D. Broekmans, Onno D. Broekmans, Géraldine Farge, Géraldine Farge, Carolin Menges, Carolin Menges, Wolfgang Wende, Wolfgang Wende, Stefan W. Hell, Stefan W. Hell, Erwin J.G. Peterman, Erwin J.G. Peterman, Gijs J. Wuite, Gijs J. Wuite, } "STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA (presentation video)", Proc. SPIE 9164, Optical Trapping and Optical Micromanipulation XI, 916416 (14 November 2014); doi: 10.1117/12.2062819; https://doi.org/10.1117/12.2062819
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