STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA
- Heller, I.
- Sitters, G.
- Broekmans, O.D.
- Farge, G.A.
- Menges, C.
- Wende, W.
- Hell, S.W.
- Peterman, E.J.G.
- Wuite, G.J.L.
DOIAbstract
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...
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