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Proceedings Paper

STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA (presentation video)
Author(s): Iddo Heller; Gerrit Sitters; Onno D. Broekmans; Géraldine Farge; Carolin Menges; Wolfgang Wende; Stefan W. Hell; Erwin J.G. Peterman; Gijs J. Wuite
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Paper 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.

Paper Details

Date Published: 14 November 2014
PDF: 1 pages
Proc. SPIE 9164, Optical Trapping and Optical Micromanipulation XI, 916416 (14 November 2014); doi: 10.1117/12.2062819
Show Author Affiliations
Iddo Heller, Vrije Univ. Amsterdam (Netherlands)
Gerrit Sitters, VU Univ. Amsterdam (Netherlands)
Onno D. Broekmans, VU Univ. Amsterdam (Netherlands)
Géraldine Farge, Vrije Univ. Amsterdam (Netherlands)
Carolin Menges, Justus-Liebig-Univ. Giessen (Germany)
Wolfgang Wende, Justus-Liebig-Univ. Giessen (Germany)
Stefan W. Hell, Max-Planck-Institut für Biophysikalische Chemie (Germany)
Erwin J.G. Peterman, Vrije Univ. Amsterdam (Netherlands)
Gijs J. Wuite, Vrije Univ. Amsterdam (Netherlands)

Published in SPIE Proceedings Vol. 9164:
Optical Trapping and Optical Micromanipulation XI
Kishan Dholakia; Gabriel C. Spalding, Editor(s)

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