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Mobility Analysis of Super‐Resolved Proteins on Optically Stretched DNA: Comparing Imaging Techniques and Parameters
Author(s) -
Heller Iddo,
Sitters Gerrit,
Broekmans Onno D.,
Biebricher Andreas S.,
Wuite Gijs J. L.,
Peterman Erwin J. G.
Publication year - 2014
Publication title -
chemphyschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.016
H-Index - 140
eISSN - 1439-7641
pISSN - 1439-4235
DOI - 10.1002/cphc.201300813
Subject(s) - sted microscopy , dna , biological system , optical tweezers , temporal resolution , confocal , biophysics , diffusion , microscopy , confocal microscopy , fluorescence lifetime imaging microscopy , resolution (logic) , chemistry , image resolution , superresolution , fluorescence , physics , computer science , optics , artificial intelligence , biology , thermodynamics , biochemistry , image (mathematics)
Fluorescence microscopy in conjunction with optical tweezers is well suited to the study of protein mobility on DNA. Here, we evaluate the benefits and drawbacks of super‐resolution and conventional imaging techniques for the analysis of one‐dimensional (1D) protein diffusion as commonly observed for DNA‐binding proteins. In particular, we demonstrate the visualization of DNA‐bound proteins using wide‐field, confocal, and stimulated emission depletion (STED) microscopy. We review the suitability of these techniques to conditions of high protein density, and quantify their performance in terms of spatial and temporal resolution. Tracking proteins on DNA forces one to make a choice between localization precision on the one hand, and the number and rate of localizations on the other, by altering imaging modality, excitation intensity, and acquisition rate. Using simulated diffusion data, we quantify the effect of these imaging conditions on the accuracy of 1D diffusion analysis. In addition, we consider the case of diffusion confined between local roadblocks, a case particularly relevant for proteins bound to DNA. Together these results provide guidelines that can assist in judiciously optimizing the experimental conditions required for the analysis of protein mobility on DNA and other 1D systems.