Regensburg 2013 – scientific programme
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BP: Fachverband Biologische Physik
BP 13: Imaging
BP 13.2: Talk
Tuesday, March 12, 2013, 10:00–10:15, H44
Probing Transcription Factor DNA Binding at the Single Molecule Level in Live Mammalian Cells — •J. Christof M. Gebhardt1,4, David M. Suter1,4, Rahul Roy1,2, Ziqing W. Zhao1, Alec Chapman1, Srinjan Basu1,3, Tom Maniatis3, and X. Sunney Xie1 — 1Harvard University, Cambridge MA, USA — 2Indian Institute of Science, Bangalore, India — 3Columbia University Medical Center, New York NY, USA — 4equal contribution
Imaging single fluorescent proteins in living mammalian cells is challenging due to out-of-focus fluorescence excitation by common microscopy schemes. We report the development of a novel fluorescence microscopy method, reflected light sheet microscopy (RLSM), which allows selective plane illumination throughout the nucleus of living mammalian cells, for reducing out-of-focus fluorescence signal. Generation of a thin light sheet parallel to the imaging plane and close to the sample surface is achieved by reflecting an elliptical laser beam incident from the top by 45° with a small mirror. The thin light sheet allows for an increased signal-to-background ratio superior to previous illumination schemes and enables imaging of single fluorescent proteins with up to 100 Hz time resolution. We demonstrate the sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determine the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor (ER), enabling us to resolve different modes of DNA binding of GR. Finally, we demonstrate two-color single molecule imaging by observing the spatio-temporal co-localization of interacting protein pairs.