Journal
NATURE METHODS
Volume 10, Issue 5, Pages 421-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH.2411
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Funding
- US National Institutes of Health [5RO1EB010244-3, 5R01GM096450-02]
- Human Frontier Science Program [R01NS043915, DP1OD003930-03]
- Swiss National Science Foundation
- Agency of Science, Technology and Research (A*STAR) of Singapore
- Jane Coffin Childs
- Molecular Biophysics Training Grant Agency
- US National Institutes of Health-National Institute of General Medical Sciences [T32 GM008313]
- US National Science Foundation [ECS-0335765]
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Imaging single fluorescent proteins in living mammalian cells is challenged by out-of-focus fluorescence excitation. To reduce out-of-focus fluorescence we developed reflected light-sheet microscopy (RLSM), a fluorescence microscopy method allowing selective plane illumination throughout the nuclei of living mammalian cells. A thin light sheet parallel to the imaging plane and close to the sample surface is generated by reflecting an elliptical laser beam incident from the top by 90 with a small mirror. The thin light sheet allows for an increased signal-to-background ratio superior to that in previous illumination schemes and enables imaging of single fluorescent proteins with up to 100-Hz time resolution. We demonstrated the single-molecule sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determining the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor-alpha (ERER), which permitted us to resolve different modes of DNA binding of GR. We demonstrated two-color single-molecule imaging by observing the spatiotemporal colocalization of two different protein pairs. Our single-molecule measurements and statistical analysis revealed dynamic properties of transcription factors.
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