Single-molecule imaging of transcription factor binding to DNA in live mammalian cells

Abstract

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 two different protein pairs. The combination of our single molecule measurements and statistical analysis reveals dynamic properties of transcription factors in live mammalian cells.

Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy

“…However, due to the steric hindrance that arises from placing high numerical aperture objectives in close proximity for narrow optical sectioning, these studies were limited to only very large cells or cell clusters. We recently circumvented this constraint by reflecting the light sheet off a miniature mirror placed next to the cell to be imaged (21). This reflected light-sheet (RLS) microscopy technique allows single-molecule imaging with superior signal-to-background ratio in the nucleus of a live, normal-sized mammalian cell at faster than video rate.…”

“…Here, we develop an SRM technique combining reflected light-sheet illumination (21) and superresolution microscopy (RLS-SRM) to probe the spatial organization of RNAP II in mammalian cell nuclei. Leveraging on the blinking photophysics of rhodamine-based dyes, we also develop a density-based clustering algorithm that pools multiple localizations in superresolution images based on their spatial and temporal proximity in order to accurately count the copy number of RNAP II molecules in transcription foci.…”

“…Among the various approaches, stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM) take advantage of the ability to detect single fluorescent probes attached to biomolecules while they transiently reside in their fluorescent "on" state and subsequently reconstruct a location map of their centroid positions (17)(18)(19). Although STORM/PALM is easier to implement with total internal reflection or highly inclined illumination schemes (20), the former has limited penetration depth whereas the latter has limited signal-to-background ratio due to auto-fluorescence background and out-of-focus fluorescent molecules, especially in highly dense structures (21).…”

Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

“…et al [55]. AFM cantilever lightsheet (by Gebhardt, J.C. et al [11]), lattice light-sheet (by Chen B.C. et al [56]), multi-focus (by Abrahamsson S. et al [57].…”

“…Previously, single-molecule fluorescence microscopy has been used to study TF localization in living cells across a range of model organisms, including bacteria, yeast and multi-cellular organisms [10–16]. Many studies suggest complexities in diffusion and binding [4,11,12,15,17] which may include intersegmental transfer [4,11,17]. However, until now, the direct experimental evidence for intersegmental transfer has been limited.…”