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-α (ER), 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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We acknowledge W. Min for his contribution in the early stage of this work. The pLV-tetO-Oct4 plasmid was provided by K. Hochedlinger (Howard Hughes Medical Institute and Harvard Stem Cell Institute), and MD2G and PAX2 plasmids were provided by D. Trono (Ecole Polytechnique Fédérale de Lausanne). We acknowledge funding from the US National Institutes of Health (X.S.X. (5RO1EB010244-3 and 5R01GM096450-02) and T.M. (R01NS043915 and DP1OD003930-03)), Human Frontier Science Program (J.C.M.G.), fellowship for advanced researchers from the Swiss National Science Foundation (D.M.S.), Jane Coffin Childs postdoctoral fellowship (R.R.), National Science Scholarship from the Agency of Science, Technology and Research (A*STAR) of Singapore (Z.W.Z.) and Molecular Biophysics Training Grant Agency, US National Institutes of Health–National Institute of General Medical Sciences T32 GM008313 (A.R.C.). This work was performed in part at the Harvard Center for Nanoscale Systems, a member of the National Nanotechnology Infrastructure Network, which is supported by the US National Science Foundation under award ECS-0335765.
The authors declare no competing financial interests.
Supplementary Figures 1–10 and Supplementary Tables 1 and 2 (PDF 12902 kb)
The vertical arrangement of illumination and detection objectives combined with a small mirror next to the cell enables selective plane illumination of the cell nucleus with a thin sheet of light. z sectioning is achieved by piezo-driven movement of the sample. Common glass bottom cell culture dishes can be used to grow and image the cells. (MOV 2828 kb)
SNAP protein, labeled with TMR, expressed in an MCF-7 cell and imaged by RLSM. TMR was excited with a 560-nm laser. The video shows 200 frames taken at 33 Hz. (MOV 8845 kb)
Halo protein, labeled with TMR, expressed in a U2-OS cell and imaged by RLSM. TMR was excited with a 560-nm laser. The video shows 200 frames taken at 33 Hz. (MOV 8535 kb)
mEos2 fluorescent protein, expressed in a MCF-7 cell and imaged by RLSM. mEos2 was activated with a 405-nm laser, and excited with a 560-nm laser. The video shows 200 frames taken at 28 Hz. (MOV 7271 kb)
YPet fluorescent protein, expressed in a U2-OS cell and imaged by RLSM. YPet was excited with a 514-nm laser. The video shows 200 frames taken at 20 Hz. (MOV 8229 kb)
Glucocorticoid receptor fused to mEos2, expressed in an MCF-7 cell in the presence of 100 nM dexamethasone and imaged by RLSM. mEos2 was activated with a 405-nm laser and excited with a 560-nm laser. The video shows 200 frames taken at 100 Hz. (MOV 8518 kb)
Glucocorticoid receptor fused to YPet, expressed in an MCF-7 cell in the presence of 100 nM dexamethasone and imaged by RLSM. YPet was excited with a 514-nm laser. The video shows 200 frames taken at 20 Hz. (MOV 4610 kb)
GRIP1 fused to EGFP and GCR fused to TagRFP-T, expressed in a U2-OS cell in the presence of 100 nM dexamethasone and imaged by RLSM. Excitation alternated every 50 ms between a 488-nm laser and a 560-nm laser. For better representation, successive EGFP (false-color green) and TagRFP-T (false-color red) images were overlaid. The red arrowhead highlights a GCR-GRIP1 colocalization event; the blue arrowhead highlights a GRIP1 binding event. The video shows 100 frames (50 overlaid frames) at 20 Hz. (MOV 2224 kb)
CLOCK fused to EGFP and BMAL1 fused to TagRFP-T, expressed in a U2-OS cell and imaged by RLSM 24 h after serum shock. Excitation alternates every 50 ms between a 488-nm laser and a 560-nm laser. For better representation, successive EGFP (false-color green) and TagRFP-T (false-color red) images were overlaid. The red arrowhead highlights a BMAL1-CLOCK colocalization event; the blue arrowhead highlights a CLOCK binding event. The video shows 100 frames (50 overlaid frames) at 20 Hz. (MOV 3025 kb)
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Gebhardt, J., Suter, D., Roy, R. et al. Single-molecule imaging of transcription factor binding to DNA in live mammalian cells. Nat Methods 10, 421–426 (2013). https://doi.org/10.1038/nmeth.2411
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