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Single-molecule imaging of non-equilibrium molecular ensembles on the millisecond timescale

Nature Methods volume 13, pages 341344 (2016) | Download Citation

Abstract

Single-molecule fluorescence microscopy is uniquely suited for detecting transient molecular recognition events, yet achieving the time resolution and statistics needed to realize this potential has proven challenging. Here we present a single-molecule imaging and analysis platform using scientific complementary metal-oxide semiconductor (sCMOS) detectors that enables imaging of 15,000 individual molecules simultaneously at millisecond rates. This system enabled the detection of previously obscured processes relevant to the fidelity mechanism in protein synthesis.

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Acknowledgements

We thank J. Munro and P. Geggier for contributing to the design and implementation of analysis software; F. Sachs and C. Nicholi for help using QuB; P. Schultz (Scripps) for the Escherichia coli S12 knockout strain; Q. Zheng for preparing labeled DNA duplexes; and J. Sims, D. Butts, M. Feldman, F. Huang and the members of the Blanchard lab for helpful discussions. This work was supported by the US National Institutes of Health (grants 1R01GM079238 and 1R01GM098859 to S.C.B.). M.F.J. was supported by a postdoctoral fellowship from the German Academic Exchange Service (DAAD).

Author information

Author notes

    • Manuel F Juette
    •  & Daniel S Terry

    These authors contributed equally to this work.

Affiliations

  1. Department of Physiology and Biophysics, Weill-Cornell Medical College, New York, New York, USA.

    • Manuel F Juette
    • , Daniel S Terry
    • , Michael R Wasserman
    • , Roger B Altman
    • , Zhou Zhou
    • , Hong Zhao
    •  & Scott C Blanchard

Authors

  1. Search for Manuel F Juette in:

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  3. Search for Michael R Wasserman in:

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  5. Search for Zhou Zhou in:

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Contributions

M.F.J. and D.S.T. designed and implemented the sCMOS TIRF (total internal reflection fluorescence) microscope and analyzed the data. M.F.J. and M.R.W. performed smFRET experiments. D.S.T. designed and implemented the analysis software, with help from S.C.B., M.F.J. and M.R.W. M.F.J. designed and implemented data-acquisition software. M.R.W. developed the S12 ribosome-labeling strategy. M.R.W., R.B.A. and M.F.J. prepared ribosome and smFRET reagents. Z.Z. and H.Z. synthesized fluorophores. All authors contributed to experimental design and writing of the manuscript.

Competing interests

S.C.B. and R.B.A. have an equity interest in Lumidyne Technologies.

Corresponding author

Correspondence to Scott C Blanchard.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–3 and Supplementary Table 1

Zip files

  1. 1.

    Supplementary Software 1

    Compiled MATLAB software for smFRET data analysis, including detailed documentation. Requires 64-bit Windows.

  2. 2.

    Supplementary Software 2

    Compiled DLL (written in C) for conversion of Hamamatsu raw image format to BigTIFF with example client code for LabVIEW 8.2 or higher. Requires 64-bit Windows.

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DOI

https://doi.org/10.1038/nmeth.3769

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