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Simultaneous, coincident optical trapping and single-molecule fluorescence

Nature Methods volume 1, pages 133–139 (2004)Cite this article

Abstract

We constructed a microscope-based instrument capable of simultaneous, spatially coincident optical trapping and single-molecule fluorescence. The capabilities of this apparatus were demonstrated by studying the force-induced strand separation of a dye-labeled, 15-base-pair region of double-stranded DNA (dsDNA), with force applied either parallel ('unzipping' mode) or perpendicular ('shearing' mode) to the long axis of the region. Mechanical transitions corresponding to DNA hybrid rupture occurred simultaneously with discontinuous changes in the fluorescence emission. The rupture force was strongly dependent on the direction of applied force, indicating the existence of distinct unbinding pathways for the two force-loading modes. From the rupture force histograms, we determined the distance to the thermodynamic transition state and the thermal off rates in the absence of load for both processes.

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Figure 1: Schematic optical layout of the instrument (not all components displayed).
Figure 2: Simultaneous traces of force (red) and photon counts (blue) recorded for four experimental geometries (insets).
Figure 3: Cartoon of the experimental geometry for both pulling geometries.
Figure 4: Control trace demonstrating complete overlap of excitation, detection and trapping beams.
Figure 5: Histograms of unbinding and shearing forces with fits to the probability distribution function for breakage force developed by Evans and Ritchie28 (see Supplementary Note online).

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Acknowledgements

We thank the entire Block lab for helpful discussions and J. Shaevitz for assistance with energy landscape calculations. This work was supported by grants to S.M.B. from the National Institutes of Health. P.M.F. acknowledges support from a National Science Foundation predoctoral fellowship and a Lieberman Award Fellowship; A.M.E. was supported by the Stanford Biophysics Training Grant from the National Institutes of Health; and M.J.L. was supported by a postdoctoral fellowship from the Jane Coffin Childs Foundation.

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Author notes

  1. Matthew J Lang

    Present address: Biological Engineering and Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts, 02139-4307, USA

  2. Matthew J Lang and Polly M Fordyce: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, Stanford University, Stanford, 94305, California, USA

    Matthew J Lang, Keir C Neuman & Steven M Block

  2. Department of Applied Physics, Stanford University, Stanford, 94305, California, USA

    Matthew J Lang, Keir C Neuman & Steven M Block

  3. Department of Physics, Stanford University, Stanford, 94305, California, USA

    Polly M Fordyce

  4. Graduate Program in Biophysics, Stanford University, Stanford, 94305, California, USA

    Anita M Engh

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Correspondence to Steven M Block.

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Lang, M., Fordyce, P., Engh, A. et al. Simultaneous, coincident optical trapping and single-molecule fluorescence. Nat Methods 1, 133–139 (2004). https://doi.org/10.1038/nmeth714

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