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On/off blinking and switching behaviour of single molecules of green fluorescent protein

Nature volume 388, pages 355–358 (1997)Cite this article

Abstract

Optical studies of individual molecules at low and room temperature can provide information about the dynamics of local environments in solids, liquids and biological systems unobscured by ensemble averaging1,2,3,4,5,6,7,8,9,10,11,12,13,14. Here we present a study of the photophysical behaviour of single molecules of the green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria. Wild-type GFP and its mutant have attracted interest as fluorescent biological labels because the fluorophore may be formed in vivo15,16. GFP mutants immobilized in aereated aqueous polymer gels and excited by 488-nm light undergo repeated cycles of fluorescent emission (‘blinking’) on a timescale of several seconds—behaviour that would be unobservable in bulk studies. Eventually the individual GFP molecules reach a long-lasting dark state, from which they can be switched back to the original emissive state by irradiation at 405 nm. This suggests the possibility of using these GFPs as fluorescent markers for time-dependent cell processes, and as molecular photonic switches or optical storage elements, addressable on the single-molecule level.

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Figure 1: Bulk spectra of GFP mutants.
Figure 2: The switching behaviour of a single T203F molecule, illustrated by a series of ten consecutive experiments on the same molecule.
Figure 3: Traces of typical fluorescence intensity versus time for T203Y (a) and T203F (b) with an excitation intensity of 2,000 W cm−2.
Figure 4: Histograms of single-molecule autocorrelation times τc as a function of incident intensity; average autocorrelation times, τcavg, are calculated from the distributions.
Figure 5: Schematic diagram of the various states consistent with the experimental observations as described in the text.

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Acknowledgements

We thank L. S. B. Goldstein and S. Kummer for discussions. This work was supported by the NSF.

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Authors and Affiliations

  1. *Department of Chemistry and Biochemistry 0340, University of California San Diego, La Jolla, 92093-0340, California, USA

    Robert M. Dickson & W. E. Moerner

  2. †Aurora Biosciences, 11149 North Torrey Pines Road, La Jolla, 92037, California, USA

    Andrew B. Cubitt

  3. ‡Department of Pharmacology and Howard Hughes Medical Institute 0647, University of California San Diego, La Jolla, 92093-0647, California, USA

    Roger Y. Tsien

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  1. Robert M. Dickson
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  4. W. E. Moerner
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Correspondence to W. E. Moerner.

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Dickson, R., Cubitt, A., Tsien, R. et al. On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388, 355–358 (1997). https://doi.org/10.1038/41048

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