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letters to nature 358 NATURE | VOL 388 | 24 JULY 1997 nucleotides encoding an amino-terminal polyhistidine tag. Sequence changes were introduced by site-directed mutagenesis using the Bio-Rad mutagenesis kit 28 and confirmed by sequencing. The recombinant proteins were expressed in the bacterial strain BL21(DE3) after induction with IPTG (0.5 mM) at room temperature and purified by Ni affinity chromatography. T203F/T203Y imaging. Samples were prepared by the methods of ref. 21 from 10 −10 M solutions of protein diluted in 1 mg ml −1 BSA. Polyacrylamide gels (T ¼ 15%, C ¼ 5% without SDS) were prepared in pH 7 phosphatebuffered saline doped with protein (here T is the total concentration of monomer in g per 100 ml, C is the wt% of total monomer which is crosslinker, and SDS indicates sodium dodecyl sulphate). The gel host provided pore sizes small enough for convenient (and complete) immobilization of each protein molecule, while maintaining its naturally fluorescent, native conformation 21,29 . Excitation with a 488-nm laser (100-2,000 W cm −2 at the gel/coverslip interface) occurred in the total-internal-reflection geometry; the emission was imaged with a Nikon inverted microscope (250-nm resolution) with an Omega 535DF55 filter and a Princeton Instruments intensified frame transfer CCD (100 ms time resolution, 90 s collection time). Oxygen was not removed from samples for which data is shown, but samples prepared with ϳ10 min helium bubbling showed similar on/off behaviour. 405-nm irradiation was produced by a Hg arc lamp with line filter through the epi-illumination port (1 W cm −2 ). The linear increase of detected photons as a function of laser intensity (100-2,000 W cm −2 ) indicated that saturation and multiphoton processes were negligible in these studies. Typical detected count rates of 5,000-6,000 photons s −1 at 2,000 W cm −2 pumping intensity (ϳ150,000 excitations s −1 ) were achieved, with most of the molecules emitting several millions of photons without irreversible bleaching. Autocorrelation analysis. We define the autocorrelation function, C(t), for discrete data points;where I is the average intensity, t is the time summed from 0 to N 100-ms intervals, and I(t) is the time-dependent fluorescence intensity. Confidence limits were generated on the autocorrelations such that any values within the limits were consistent with zero 30 . Exponential fits of the autocorrelations were generated only for the statistically significant portions of the curves beyond the short time correlation spike arising from band-limited noise.

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