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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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Models of the production of cosmogenic nuclides typically incorporate an adjustable production rate parameter that is scaled for variations in production with latitude and altitude. In practice, this production rate parameter is set by calibration of the model using cosmogenic nuclide data from sites with independent age constraints. In this paper, we describe a calibration procedure developed during the Cosmic-Ray Produced Nuclide Systematics on Earth (CRONUS-Earth) project and its application to an extensive data set that included both new CRONUS-Earth samples and samples from pre

We have studied the concentrations of 10Be and 26Al in quartz crystals extracted from glacially polished granitic surfaces from the Sierra Nevada range. These surfaces were identified with the glacial advance during the Tioga period ∼11,000 years ago. Our measurements yield the most accurate estimates to date for the absolute production rates of these nuclides in SiO2 due to cosmic ray nucleons and muons for geomagnetic latitudes 43.8°–44.6°N and altitudes 2.1–3.6 km. The estimates are relatively free from uncertainties in snow cover since we studied a suite of rock surfaces inclined 0°–75° with respect to the horizontal. The principal uncertainty arises due to the lack of a precise date for the glacial retreat event, about ±10%. The 26Al/10Be ratio at production (6.0) is determined more accurately since the exposure age of the samples is 2 orders of magnitude smaller than the mean lives of the two nuclides. Production rates of 10Be and 26Al in quartz at other latitudes and altitudes in the troposphere can be determined from the present measurements by scaling, using the known altitude and latitude dependence of cosmic ray fluxes of nucleons and negative muons. Knowledge of the production rates of these nuclides is a prerequisite for their application in erosion and geomorphological studies.