Large biological molecules including RNA, DNA and proteins have been studied at the ensemble level for many years in disciplines like biochemistry and molecular biology.Over the last few years, technological advances have opened up the field of optical single-molecule biophysics. In our experiments, pairs of dye molecules with distinct spectra are site-specifically attached to a host DNA molecule. Fluorescence from the dye molecules is coupled by Fluorescence Resonance Energy Transfer (FRET), a strongly distance-dependent, near-field dipole coupling of fluorophores with overlapping spectra. The ratio of fluorescence intensities from the dyes is strongly dependent on the distance between them (in the few nanometer range), so that this ratio reports on the overall conformation of the larger DNA molecule. Recent experiments have pioneered the use of single-pair FRET for observing conformational fluctuations and reaction kinetics in individual biological molecules [1-3]

In our group, we apply techniques from quantum optics to the observation of individual dye-labeled biomolecules, particularly DNA. Above is a diagram and a photograph of our experimental apparatus for observing single-molecule fluoresence. We use a home-built confocal microscope, image fluorescence light onto single-photon counters, and record data at Time-Interval Analyzer (TIA) boards. We measure statistics of fluorescence photon arrival times from FRET-coupled dye pairs. These photon statistics depend not only on the photophysics of the FRET coupling, but also on the underlying molecular dynamics which determine the conformation and local chemical environment of the dye pair. These statistical measurements are useful since the dynamics we are interested in observing (fluorescence, conformational fluctuations, diffusion, reaction kinetics) are non-deterministic random processes.

To the left is an example of autocorrelation and cross-correlation measurements of the fluorescence from pairs of dye molecules tethered to a host DNA molecule and coupled by FRET: these high time-resolution measurements reveal some properties of the fluorescent dyes as well as characteristic timescales of fast molecular fluctuations [4]. One of the goals of our research is a quantititave understanding of the flow of information from underlying stochastic dynamics to photon arrival time measurement records.

There are several biophysics experiments in progress in our group, including development of novel algorithms for statistical estimation in biomolecular systems [5], high time-resolution, single-particle tracking [6], measurement of sequence-dependent elasticity of double-stranded DNA (with R. Phillips), observation of stochastic kinetics in DNA hybridization (with N. Pierce and E. Winfree). Many of the experimental and theoretical techniques in these experiments overlap with traditional techniques from quantum optics: lasers, optics, electronics, quantum mechanics, probability/statistics, and stochastic processes.

(last updated 7/2004)

References

Group publications