Single-molecule spectral fluctuations at room temperature

Abstract

RECENT advances in near-field1and far-field2,3 fluorescence microscopy have made it possible to image single molecules and measure their emission3,4 and excitation5 spectra and fluorescence lifetimes3,6–8 at room temperature. These studies have revealed spectral shifts4 and intensity fluctuations6,7, the origins of which are not clear. Here we show that spontaneous fluctuations in the spectra of immobilized single dye molecules occur on two different timescales: hundreds of milliseconds and tens of seconds, indicating that these fluctuations have two distinct activation energies. In addition, we see photoinduced spectral fluctuations on repeated photoexcitation of single molecules. We suggest that all of these fluctuations can be understood as transitions between metastable minima in the molecular potential-energy surface.