We investigate the fluorescence intensity of rhodamine 6G in poly(vinyl alcohol) as a function of excitation intensity, illumination time, the presence of oxygen, and temperature. The variations in emissivity (or fluorescence brightness) are attributed to a dark state, which shows populating kinetics resembling those of the triplet state, but a much longer lifetime. We simulate the observed kinetics by a four-level model, in which a long-lived dark state is formed through the triplet as an intermediate state. The weak temperature dependence of the lifetime of the dark state points to electron tunneling as the main recovery process. This intermolecular mechanism also explains the observed broad distribution of lifetimes. An electron-spin-resonance experiment confirms the assignment of the dark state to a radical. For the first time, photoinduced charge transfer is identified as a source of blinking in single-molecule measurements.

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We probe the rotational diffusion of a perylene dye in supercooled glycerol, 5-25 K above the glass-transition temperature (T g ‫؍‬ 190 K) at the ensemble and the single-molecule level. The singlemolecule results point to a broad distribution of local viscosities that vary by a factor of five or more for different individual fluorophores at a given temperature. By following the same single molecules at various temperatures, we find that the distribution of local viscosities itself broadens upon approaching T g. This spatial heterogeneity is found to relax extremely slowly, persisting over hours or even days. These results convey a picture of heterogeneous liquid pockets separated by solid-like walls, which exist already well above the viscosimetric glass transition.fluorescence ͉ glass transition ͉ anisotropy ͉ correlation G lycerol is an archetypal molecular glass former. Although glycerol crystals can be grown from solution, the pure liquid readily supercools because of its high viscosity [10 3 times that of water at room temperature (1)], which is caused by the formation of a highly branched network of intermolecular hydrogen bonds. The viscosity of supercooled glycerol increases by 10 orders of magnitude between 291.8 K, the melting temperature of crystalline glycerol, and its viscosimetric glass-transition temperature T g ϭ 190 K. Throughout this range of temperatures, macroscopic rheology measurements seem consistent with the expected behavior of a Newtonian liquid, although they also reveal highly nonexponential kinetics (1-3).The necessity for cooperative motion at larger and larger scales on approaching the glass transition was recognized a long time ago by Adam and Gibbs (4). In their view, glass formation arises from dynamical arrest of cooperative domains, and their spread in sizes leads to an overall nonexponential relaxation. Indeed, spatially inhomogeneous dynamics (5) have been observed with a variety of techniques in many glass formers, including simple liquids (6-10) and polymers (11-15). Inhomogeneities have been found in the particular case of supercooled glycerol by dielectric hole burning (16), NMR (17), x-ray (18), light scattering (19), and stimulated Brillouin gain spectroscopy experiments (20, 21). These observations have to be reconciled with the common view of a supercooled liquid, which is supposed to remain a normal ergodic liquid until the glass transition. Heterogeneous regions with different relaxation dynamics are therefore expected to exchange with one another to restore ergodicity, by processes called environmental exchanges (6,9,10,12,(14)(15)(16). The typical mechanisms, length scales, and time scales of environmental exchanges remain largely unclear. NMR and dielectric relaxation point to times comparable to the molecular rotation times (16,22), whereas light scattering and fluorescence show inhomogeneities with exceedingly slow relaxation (9,12,(19)(20)(21).Single-molecule fluorescence is a well established method for studying rotational diffusion (23-26). Linear dichroism...

Photobleaching is a severely limiting factor in the optical study of single biomolecules. We investigate the photobleaching of rhodamine 6G (R6G) ensembles in poly(vinyl alcohol) (PVA) as a function of illumination time, excitation intensity, the presence of oxygen, and temperature. We observe nonexponential kinetics related to primary photobleaching through two dark statesthe triplet state and a radical anionand to secondary photobleaching after the optical excitation of those dark states. Reactions of the metastable states with oxygen can lead either to photoproducts or to a recovery of the ground state. Oxygen can therefore enhance or reduce photobleaching, depending on the experimental conditions. At low temperature, photobleaching is reduced although not completely suppressed. Despite the presence of the long-lived radical anion, we are able to observe single R6G molecules in PVA. At room temperature, only relatively bleaching-resistant molecules are resolved as individuals. At low temperature, the observation times become considerably longer. Our study shows that metastable states other than the triplet drastically affect photobleaching.

We probe the mechanical response of two supercooled liquids, glycerol and ortho-terphenyl, by conducting rheological experiments at very weak stresses. We find a complex fluid behavior suggesting the gradual emergence of an extended, delicate solidlike network in both materials in the supercooled state-i.e., above the glass transition. This network stiffens as it ages, and very early in this process it already extends over macroscopic distances, conferring all well known features of soft glassy rheology (yieldstress, shear thinning, aging) to the supercooled liquids. Such viscoelastic behavior of supercooled molecular glass formers is difficult to observe because the large stresses in conventional rheology can easily shear-melt the solid-like structure. glass transition ͉ glycerol ͉ ortho-terphenyl ͉ yield-stress

We demonstrate a novel technique to achieve fast thermal cycles of a small sample (a few femtoliters). Modulating a continuous near-infrared laser focused on a metal film, we can drive the local temperature from 130 to 300 K and back, within a few microseconds. By fluorescence microscopy of dyes in a thin glycerol film, we record images of the hot spot, calibrate its temperature, and follow its variations in real time. The temperature dependence of fluorescence anisotropy, due to photophysics and rotational diffusion, gives a steady-state temperature calibration between 200 and 350 K. From 200 to 220 K, we monitor temperature more accurately by fluorescence autocorrelation, a probe for rotational diffusion. Time-resolved measurements of fluorescence anisotropy give heating and cooling times of a few microseconds, short enough to supercool pure water. We designed our method to repeatedly cycle a single (bio)molecule between ambient and cryostat temperatures with microsecond time resolution. Successive measurements of a structurally relevant variable will decompose a dynamical process into structural snapshots. Such temperature-cycle experiments, which combine a high time resolution with long observation times, can thus be expected to yield new insights into complex processes such as protein folding.

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