Rob Zondervan

Local viscosity of supercooled glycerol near Tg probed by rotational diffusion of ensembles and single dye molecules

We probe the rotational diffusion of a perylene dye in supercooled glycerol, 5–25 K above the glass-transition temperature (Tg = 190 K) at the ensemble and the single-molecule level. The single-molecule 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 Tg. 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.

Soft glassy rheology of supercooled molecular liquids

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 solid-like 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 (yield-stress, 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. The work presented here, combined with evidence for long-lived heterogeneity from previous single-molecule studies [Zondervan R, Kulzer F, Berkhout GCG, Orrit M (2007) Local viscosity of supercooled glycerol near Tg probed by rotational diffusion of ensembles and single dye molecules. Proc Natl Acad Sci USA 104:12628–12633], has a profound impact on the understanding of the glass transition because it casts doubt on the widely accepted assumption of the preservation of ergodicity in the supercooled state.

Spin polarization induced by optical and microwave resonance radiation in a Si vacancy in SiC: A promising subject for the spectroscopy of single defects

Depending on the temperature, crystal polytype, and crystal position, two opposite schemes have been observed for the optical alignment of the populations of spin sublevels in the ground state of a Si vacancy in SiC upon irradiation with unpolarized light at frequencies of zero-phonon lines. A giant change by a factor of 2–3 has been found in the luminescence intensity of zero-phonon lines in zero magnetic field upon absorption of microwave radiation with energy equal to the fine-structure splitting of spin sublevels of the vacancy ground state, which opens up possibilities for magnetic resonance detection at a single vacancy.

Single Biomolecules at Cryogenic Temperatures: From Structure to Dynamics

Elucidating the dynamics of proteins remains a central and daunting challenge of molecular biology. In our contribution we discuss the relevance of low- temperature observations not only to structure, but also to dynamics, and thereby to the function of proteins. We first review investigations on light-harvesting complexes to illustrate how increased photostability at low temperatures and spectral selection provide a deeper insight into the excitonic interactions of the chromophores and the dynamics of the protein scaffold. Furthermore, we introduce a novel technique that achieves controlled, reproducible temperature cycles of a microscopic sample on microsecond timescales. We discuss the potential of this technique as a tool to achieve repeatable single-molecule freeze-trapping and to overcome some of the limitations of single-molecule experiments at room temperature.

Non-exponential Kinetics of Photoblinking and Photobleaching of Rhodamine 6G in Polyvinylalcohol

Photobleaching and photoblinking have proven to be the main bottleneck for single-molecule microscopy and spectroscopy at room temperature. Here, a quantitative ensemble study of the kinetics of photoblinking and photobleaching at room temperature of a typical fluorescent label, Rhodamine 6G, in a polar, hydrogen bonding, solid matrix of polyvinylalcohol is presented as a function of the excitation intensity and the presence of oxygen. To achieve uniform irradiation of all molecules present in the excitation focus, the sample (2.0 x 10–5 M R6G in PVA spin-coated on a quartz substrate) is covered by a pinhole array mask with holes of diameter 40 ∝m, each addressable as an individual sample. The experiments are performed at intensities between 65 mW/cm2 and 320 W/cm2 and the measured emissivity of the system is normalized to that at 65 mW/cm2. The emissivity is shown to decrease by a factor of up to 20 at high intensity indicating the presence of a dark state, which would lead to photoblinking of single molecules. The triplet state of R6G cannot be this dark state, as it is hardly populated at excitation intensities below 1 kW/cm2. However, our data suggest that this state might be an intermediate between the singlet excited state and the dark state, which could be for instance a radical. Fig. 1 shows long-term fluorescence traces obtained at room temperature in an air atmosphere, displaying photobleaching. The rates governing blinking and bleaching are found to be widely distributed. Bleaching is shown to be more efficient in air, while blinking is more pronounced in the nitrogen atmosphere, because the lifetime of the dark state becomes longer.

Perspectives for variable-temperature investigations of single-protein dynamics

The conformational dynamics of proteins, a key issue in molecular biology, are characterized by the existence of a complex energy landscape, leading to a potentially huge number of possible folding routes for a given protein. This presents the experimenter with the challenge of following simultaneous rapid transitions between many different conformational states. Fluorescent labeling allows one to conduct optical experiments on individual protein molecules which naturally avoids the difficulties associated with unsynchronized ensembles. However, single-molecule experiments on biomolecules at room temperature only give access to their dynamics on a limited timescale