Florian Kulzer

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.

Single molecules as optical nanoprobes for soft and complex matter.

The optical signals of single molecules provide information about structure and dynamics of their nanoscale environment, free from space and time averaging. These new data are particularly useful whenever complex structures or dynamics are present, as in polymers or in porous oxides, but also in many other classes of materials, where heterogeneity is less obvious. We review the main uses of single molecules in studies of condensed matter at nanometer scales, especially in the fields of soft matter and materials science. We discuss several examples, including the orientation distribution of molecules in crystals, rotational diffusion in glass-forming molecular liquids, polymer studies with probes and labeled chains, porous and heterogeneous oxide materials, blinking of single molecules and nanocrystals, and the potential of surface-enhanced Raman scattering for local chemical analysis. All these examples show that static and dynamic heterogeneities and the spread of molecular parameters are much larger than previously imagined.

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.

Photothermal detection of individual gold nanoparticles: perspectives for high-throughput screening.

We use photothermal microscopy to detect and image individual gold nanoparticles that are either embedded in a polymer film or immobilized in an aqueous environment. Reducing the numerical aperture of the detection optics allows us to achieve a 200-fold-enlarged detection volume while still retaining sufficient detectivity. We characterize the capabilities of this approach for the detection of gold colloids with a diameter of 20 nm, with emphasis on practical aspects that are important for high-throughput-screening applications. The extended detection volume in combination with the stability of the photothermal signal are major advantages compared to fluorescence-based approaches, which are limited by photoblinking and photobleaching. Careful consideration is given to the trade-off between the maximum increase in local temperature that can be tolerated by a biological specimen and the minimum integration time needed to reliably determine whether a given volume contains a target species. We find that our approach has the potential to increase the detection-limited flow rate (i.e. the limit given by the detection volume divided by the minimum detection time) by two to three orders of magnitude.

Local refractive index probed via the fluorescence decay of semiconductor quantum dots.

We present a novel approach for convenient tuning of the local refractive index around nanostructures. We apply this technique to study the influence of the local refractive index on the radiative decay time of CdSe/ZnS quantum dots with three distinct emission wavelengths. The dependence of the luminescence decay time on the environment is well described by an effective medium approach. A critical distance of about 80 nm is found for the determination of the effective local index of refraction. An estimation for the emitting-state quantum efficiency can be extracted.

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.

Nondestructive Encapsulation of CdSe/CdS Quantum Dots in an Inorganic Matrix by Pulsed Laser Deposition

We report the successful encapsulation of colloidal quantum dots in an inorganic matrix by pulsed laser deposition. Our technique is nondestructive and thus permits the incorporation of CdSe/CdS core/shell colloidal quantum dots in an amorphous yttrium oxide matrix (Y2O3) under full preservation of the advantageous optical properties of the nanocrystals. We find that controlling the kinetic energy of the matrix precursors by means of the oxygen pressure in the deposition chamber facilitates the survival of the encapsulated species, whose well-conserved optical properties such as emission intensity, luminescence spectrum, fluorescence lifetime, and efficiency as single-photon emitters we document in detail. Our method can be extended to different types of nanoemitters (e.g., nanorods, dots-in-rods, nanoplatelets) as well as to other matrices (oxides, semiconductors, metals), opening up new vistas for the realization of fully inorganic multilayered active devices based on colloidal nano-objects.

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