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Supercooled liquids demonstrate stretched exponential relaxations consistent with the presence of spatially heterogeneous dynamics. Many experimental results are consistent with this picture, but differences in experimental approach may lead to different conclusions about the degree of heterogeneity in a given system. Here, we investigate whether observables accessible with single molecule (SM) approaches are consistent with each other and with ensemble measurements. In particular, the distribution of rotational relaxation times, τ(c), obtained from SM measurements is compared with the stretching exponent determined from a quasi-ensemble treatment of the same data. It is shown that the time-limited trajectories typical of SM experiments can lead to a stretching exponent that suggests homogeneous dynamics even in the presence of heterogeneous dynamics. After correction for the time-limited trajectories, additional discrepancy remains between stretching exponents measured via SM experiments and ensemble techniques. The remaining difference is attributed to the limited dynamic range of the SM experiments.

Spatially heterogeneous dynamics in supercooled glycerol over the temperature range 198 K (1.04T(g))-212 K (1.12T(g)) is investigated using widefield single molecule (SM) fluorescence microscopy. Measurements are performed using three different perylenedicarboximide probes to investigate whether probe size and probe-host interactions affect breadth of heterogeneity reported in the glassy host by such SM experiments. Rotational relaxation times of single probe molecules are measured, and for all probes, log-normal distributions of relaxation times are found. No significant change in relaxation time distribution as a function of temperature is evident for a given probe. However, across probes, probe rotational relaxation time is correlated with breadth of heterogeneous dynamics reported. Molecules that undergo changes in dynamics are identified using two complementary approaches that interrogate time scales between 10(3) and 10(6) τ(α), with τ(α) the structural relaxation time of glycerol. Exchange is found on the shortest time scales probed (~30 τ(c), with τ(c) the rotational correlation time of the probe) and is relatively temperature and probe independent. No evidence is found for additional exchange occurring on the longest time scales interrogated. Taken together with the fact that probes that rotate the fastest report the greatest breadth of spatially heterogeneous dynamics in the system, this indicates that exchange times reported from analysis of SM linear dichroism trajectories as described here are upper bounds on the average exchange time in the system.

Gelation temperatures from 22 degrees C to 37 degrees C were used to control the pore size of collagen matrices independent of collagen concentration. To limit cell exposure to temperatures lower than physiological temperature, the putative nucleation and growth mechanism of collagen was investigated to determine the time at which gel fibril and network structure becomes independent of temperature. It was found that the temperature dependent portion of collagen gelation ends close to the time at which fibrils first form a network spanning structure. These findings were then exploited to prepare cell-embedded gels nucleated at 22, 27, or 32 degrees C and then incubated at 37 degrees C. This achieves fibrillar and network structure characteristic of gels formed solely at the nucleation temperature. Proof of principle studies of glioma invasion in these gels suggested pore size is a key determinant of glioma invasive speed in collagen gels.

Single molecule fluorescence experiments, with their associated low signals, require very low background fluorescence in the sample. Even high purity liquids will often possess large numbers of fluorescent impurities that are difficult to completely remove through standard purification techniques such as distillation and recrystallization. We have constructed a simple setup in which such impurities can be photobleached before final sample preparation. The instrument consists of high power light emitting diodes, and it delivers almost 10 W of light to the sample without the heating associated with more conventional light sources or the cost and safety concerns associated with a high power laser.

We report the observation of imaging beyond the diffraction limit due to bound surface states in negative refraction photonic crystals. We achieve an effective negative index figure-of-merit [-Re(n)/Im(n)] of at least 380, ~125x improvement over recent efforts in the near-infrared, with a 0.4 THz bandwidth. Supported by numerical and theoretical analyses, the observed near-field resolution is 0.47 lambda, clearly smaller than the diffraction limit of 0.61 lambda. Importantly, we show this sub-diffraction imaging is due to the resonant excitation of surface slab modes, allowing refocusing of non-propagating evanescent waves.

Supercooled liquids are proposed to be dynamically heterogeneous, with regions exhibiting relaxation time scales that vary in space and time. Measurement of the distribution of such time scales could be an important test of various proposed theories of vitrification. Single molecule fluorescence experiments attempt to uncover this distribution, typically by embedding single molecule probes into these systems and monitoring their individual rotational relaxations from a computed autocorrelation function (ACF). These ACFs may exhibit stretched exponential decays, with the value of the stretching exponent assumed to report the set of dynamical environments explored by the probe. Here, we use simulated trajectories of rotation to investigate how the time scale of dynamic exchange relative to underlying relaxation time scales in the system affects probe ability to report the distribution relaxation of time scales present. We find that dynamically heterogeneous regions must persist for approximately 50 times the median relaxation time scale for a single molecule to accurately report the full distribution of time scales it has experienced. In systems with faster dynamic exchange, single molecule ACFs average over successive environments, limiting the reported heterogeneity of the system. This leads to degeneracies in stretching exponent for systems with different underlying relaxation time distributions. We show that monitoring single molecule median stretching exponent as a function of trajectory length or simultaneously measuring median stretching exponent and measured relaxation time distribution at a given trajectory length can resolve these degeneracies, revealing the underlying set of relaxation times as well as median exchange time.

We investigate by molecular dynamics simulations the validity of the frequency-dependent Stokes-Einstein (SE) relation in supercooled liquids at different temperatures. The results indicate that the SE relation holds at intermediate frequencies that correspond to the beta -relaxation and the onset of the alpha -relaxation regimes. Large deviations, which increase as the temperature decreases, are observed at frequencies well below the frequency at which the non-Gaussian parameter alpha2 is maximum. We argue that the breakdown of the SE relation in supercooled liquids arises from underestimation of the diffusion coefficient due to neglect of correlated motions.

We study the growth and invasion of glioblastoma multiforme (GBM) in three-dimensional collagen I matrices of varying collagen concentration. Phase-contrast microscopy studies of the entire GBM system show that invasiveness at early times is limited by available collagen fibers. At early times, high collagen concentration correlates with more effective invasion. Conversely, high collagen concentration correlates with inhibition in the growth of the central portion of GBM, the multicellular tumor spheroid. Analysis of confocal reflectance images of the collagen matrices quantifies how the collagen matrices differ as a function of concentration. Studying invasion on the length scale of individual invading cells with a combination of confocal and coherent anti-Stokes Raman scattering microscopy reveals that the invasive GBM cells rely heavily on cell-matrix interactions during invasion and remodeling.

We present actively phase-locked heterodyne detected fifth-order data. Nodal lines are found in several tensor elements, and these are also present in molecular dynamics simulations of the signal. The nodes are a result of anharmonic coupling between modes.

One of the major unsolved questions in ultrafast liquid dynamics is how diffusive dynamics (i.e., those involving structural change) emerge from the fluctuations of individual molecules around their equilibrium positions. This question is intimately related to that of energy flow between different degrees of freedom in the liquid and ultimately to the flow of energy between solutes and solvents which initiate chemical reactions and stabilize products once barriers are crossed. The challenge of developing a spectroscopic technique that can probe the intermolecular frequencies characteristic of liquid motions, and their couplings with each other, has proved technically demanding. The need for a nonresonant form of photon echo spectroscopy, capable of eliminating the ensemble average broadening inherent in linear spectroscopy, was recognized by Tanimura and Mukamel, who proposed a fifth-order Raman measurement, initially as a probe of spatial heterogeneity [1,2]. In the experiment, two pump pulses prepare a vibrational coherence. After a time t1 ,a second set of pulses transfers this vibrational coherence to a second vibrational coherence. After a time t2, the second coherence is probed. It was quickly realized that this measurement also probes couplings between different nuclear motions and thus possesses the potential to provide qualitatively new insights into the anharmonic dynamics of liquid motion. The technique has, in principle, wide applicability to condensed phase dynamical problems, such as the question of spatial heterogeneity of liquids near the glass transition, or determination of biopolymer structure (with femtosecond time resolution) through space coupling of specific vibrational modes. The full potential of nonlinear optical spectroscopy is realized only when the full signal field, rather than the signal intensity, is measured, since only then can sign changes be determined and linear combinations of different signals be created to characterize the response [3]. In this Letter, we report actively phase-locked heterodyne detection of the fifth-order Raman signal from liquid CS2. We believe that this is the first measurement to obtain the fully nonresonant R 5 response, avoiding complications from the presence of either R 3 and absorptive portions of third-order cascade

We have successfully used phase matching considerations to provide significant discrimination against cascaded lower-order responses and measured the direct fifth-order response from the intermolecular motions in liquid CS2. Here we report the measured polarized and magic angle responses and discuss comparisons with simulations.

Three dimensional in vitro approximations to extracellular matrix (ECM) are increasingly being used in biophysical experiments investigating cell behavior. One advantage to using collagen I gels as ECM approximations in such experiments is the ability to image the collagen fibers within the gel. This allows simultaneous imaging of cells and their local environment simultaneously with three dimensional (3D) resolution. In this study, both pure and fluorescently labeled (3% by weight) collagen gels are assembled over a range of concentrations (0.1, 0.5, 1.0, 2.0, 5.0, and 5.5 mg/mL) and at various temperatures (37, 32, 27, and 22°C). The networks are investigated using confocal reflectance microscopy (CRM) and confocal fluorescence microscopy (CFM). Comparison between CRM and CFM reveals that they are not equally sensitive to details of network structure, with CRM (CFM) displaying higher sensitivity to fibers perpendicular (parallel) to the optical axis. Furthermore, analysis of background signal in CFM images suggests the existence of small fibrillar structures that are not resolved by CRM. Despite these differences, image analyses performed on 2D slices of CFM and CRM images to quantify mesh size, number of fibers, and fiber length reveal identical trends as a function of gel concentration and gelation temperature. Fiber width approximated from both CRM and CFM is in good accord with fiber width determination using electron microscopy. Overall network structures (as quantified via mesh size, fiber number, fiber length and fiber width) are related to bulk mechanical properties measured by rheology. Finally, we demonstrate the ability to form collagen gels of varied mesh size at identical collagen concentration, all of which are compatible with cell health and 3D cell studies.

The rotational dynamics of three perylene diimide dyes are studied on the single molecule (SM) level in ortho-terphenyl (OTP) near the glass transition temperature (T(g)). At all temperatures probed, spanning 1.03-1.06 T(g), each of the three probes exhibits rotational correlation times, τ(c), that span more than a decade, consistent with the presence of spatially heterogeneous dynamics in OTP. No trend is found as a function of temperature, but a trend as a function of probe is observed: Average probe rotational correlation time scales inversely with breadth of SM τ(c) distribution, with faster probes exhibiting broader τ(c) distributions. This implies that dynamic exchange occurs on and below time scales associated with probe rotation. Extrapolating FWHM of rotational relaxation times to the structural relaxation time of the host shows that the τ(c) distribution would span nearly two decades in the limit of no probe temporal averaging. Comparison with SM measurements in glycerol suggests that OTP demonstrates a greater degree of spatially heterogeneous dynamics in this temperature range than does glycerol.

We quantify spatial and temporal heterogeneity in supercooled glycerol at T=T(g)+14 K employing a widefield detection scheme and using rubrene as the probe molecule. We describe how microscopy configuration affects measured intensity, linear dichroism, and the resulting autocorrelation function. Rotational relaxation times tau(c) of 241 probe molecules are measured, and we find spatial heterogeneity over almost three orders of magnitude present at this temperature. An approach for detecting temporally heterogeneous molecules and quantifying exchange times is introduced. Of molecules that can be assessed, approximately 15% display evidence of temporal heterogeneity-changes of tau(c) during the measurement-that are detected with the analysis technique employed. Exchanges between dynamically disparate environments occur rarely but in the proportion expected given the rarity of very slowly rotating molecules present. Heterogeneous molecules are characterized by persistence and exchange times. Median exchange and persistence times of the molecules identified as heterogeneous relative to glycerol's structural relaxation time tau(alpha) are found to be tau(ex)/tau(alpha)=202 and tau(pers)/tau(alpha)=405, respectively. These results are discussed in the context of values of exchange times that have been determined in other experiments.

The authors present results from molecular dynamics simulations on the effect of smooth and rough probes on the dynamics of a supercooled Lennard-Jones (LJ) mixture. The probe diameter was systematically varied from one to seven times the diameter of the large particles of the LJ mixture. Mean square displacements show that in the presence of a large smooth probe the supercooled liquid speeds up, while in the presence of a large rough probe, the supercooled liquid slows down. Non-Gaussian parameters indicate that with both smooth and rough probes, the heterogeneity of the supercooled system increases. From the analysis of local Debye-Waller factors, it is evident that the change in the dynamics of the LJ system is heterogeneous, with the largest perturbations close to the probes. Large smooth and rough probes appear to set up heterogeneities in these supercooled systems that would otherwise not occur, and these heterogeneities persist for long times.

Coherent anti-Stokes Raman scattering microscopy is performed on glassy systems of poly(methylmethacrylate) colloidal particles in density- and refractive-index-matched solvents. Samples are prepared with varying amounts of linear polystyrene, which induces a depletion driven attraction between the nearly hard-sphere particles. Images collected over several hours confirm the existence of a reentrant glass transition. The images also reveal that the dynamics of repulsive and attractive glasses are qualitatively different. Colloidal particles in repulsive glasses exhibit cage rattling and escape, while those in attractive glasses are nearly static while caged but exhibit large displacements upon (infrequent) cage escape.

Type I collagen gels are routinely used in biophysical studies and bioengineering applications. The structural and mechanical properties of these fibrillar matrices depend on the conditions under which collagen fibrillogenesis proceeds, and developing a fuller understanding of this process will enhance control over gel properties. Turbidity measurements have long been the method of choice for monitoring developing gels, whereas imaging methods are regularly used to visualize fully developed gels. In this study, turbidity and confocal reflectance microscopy (CRM) were simultaneously employed to track collagen fibrillogenesis and reconcile the information reported by the two techniques, with confocal fluorescence microscopy (CFM) used to supplement information about early events in fibrillogenesis. Time-lapse images of 0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml acid-solubilized collagen I gels forming at 27°C, 32°C, and 37°C were collected. It was found that in situ turbidity measured in a scanning transmittance configuration was interchangeable with traditional turbidity measurements using a spectrophotometer. CRM and CFM were employed to reveal the structures responsible for the turbidity that develops during collagen self-assembly. Information from CRM and transmittance images was collapsed into straightforward single variables; total intensity in CRM images tracked turbidity development closely for all collagen gels investigated, and the two techniques were similarly sensitive to fibril number and dimension. Complementary CRM, CFM, and in situ turbidity measurements revealed that fibril and network formation occurred before substantial turbidity was present, and the majority of increasing turbidity during collagen self-assembly was due to increasing fibril thickness.

We investigate the structural, dynamical, and rheological properties of colloid-polymer mixtures in a volume fraction range of φ=0.15-0.35. Our systems are density-matched, residual charges are screened, and the polymer-colloid size ratio is ∼0.37. For these systems, the transition to kinetically arrested states, including disconnected clusters and gels, coincides with the fluid-fluid phase separation boundary. Structural investigations reveal that the characteristic length, L, of the networks is a strong function of the quench depth: for shallow quenches, L is significantly larger than that obtained for deep quenches. By contrast, L is for a given quench depth almost independent of φ; this indicates that the strand thickness increases with φ. The strand thickness determines the linear rheology: the final relaxation time exhibits a strong dependence on φ, whereas the high frequency modulus does not. We present a simple model based on estimates of the strand breaking time and shear modulus that semiquantitatively describes the observed behavior. © 2010 The Society of Rheology.