Controlling morphological order of conjugated polymers over mesoscopic and microscopic scales could yield critical improvements in the performance of organic electronics. Here, we utilize a multimodal apparatus allowing for controlled solvent vapor annealing and simultaneous wide-field epifluorescence microscopy to demonstrate bottom-up growth of morphologically ordered anisotropic aggregates prepared from single poly(2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene) (MEH-PPV) chains, with length scales controllable from tens of nanometers to several micrometers. Preparation of micrometer-scale fiber aggregates that interconnect to form spanning networks is also demonstrated. We quantify aggregate physical and optical anisotropy, degree of quenching, and exciton diffusion characteristics as a function of aggregate size. The demonstration of controlled preparation of highly anisotropic aggregates provides a path for controlled postprocessing of organic thin films at length scales relevant to the operation of devices.

Fibrillar type I collagen-based hydrogels are commonly used in tissue engineering and as matrices for biophysical studies. Mechanical and structural properties of these gels are known to be governed by the conditions under which fibrillogenesis occurs, exhibiting variation as a function of protein concentration, temperature, pH, and ionic strength. Deeper understanding of how macroscopic structure affects viscoelastic properties of collagen gels over the course of fibrillogenesis provides fundamental insight into biopolymer gel properties and promises enhanced control over the properties of such gels. Here, we investigate type I collagen fibrillogenesis using confocal rheology—simultaneous confocal reflectance microscopy, confocal fluorescence microscopy, and rheology. The multimodal approach allows direct comparison of how viscoelastic properties track the structural evolution of the gel on fiber and network length scales. Quantitative assessment and comparison of each imaging modality and the simultaneously collected rheological measurements show that the presence of a system-spanning structure occurs at a time similar to rheological determinants of gelation. Although this and some rheological measures are consistent with critical gelation through percolation, additional rheological and structural properties of the gel are found to be inconsistent with this theory. This study clarifies how structure sets viscoelasticity during collagen fibrillogenesis and more broadly highlights the utility of multimodal measurements as critical test-beds for theoretical descriptions of complex systems.

In a polymeric material near its glass transition temperature, segmental dynamics of a given spatial region may differ considerably from that of neighboring regions without apparent structural origin, analogous to the supercooled liquid state of low molecular weight glass formers. Given that the supercooled liquid state is a (metastable) equilibrium state, spatial variations in dynamics are expected to average out in time, consistent with ergodicity of the system. By probing the rotations of fluorescent guest molecules, local segmental dynamics of polystyrene was scrutinized molecule by molecule. Two perylene dicarboximide dyes were investigated as potential reporters, and one of these was found to report a substantial proportion of the dynamic heterogeneity of the host polystyrene. Using this probe, we demonstrate that the polystyrene system is ergodic and characterize time scales over which molecules experience changes to their dynamics. We identify a characteristic time scale of exchange much longer than the structural relaxation of the host segmental dynamics, consistent with both previous studies on polystyrene and studies on small molecule glass formers. Moreover, we show that dynamic exchange spans a wide range of time scales from <150 to ∼35 000 times the segmental relaxation time of the polystyrene.

The relationship between photostability and conformation of 2-methoxy-5-(2′-ethylhexyloxy)-1,4-phenylenevinylene (MEH-PPV) conjugated polymers was studied via excitation polarization modulation depth (M) measurements. Upon partial photobleaching, M distributions of collapsed, highly ordered MEH-PPV molecules shifted toward lower values. Conversely, M distributions of MEH-PPV molecules with random coil conformations moved toward higher values after partial photobleaching. Monte Carlo simulations of randomly distributed dipole moments along polymer chains subjected to partial photobleaching revealed that a statistical effect leads to an increase in peak M value. Decreases in M values seen experimentally in the population of MEH-PPV molecules with high M values, however, are due to conformation-dependent photostability within single MEH-PPV polymers. We show that, while folded MEH-PPV molecules are relatively more photostable than extended MEH-PPV molecules in an ensemble, extended portions of particular molecules are more photostable than folded domains within single MEH-PPV molecules.

The concept of dynamic heterogeneity and the picture of the supercooled liquid as a mosaic of environments with distinct dynamics that interchange in time have been invoked to explain the nonexponential relaxations measured in these systems. The spatial extent and temporal persistence of these regions of distinct dynamics have remained challenging to identify. Here, single-molecule fluorescence measurements using a probe similar in size and mobility to the host o-terphenyl unambiguously reveal exponential relaxations distributed in time and space and directly demonstrate ergodicity of the system down to the glass transition temperature. In the temperature range probed, at least 200 times the structural relaxation time of the host is required to recover ensemble-averaged relaxation at every spatial region in the system.

To thoroughly elucidate how molecular conformation and photophysical properties of conjugated polymers (CPs) are related requires simultaneous probing of both. Previous efforts used fluorescence imaging with one nanometer accuracy (FIONA) to image CPs, which allowed simultaneous estimation of molecular conformation and probing of fluorescence intensity decay. We show that calculating the molecular radius of gyration for putative folded and unfolded poly(2-methoxy-5-(2′-ethylhexyloxy)1,4-phenylenevinylene) (MEH-PPV) molecules using FIONA underestimates molecular extension by averaging over emitters during localization. In contrast, employing algorithms based on single molecule high resolution imaging with photobleaching (SHRImP), including an approach we term all-frames SHRImP, allows localization of individual emitters. SHRImP processing corroborates that compact MEH-PPV molecules have distinct photophysical properties from extended ones. Estimated radii of gyration for isolated 168 kDa MEH-PPV molecules immobilized in polystyrene and exhibiting either stepwise or continuous intensity decays are found to be 12.6 and 25.3 nm, respectively, while the distance between exciton recombination sites is estimated to be ∼10 nm independent of molecular conformation.Keywords: conjugated polymer; emission site localization; exciton recombination; FIONA; fluorescence imaging; MEH-PPV; SHRImP; super-resolution microscopy;

Much of the interesting behavior that has been observed in supercooled liquids appears to be related to dynamic heterogeneity, the presence of distinct dynamic environments – with no apparent underlying structural basis – in these systems. To most directly interrogate these environments, proposed to span regions just a few nanometers across, molecular length scale probes are required. Single molecule fluorescent microscopy was introduced to the field a decade ago and has provided strong evidence of dynamic heterogeneity in supercooled systems. However, only more recently has the full set of challenges associated with interpreting results of these experiments been described. With a fuller understanding of these challenges in hand, single molecule measurements can be employed to provide a more precise picture of dynamic heterogeneity in supercooled liquids and other complex systems. In this tutorial review, experimental and data analysis details are presented for the most commonly employed single molecule approach to studying supercooled liquids, the measurement of rotational dynamics of single molecule probes. Guidance is provided in experimental set-up and probe selection, with a focus on choices that affect data interpretation and probe sensitivity to dynamic heterogeneity.

Monitoring single molecule probe rotations is an increasingly common approach to studying dynamics of complex systems, including supercooled liquids. Even with advances in fluorophore design and detector sensitivity, such measurements typically exhibit low signal to noise and signal to background ratios. Here, we simulated and analyzed orthogonally decomposed fluorescence signals of single molecules undergoing rotational diffusion in a manner that mimics experimentally collected data of probes in small molecule supercooled liquids. The effects of noise, background, and trajectory length were explicitly considered, as were the effects of data processing approaches that may limit the impact of noise and background on assessment of environmental dynamics. In many cases, data treatment that attempts to remove noise and background were found to be deleterious. However, for short trajectories below a critical signal to background threshold, a thresholding approach that successfully removed data points associated with noise and spared those associated with signal allowed for assessment of environmental dynamics that was as accurate and precise as would be achieved in the absence of noise.

Spatially heterogeneous dynamics in supercooled glycerol over the temperature range 198 K (1.04Tg) − 212 K (1.12Tg) 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 103 and 106τα,with τα the structural relaxation time of glycerol. Exchange is found on the shortest time scales probed (∼30 τc, with τc 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.

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, τc, 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.Keywords: glass transition; rotational relaxation; simulations; single molecule; supercooled liquids;

Microcapsules fabricated by layer-by-layer self-assembly have unique physicochemical properties that make them attractive for drug delivery applications. This study chiefly investigated the biocompatibility of one of the most stable types of microcapsules, those composed of poly-(sodium 4-styrene sulfonate) [PSS] and poly-(allylamine hydrocholoride) [PAH], with cells cultured on two-dimensional (2D) substrates and in three-dimensional (3D) matrices. C6 glioma and 3T3 fibroblast cell morphology was observed after 24 h of co-culture with PSS/PAH microcapsules on a 2D substrate. Cells were also cultured with four other types of microcapsules, each composed of at least one naturally occurring polyelectrolyte. At microcapsule to cell ratios up to 100:1, it was found that PSS/PAH microcapsules do not affect number of viable cells more substantially than do the other microcapsules investigated. However, differences in number of viable cells were found as a function of microcapsule composition, and our results suggest particular biochemical interactions between cells and internalized microcapsules, rather than mechanical effects, are responsible for these differences. We then investigated the effects of PSS/PAH microcapsules on cells embedded in 3D collagen matrices, which more closely approximate the tumor environments in which microcapsules may be useful drug delivery agents. Matrix structure, cell invasion, and volumetric spheroid growth were investigated, and we show that these microcapsules have a negligible effect on cell invasion and tumor spheroid growth even at high concentration. Taken together, this work suggests that PSS/PAH microcapsules have sufficiently high biocompatibility with at least some cell lines for use as proof of principle drug delivery agents in in vitro studies.

Invasive breast cancer and other tumors of epithelial origin must breach a layer of basement membrane (BM) that surrounds the primary tumor before invading into the adjacent extracellular matrix. To analyze invasive strategies of breast cancer cells during BM breaching and subsequent invasion into a collagen I-rich extracellular matrix (ECM), we developed a physiologically relevant 3D in vitro model that recreates the architecture of a solid tumor with an intact, degradable, cell-assembled BM layer embedded in a collagen I environment. Using this model we demonstrate that while the BM layer fully prevents dissemination of non-malignant cells, cancer cells are capable of breaching it and invading into the surrounding collagen, indicating that the developed system recreates a hallmark of invasive disease. We demonstrate that cancer cells exhibiting individual invasion in collagen matrices preferentially adopt a specific mode of collective invasion when transmigrating a cell-assembled BM that is not observed in any other tested fibrillar, non-fibrillar, or composite ECM. Matrix-degrading enzymes are found to be crucial during BM breaching but not during subsequent invasion in the collagen matrix. It is further shown that multicellular transmigration of the BM is less susceptible to pharmacological MMP inhibition than multicellular invasion in composite collagen/basement membrane extract matrices. The newly developed in vitro model of metastasis allows 3D cancer cell invasion to be studied not only as a function of a particular tumor's genetics but also as a function of its heterogeneous environment and the different stages of invasion. As such, this model is a valuable new tool with which to dissect basic mechanisms of invasion and metastasis and develop new therapeutic approaches in a physiologically relevant, yet inexpensive and highly tunable, in vitro setting.

Invasive breast cancer and other tumors of epithelial origin must breach a layer of basement membrane (BM) that surrounds the primary tumor before invading into the adjacent extracellular matrix. To analyze invasive strategies of breast cancer cells during BM breaching and subsequent invasion into a collagen I-rich extracellular matrix (ECM), we developed a physiologically relevant 3D in vitro model that recreates the architecture of a solid tumor with an intact, degradable, cell-assembled BM layer embedded in a collagen I environment. Using this model we demonstrate that while the BM layer fully prevents dissemination of non-malignant cells, cancer cells are capable of breaching it and invading into the surrounding collagen, indicating that the developed system recreates a hallmark of invasive disease. We demonstrate that cancer cells exhibiting individual invasion in collagen matrices preferentially adopt a specific mode of collective invasion when transmigrating a cell-assembled BM that is not observed in any other tested fibrillar, non-fibrillar, or composite ECM. Matrix-degrading enzymes are found to be crucial during BM breaching but not during subsequent invasion in the collagen matrix. It is further shown that multicellular transmigration of the BM is less susceptible to pharmacological MMP inhibition than multicellular invasion in composite collagen/basement membrane extract matrices. The newly developed in vitro model of metastasis allows 3D cancer cell invasion to be studied not only as a function of a particular tumor's genetics but also as a function of its heterogeneous environment and the different stages of invasion. As such, this model is a valuable new tool with which to dissect basic mechanisms of invasion and metastasis and develop new therapeutic approaches in a physiologically relevant, yet inexpensive and highly tunable, in vitro setting.

We quantitatively compare data obtained from imaging two-dimensional slices of three-dimensional unlabeled and fluorescently labeled collagen gels with confocal reflectance microscopy (CRM) and/or confocal fluorescence microscopy (CFM). Different network structures are obtained by assembling the gels over a range of concentrations at various temperatures. Comparison between CRM and CFM shows that the techniques are not equally sensitive to details of network structure, with CFM displaying higher fidelity in imaging fibers parallel to the optical axis. Comparison of CRM of plain and labeled collagen gels shows that labeling itself induces changes in gel structure, chiefly through inhibition of fibril bundling. Despite these differences, image analyses carried out on two-dimensional CFM and CRM slices of collagen gels reveal identical trends in structural parameters as a function of collagen concentration and gelation temperature. Fibril diameter approximated from either CRM or CFM is in good accord with that determined via electron microscopy. Two-dimensional CRM images are used to show that semiflexible polymer theory can relate network structural properties to elastic modulus successfully. For networks containing bundled fibrils, it is shown that average structural diameter, rather than fibril diameter, is the length scale that sets the magnitude of the gel elastic modulus.

In this work, the gelation of three-dimensional collagen and collagen/hyaluronan (HA) composites is studied by time sweep rheology and time lapse confocal reflectance microscopy (CRM). To investigate the complementary nature of these techniques, first collagen gel formation is investigated at concentrations of 0.5, 1.0, and 1.5 mg/mL at 37°C and 32°C. The following parameters are used to describe the self-assembly process in all gels: the crossover time (tc), the slope of the growth phase (kg), and the arrest time (ta). The first two measures are determined by rheology, and the third by CRM. A frequency-independent rheological measure of gelation, tg, is also measured at 37°C. However, this quantity cannot be straightforwardly determined for gels formed at 32°C, indicating that percolation theory does not fully capture the dynamics of collagen network formation. The effects of collagen concentration and gelation temperature on kg, tc, and ta as well as on the mechanical properties and structure of these gels both during gelation and at equilibrium are elucidated. Composite collagen/HA gels are also prepared, and their properties are monitored at equilibrium and during gelation at 37°C and 32°C. We show that addition of HA subtly alters mechanical properties and structure of these systems both during the gelation process and at equilibrium. This occurs in a temperature-dependent manner, with the ratio of HA deposited on collagen fibers versus that distributed homogeneously between fibers increasing with decreasing gelation temperature. In addition to providing information on collagen and collagen/HA structure and mechanical properties during gelation, this work shows new ways in which rheology and microscopy can be used complementarily to reveal details of gelation processes.

In this work, a cellular Potts model based on the differential adhesion hypothesis is employed to analyze the relative importance of select cell-cell and cell-extracellular matrix (ECM) contacts in glioma invasion. To perform these simulations, three types of cells and two ECM components are included. The inclusion of explicit ECM with an inhomogeneous fibrous component and a homogeneously dispersed afibrous component allows exploration of the importance of relative energies of cell-cell and cell-ECM contacts in a variety of environments relevant to in vitro and in vivo experimental investigations of glioma invasion. Simulations performed here focus chiefly on reproducing findings of in vitro experiments on glioma spheroids embedded in collagen I gels. For a given range and set ordering of energies associated with key cell-cell and cell-ECM interactions, our model qualitatively reproduces the dispersed glioma invasion patterns found for most glioma cell lines embedded as spheroids in collagen I gels of moderate concentration. In our model, we find that invasion is maximized at intermediate collagen concentrations, as occurs experimentally. This effect is seen more strongly in model gels composed of short collagen fibers than in those composed of long fibers, which retain significant connectivity even at low density. Additional simulations in aligned model matrices further elucidate how matrix structure dictates invasive patterns. Finally, simulations that allow invading cells to both dissolve and deposit ECM components demonstrate how Q-Potts models may be elaborated to allow active cell alteration of their surroundings. The model employed here provides a quantitative framework with which to bound the relative values of cell-cell and cell-ECM interactions and investigate how varying the magnitude and type of these interactions, as well as ECM structure, could potentially curtail glioma invasion.

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.

Spatially heterogeneous dynamics in supercooled glycerol over the temperature range 198 K (1.04Tg) − 212 K (1.12Tg) 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 103 and 106τα,with τα the structural relaxation time of glycerol. Exchange is found on the shortest time scales probed (∼30 τc, with τc 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.

Much of the interesting behavior that has been observed in supercooled liquids appears to be related to dynamic heterogeneity, the presence of distinct dynamic environments – with no apparent underlying structural basis – in these systems. To most directly interrogate these environments, proposed to span regions just a few nanometers across, molecular length scale probes are required. Single molecule fluorescent microscopy was introduced to the field a decade ago and has provided strong evidence of dynamic heterogeneity in supercooled systems. However, only more recently has the full set of challenges associated with interpreting results of these experiments been described. With a fuller understanding of these challenges in hand, single molecule measurements can be employed to provide a more precise picture of dynamic heterogeneity in supercooled liquids and other complex systems. In this tutorial review, experimental and data analysis details are presented for the most commonly employed single molecule approach to studying supercooled liquids, the measurement of rotational dynamics of single molecule probes. Guidance is provided in experimental set-up and probe selection, with a focus on choices that affect data interpretation and probe sensitivity to dynamic heterogeneity.