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The extracellular matrix protein collagen I has been used extensively in the field of biomaterials due to its inherent biocompatibility and unique viscoelastic and mechanical properties. Collagen I self-assembly into fibers and networks is environmentally sensitive to gelation conditions such as temperature, resulting in gels with distinct network architectures and mechanical properties. Despite this, collagen gels are not suitable for many applications given their relatively low storage modulus. We have prepared collagen-poly(ethylene glycol) [PEG] interpenetrating network (IPN) hydrogels to reinforce the collagen network, which also induces changes to network plasticity, a recent focus of study in cell-matrix interactions. Here, we prepare collagen/PEG IPNs, varying collagen concentration and collagen gelation temperature to assess changes in microarchitecture and mechanical properties of these networks. By tuning these parameters, IPNs with a range of stiffness, plasticity and pore size are obtained. Cell studies suggest that matrix plasticity is a key determinant of cell behavior, including cell elongation, on these gels. This work presents a natural/synthetic biocompatible matrix that retains the unique structural properties of collagen networks with increased storage modulus and tunable plasticity. The described IPN materials will be of use for applications in which control of cell spreading is desirable, as only minimal changes in sample preparation lead to changes in cell spreading and circularity. Additionally, this study contributes to our understanding of the connection between collagen self-assembly conditions and matrix structural and mechanical properties and presents them as useful tools for the design of other collagen based biomaterials. STATEMENT OF SIGNIFICANCE: We developed a collagen-poly(ethylene glycol) interpenetrating network (IPN) platform that retains native collagen architecture and biocompatibility but provides higher stiffness and tunable plasticity. With minor changes in collagen gelation temperature or concentration, IPN gels with a range of plasticity, storage modulus, and pore size can be obtained. The tunable plasticity of the gels is shown to modulate cell spreading, with a greater proportion of elongated cells on the most plastic of IPNs, supporting the assertion that matrix plasticity is a key determinant of cell spreading. The material can be of use for situations where control of cell spreading is desired with minimal intervention, and the findings herein may be used to develop similar collagen based IPN platforms., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Multicellular tumor spheroids embedded in collagen I matrices are common in vitro systems for the study of solid tumors that reflect the physiological environment and complexities of the in vivo environment. While collagen I environments are physiologically relevant and permissive of cell invasion, studying spheroids in such hydrogels presents challenges to key analytical assays and to a wide array of imaging modalities. While this is largely due to the thickness of the 3D hydrogels that in other samples can typically be overcome by sectioning, because of their highly porous nature, collagen I hydrogels are very challenging to section, especially in a manner that preserves the hydrogel network including cell invasion patterns. Here, we describe a novel method for preparing and cryosectioning invasive spheroids in a two-component (collagen I and gelatin) matrix, a technique we term dual-hydrogel in vitro spheroid cryosectioning of three-dimensional samples (DISC-3D). DISC-3D does not require cell fixation, preserves the architecture of invasive spheroids and their surroundings, eliminates imaging challenges, and allows for use of techniques that have infrequently been applied in three-dimensional spheroid analysis, including super-resolution microscopy and mass spectrometry imaging., (© 2023. The Author(s).)

Rotational-translational decoupling in systems near T g , in which translational diffusion is apparently enhanced relative to rotation, has been observed in ensemble and single molecule experiments and has been linked to dynamic heterogeneity. Here, simulations of single molecules experiencing homogeneous diffusion and static and dynamic heterogeneous diffusion are performed to clarify the contributions of heterogeneity to such enhanced translational diffusion. Results show that time-limited trajectories broaden the distribution of diffusion coefficients in the presence of homogeneous diffusion but not when physically reasonable degrees of static heterogeneity are present. When dynamic heterogeneity is introduced, measured diffusion coefficients uniformly increase relative to input diffusion coefficients, and the widths of output distributions decrease, providing support for the idea that dynamic heterogeneity can drive apparent translational enhancement. Among simulations with dynamic heterogeneity, when the frequency of dynamic exchange is correlated with the initial diffusion coefficient, the measured diffusion coefficient behavior as a function of observation time matches that seen experimentally, the only set of simulations explored in which this occurs. Taken together with experimental results, this suggests that enhanced translational diffusion in glassy systems occurs through dynamic exchange consistent with wide underlying distributions of diffusion coefficients and exchange coupled to local spatiotemporal dynamics.

Connective tissue extracellular matrix (ECM) consists of an interwoven network of contiguous collagen fibers that regulate cell activity, direct biological function, and guide tissue homeostasis throughout life. Recently, ECM analogs have emerged as a unique ex vivo culture platform for studying healthy and diseased tissues and in the latter, enabling the screening for and development of therapeutic regimen. Since these tissue models can mitigate the concern that observations from animal models do not always translate clinically, the design and production of a collagenous ECM analogue with relevant chemistry and nano- to micro-scale architecture remains a frontier challenge in the field. Therefore, the objectives of this study are two-fold- first, to apply green electrospinning approaches to the fabrication of an ECM analog with nanoscale mimicry and second, to systematically optimize collagen crosslinking in order to produce a stable, collagen-like substrate with continuous fibrous architecture that supports human cell culture and phenotypic expression. Specifically, the "green" electrospinning solvent acetic acid was evaluated for biofabrication of gelatin-based meshes, followed by the optimization of glutaraldehyde (GTA) crosslinking under controlled ambient conditions. These efforts led to the production of a collagen-like mesh with nano- and micro-scale cues, fibrous continuity with little batch-to-batch variability, and proven stability in both dry and wet conditions. Moreover, the as-fabricated mesh architecture and native chemistry were preserved with augmented mechanical properties. These meshes supported the in vitro expansion of stem cells and the production of a mineralized matrix by human osteoblast-like cells. Collectively these findings demonstrate the potential of green fabrication in the production of a collagen-like ECM analog with physiological relevance. Future studies will explore the potential of this high-fidelity platform for elucidating cell-matrix interactions and their relevance in connective tissue healing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Brudnicki, Gonsalves, Spinella, Kaufman and Lu.)

Rotational-translational decoupling, in which translational motion is apparently enhanced over rotational motion in violation of Stokes-Einstein (SE) and Debye-Stokes-Einstein (DSE) predictions, has been observed in materials near their glass transition temperatures (T g ). This has been posited to result from ensemble averaging in the context of dynamic heterogeneity. In this work, ensemble and single molecule experiments are performed in parallel on a fluorescent probe in high molecular weight polystyrene near its T g . Ensemble results show decoupling onset at approximately 1.15T g , increasing to over three orders of magnitude at T g . Single molecule measurements also show a high degree of decoupling, with typical molecules at T g showing translational diffusion coefficients nearly 400 times higher than expected from SE/DSE predictions. At the single molecule level, higher degree of breakdown is associated with particularly mobile molecules and anisotropic trajectories, providing support for anomalous diffusion as a critical driver of rotational-translational decoupling and SE/DSE breakdown., (© 2022. The Author(s).)

Utilization of the body's regenerative potential for tissue repair is known as in situ tissue regeneration. However, the use of exogenous growth factors requires delicate control of the dose and delivery strategies and may be accompanied by safety, efficacy and cost concerns. In this study, we developed, for the first time, a biomaterial-based strategy to activate endogenous transforming growth factor beta 1 (TGFβ1) under alkaline conditions for effective in situ tissue regeneration. We demonstrated that alkaline-activated TGFβ1 from blood serum, bone marrow fluids and soaking solutions of meniscus and tooth dentin was capable of increasing cell recruitment and early differentiation, implying its broad practicability. Furthermore, we engineered an injectable hydrogel (MS-Gel) consisting of gelatin microspheres for loading strong alkaline substances and a modified gelatin matrix for hydrogel click crosslinking. In vitro models showed that alkaline MS-Gel controllably and sustainably activated endogenous TGFβ1 from tooth dentin for robust bone marrow stem cell migration . More importantly, infusion of in vivo porcine prepared root canals with alkaline MS-Gel promoted significant pulp-dentin regeneration with neurovascular stroma and mineralized tissue by endogenous proliferative cells. Therefore, this work offers a new bench-to-beside translation strategy using biomaterial-activated endogenous biomolecules to achieve in situ tissue regeneration without the need for cell or protein delivery., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

In order to gain a greater understanding of the factors that drive spatial organization in multicellular aggregates of cancer cells, we investigate the segregation patterns of 6 breast cell lines of varying degree of mesenchymal character during formation of mixed aggregates. Cell sorting is considered in the context of available adhesion proteins and cellular contractility. It is found that the primary compaction mediator (cadherins or integrins) for a given cell type in isolation plays an important role in compaction speed, which in turn is the major factor dictating preference for interior or exterior position within mixed aggregates. In particular, cadherin-deficient, invasion-competent cells tend to position towards the outside of aggregates, facilitating access to extracellular matrix. Reducing actomyosin contractility is found to have a differential effect on spheroid formation depending on compaction mechanism. Inhibition of contractility has a significant stabilizing effect on cell-cell adhesions in integrin-driven aggregation and a mildly destabilizing effect in cadherin-based aggregation. This differential response is exploited to statically control aggregate organization and dynamically rearrange cells in pre-formed aggregates. Sequestration of invasive cells in the interior of spheroids provides a physical barrier that reduces invasion in three-dimensional culture, revealing a potential strategy for containment of invasive cell types.

We report the first measurement of coherent elastic neutrino-nucleus scattering (CEvNS) on argon using a liquid argon detector at the Oak Ridge National Laboratory Spallation Neutron Source. Two independent analyses prefer CEvNS over the background-only null hypothesis with greater than 3σ significance. The measured cross section, averaged over the incident neutrino flux, is (2.2±0.7)×10^{-39}  cm^{2}-consistent with the standard model prediction. The neutron-number dependence of this result, together with that from our previous measurement on CsI, confirms the existence of the CEvNS process and provides improved constraints on nonstandard neutrino interactions.

We report on a comparison between the theoretically predicted and experimentally measured spectra of the first-forbidden nonunique β-decay transition ^{137}Xe(7/2^{-})→^{137}Cs(7/2^{+}). The experimental data were acquired by the EXO-200 experiment during a deployment of an AmBe neutron source. The ultralow background environment of EXO-200, together with dedicated source deployment and analysis procedures, allowed for collection of a pure sample of the decays, with an estimated signal to background ratio of more than 99 to 1 in the energy range from 1075 to 4175 keV. In addition to providing a rare and accurate measurement of the first-forbidden nonunique β-decay shape, this work constitutes a novel test of the calculated electron spectral shapes in the context of the reactor antineutrino anomaly and spectral bump.

The study of cancer cell invasion in 3D environments in vitro has revealed a variety of invasive modes, including amoeboid migration, characterized by primarily round cells that invade in a protease- and adhesion-independent manner. Here, we delineate a contractility-dependent migratory mode of primarily round breast cancer cells that is associated with extensive integrin-mediated extracellular matrix (ECM) reorganization that occurs at membrane blebs, with bleb necks sites of integrin clustering and integrin-dependent ECM alignment. We show that the spatiotemporal distribution of blebs and their utilization for ECM reorganization is mediated by functional β1 integrin receptors and other components of focal adhesions. Taken together, the work presented here characterizes a migratory mode of primarily round cancer cells in complex 3D environments and reveals a fundamentally new function for membrane blebs in cancer cell invasion., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

A search for neutrinoless double-β decay (0νββ) in ^{136}Xe is performed with the full EXO-200 dataset using a deep neural network to discriminate between 0νββ and background events. Relative to previous analyses, the signal detection efficiency has been raised from 80.8% to 96.4±3.0%, and the energy resolution of the detector at the Q value of ^{136}Xe 0νββ has been improved from σ/E=1.23% to 1.15±0.02% with the upgraded detector. Accounting for the new data, the median 90% confidence level 0νββ half-life sensitivity for this analysis is 5.0×10^{25}  yr with a total ^{136}Xe exposure of 234.1 kg yr. No statistically significant evidence for 0νββ is observed, leading to a lower limit on the 0νββ half-life of 3.5×10^{25}  yr at the 90% confidence level.

Many macroscopic properties of polymers depend on their molecular weight, with one notable example being glass transition temperature: polymers with higher molecular weights typically have higher glass transition temperatures than their lower molecular weight polymeric and oligomeric counterparts. Polymeric systems close to their glass transition temperatures also exhibit interesting properties, showing both high (and molecular weight dependent) fragility and strong evidence of dynamic heterogeneity. While studies have detailed the correlations between molecular weight and fragility, studies clearly detailing correlations between molecular weight and degree of heterogeneous dynamics are lacking. In this study, we use single molecule rotational measurements to investigate the impact of molecular weight on polystyrene's degree of heterogeneity near its glass transition temperature. To this end, two types of fluorescent probes are embedded in films composed of polystyrene ranging from 0.6 to 1364.0 kg mol -1 . We find correlation between polystyrene molecular weight, fragility, and degree of dynamic heterogeneity as reported by single molecule stretching exponents but do not find clear correlation between these quantities and time scales associated with dynamic exchange.

Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy., Competing Interests: The authors declare no conflict of interest.

Using extrinsic probes to study a host system relies on the probes' ability to accurately report the host properties under study. Probes have long been used to characterize dynamic heterogeneity, the phenomenon in which a liquid near its glass transition exhibits distinct dynamics as a function of time and position, with molecules within nanometers of each other exhibiting dynamics that may vary by orders of magnitude. The spatial and temporal characteristics of dynamic heterogeneity demand the selection of probes using stringent criteria on their size and dynamics. In this report, we study the dynamic heterogeneity of the prototypical molecular glass former o -terphenyl by investigating single molecule rotation of two perylene dicarboximide probe molecules that differ in size and comparing this to results obtained previously with the probe BODIPY268. It is found that a probe's ability to accurately report dynamic heterogeneity in o -terphenyl depends on whether the reported distribution of dynamics overlaps with the intrinsic dynamics of the host, which is naturally related to the width of the intrinsic dynamics and the magnitude of dynamical shift in probe dynamics relative to the host. We show that a probe that rotates ≈15 times more slowly than the intrinsic dynamics of the host o -terphenyl senses the slowest ≈5% of the full dynamic heterogeneity whereas one that rotates ≈65 times more slowly than the host fails to report dynamic heterogeneity of the host.

Polymeric systems close to their glass transition temperature are known to exhibit heterogeneous dynamics that evolve both over time and space, comparable to the dynamics of small molecule glass formers. It remains unclear how temperature influences the degree of heterogeneous dynamics in such systems. In the following report, a fluorescent perylene dicarboximide probe molecule that reflects the full breadth of heterogeneity of the host was used to examine the temperature dependence of the dynamic heterogeneity lifetime in polystyrene at several temperatures ranging from the glass transition to 10 K above this temperature via single molecule microscopy. Contrary to prior reports, no apparent temperature dependence of time scales associated with dynamic heterogeneity was detected; indeed, the probe molecules report characteristic dynamic heterogeneity lifetimes 100-300 times the average alpha-relaxation time (τ α ) of the polystyrene host at all temperatures studied.

Results from a search for neutrinoless double-beta decay (0νββ) of ^{136}Xe are presented using the first year of data taken with the upgraded EXO-200 detector. Relative to previous searches by EXO-200, the energy resolution of the detector has been improved to σ/E=1.23%, the electric field in the drift region has been raised by 50%, and a system to suppress radon in the volume between the cryostat and lead shielding has been implemented. In addition, analysis techniques that improve topological discrimination between 0νββ and background events have been developed. Incorporating these hardware and analysis improvements, the median 90% confidence level 0νββ half-life sensitivity after combining with the full data set acquired before the upgrade has increased twofold to 3.7×10^{25}  yr. No statistically significant evidence for 0νββ is observed, leading to a lower limit on the 0νββ half-life of 1.8×10^{25}  yr at the 90% confidence level.

Understanding the mechanisms that contribute to conjugated polymer aggregate formation and growth may yield enhanced control of aggregate morphology and functional properties on the mesoscopic scale. In situ optical imaging of the growth of MEH-PPV aggregates in real time in controlled swollen films shows that growth occurs through multiple mechanisms and is more complex than previously described. Direct evidence is provided for both Ostwald ripening and aggregate coalescence as operative modes of aggregate growth in solvent swollen films. These growth mechanisms have a distinct and strong impact on the evolution of morphological order of growing aggregates: while Ostwald ripening allows preservation of highly ordered morphology, aggregate coalescence occurs with no preferential orientation, leading to attenuation in degree of ordering., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The coherent elastic scattering of neutrinos off nuclei has eluded detection for four decades, even though its predicted cross section is by far the largest of all low-energy neutrino couplings. This mode of interaction offers new opportunities to study neutrino properties and leads to a miniaturization of detector size, with potential technological applications. We observed this process at a 6.7σ confidence level, using a low-background, 14.6-kilogram CsI[Na] scintillator exposed to the neutrino emissions from the Spallation Neutron Source at Oak Ridge National Laboratory. Characteristic signatures in energy and time, predicted by the standard model for this process, were observed in high signal-to-background conditions. Improved constraints on nonstandard neutrino interactions with quarks are derived from this initial data set., (Copyright © 2017, American Association for the Advancement of Science.)

Using a gelatin microbial transglutaminase (gelatin-mTG) cell culture platform tuned to exhibit stiffness spanning that of healthy and diseased glomeruli, we demonstrate that kidney podocytes show marked stiffness sensitivity. Podocyte-specific markers that are critical in the formation of the renal filtration barrier are found to be regulated in association with stiffness-mediated cellular behaviors. While podocytes typically de-differentiate in culture and show diminished physiological function in nephropathies characterized by altered tissue stiffness, we show that gelatin-mTG substrates with Young's modulus near that of healthy glomeruli elicit a pro-differentiation and maturation response in podocytes better than substrates either softer or stiffer. The pro-differentiation phenotype is characterized by upregulation of gene and protein expression associated with podocyte function, which is observed for podocytes cultured on gelatin-mTG gels of physiological stiffness independent of extracellular matrix coating type and density. Signaling pathways involved in stiffness-mediated podocyte behaviors are identified, revealing the interdependence of podocyte mechanotransduction and maintenance of their physiological function. This study also highlights the utility of the gelatin-mTG platform as an in vitro system with tunable stiffness over a range relevant for recapitulating mechanical properties of soft tissues, suggesting its potential impact on a wide range of research in cellular biophysics.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The p53-related transcription factor p63 is required for maintenance of epithelial cell differentiation. We found that activated forms of the Harvey Rat Sarcoma Virus GTPase (H-RAS) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) oncogenes strongly repress expression of ∆Np63α, the predominant p63 isoform in basal mammary epithelial cells. This regulation occurs at the transcriptional level, and a short region of the ∆Np63 promoter is sufficient for repression induced by H-RasV12. The suppression of ∆Np63α expression by these oncogenes concomitantly leads to an epithelial-to-mesenchymal transition (EMT). In addition, the depletion of ∆Np63α alone is sufficient to induce EMT. Both H-RasV12 expression and ∆Np63α depletion induce individual cell invasion in a 3D collagen gel in vitro system, thereby demonstrating how Ras can drive the mammary epithelial cell state toward greater invasive ability. Together, these results suggest a pathway by which RAS and PIK3CA oncogenes induce EMT through regulation of ∆Np63α., Competing Interests: The authors declare no conflict of interest.

The purpose of this project was to describe experiences of reservation-based American Indian (AI) veterans with the Department of Veterans Affairs (VA), and to identify opportunities for improving care and services. Focus group discussions and individual interviews were conducted with AI veterans, family members, and community members in three diverse tribes. Results showed that many veterans in tribal communities experienced challenges receiving services and benefits from the VA, including lack of culturally competent care, transportation problems, and difficulties navigating the system. Family members, often main caregivers for AI veterans, lacked necessary resources, including sources for information, support services, and financial means to procure adequate care. A number of strengths also were identified, including local leadership and a strong community commitment to improve care for veterans.

Polymer processing techniques involving solvent vapor swelling are typically challenging to control and thus reproduce. Moreover, traditional descriptions of solvent swollen films lack microscopic detail. We describe the design and use of an apparatus that facilitates macroscopic and microscopic characterization of samples undergoing solvent vapor swelling in a controlled environment. The experimental design incorporates three critical characteristics: (1) a mass-flow controlled solvent vapor delivery system allows for precise control of the amount of solvent vapor delivered to the sample, (2) a sample prepared on a quartz crystal microbalance allows for real-time assessment of the extent of sample swelling, (3) a second sample prepared and assessed in parallel on a coverslip allows real-time fluorescence microscopy during swelling. We demonstrate that this apparatus allows for single-particle tracking, which in turn facilitates in situ monitoring of local environments within the solvent-swollen film.

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.

To invade and metastasize to distant loci, breast cancer cells must breach the layer of basement membrane surrounding the tumor and then invade through the dense collagen I-rich extracellular environment of breast tissue. Previous studies have shown that breast cancer cell aggregate morphology in basement membrane extract correlated with cell invasive capacity in some contexts. Moreover, cell lines from the same aggregate morphological class exhibited similarities in gene expression patterns. To further assess the capacity of cell and aggregate morphology to predict invasive capacity in physiologically relevant environments, six cell lines with varied cell aggregate morphologies were assessed in a variety of assays including a 3D multicellular invasion assay that recapitulates cell-cell and cell-environment contacts as they exist in vivo in the context of the primary breast tumor. Migratory and invasive capacities as measured through a 2D gap assay and a 3D spheroid invasion assay reveal that breast cancer cell aggregate morphology alone is insufficient to predict migratory speed in 2D or invasive capacity in 3D. Correlations between the 3D spheroid invasion assay and gene expression profiles suggest this assay as an inexpensive functional method to predict breast cancer invasive capacity.

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.

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.

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.

We describe a system to transport and identify barium ions produced in liquid xenon, as part of R&D towards the second phase of a double beta decay experiment, nEXO. The goal is to identify the Ba ion resulting from an extremely rare nuclear decay of the isotope (136)Xe, hence providing a confirmation of the occurrence of the decay. This is achieved through Resonance Ionization Spectroscopy (RIS). In the test setup described here, Ba ions can be produced in liquid xenon or vacuum and collected on a clean substrate. This substrate is then removed to an analysis chamber under vacuum, where laser-induced thermal desorption and RIS are used with time-of-flight mass spectroscopy for positive identification of the barium decay product.

Local invasion by and dissemination of cancer cells from a primary tumor are key initial steps of metastasis, the most lethal aspect of cancer. To study these processes in vitro, the invasion of cells from multicellular breast cancer aggregates embedded in three-dimensional (3D) extracellular matrix culture systems was studied. This work showed that in 3D fibrillar environments composed of collagen I, pore size--not the viscoelastic properties of the matrix--was the biophysical characteristic controlling breast cancer cell invasion efficiency. Furthermore, it was shown that fibrillar matrix architecture is a crucial factor that allows for efficient 3D invasion. In a 3D non-fibrillar environment composed of basement membrane extract (BME), invasion efficiency was greatly diminished, the mesenchymal individual mode of 3D invasion was abolished, and establishment of cell polarity and protrusions was compromised. These effects were seen even though the BME matrix has invasion permissive viscoelasticity and suitable adhesion ligands. The altered and limited invasive behavior observed in BME was rescued through introduction of fibrillar collagen into the non-fibrillar matrix. The biophysical cues of fibrillar collagen facilitated efficient invasion of sterically disadvantageous environments through assisting cell polarization and formation of stable cell protrusions. Finally, we suggest the composite matrices employed in this study consisting of fibrillar collagen I and BME in either a liquid-like or gelled state are suitable for a wide range of 3D cell studies, as these matrices combine fibrillar features that require cells to deploy integrin-dependent mechanotransduction machinery and a tunable non-fibrillar component that may require cells to adopt alternative migratory modes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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.

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.

Bulk approaches to studying heterogeneous systems obscure important details, as they report average behavior rather than the distribution of behaviors in such environments. Small-molecule and polymeric supercooled liquids, which display heterogeneity in their dynamics without an underlying structural heterogeneity that sets those dynamics, are important constituents of this category of condensed matter systems. A variety of approaches have been devised to unravel ensemble averaging in supercooled liquids. This review focuses on the ultimate subensemble approach, single-molecule measurements, as they have been applied to the study of supercooled liquids. We detail how three key experimental observables (single-molecule probe rotation, translation, and fluorescence lifetime) have been employed to provide detail on dynamic heterogeneity in supercooled liquids. Special attention is given to the potential for, but also the challenges in, discriminating spatial and temporal heterogeneity and detailing the length scales and timescales of heterogeneity in these systems.

Biopolymer gels exhibit strain stiffening that is generally not seen in synthetic gels. Here, we investigate the strain-stiffening behavior in collagen I gels that demonstrate elasticity derived from a variety of sources including crosslinking through telopeptides, bundling through low-temperature gelation, and exogenous crosslinking with genipin. In all cases, it is found that these gels exhibit strain stiffening; in general, onset of strain stiffening occurs earlier, yield strain is lower, and degree of strain stiffening is smaller in higher concentration gels and in those displaying thick fibril bundles. Recovery after exposure to high strains is substantial and similar in all gels, suggesting that much of the stiffening comes from reversible network deformations. A key finding of this study is that collagen I gels of identical storage and loss moduli may display different nonlinear responses and different capacities to recover from high strain., (Copyright © 2012 Wiley Periodicals, Inc.)

We report on a search for neutrinoless double-beta decay of 136Xe with EXO-200. No signal is observed for an exposure of 32.5 kg yr, with a background of ∼1.5×10(-3)  kg(-1) yr(-1)  keV(-1) in the ±1σ region of interest. This sets a lower limit on the half-life of the neutrinoless double-beta decay T(1/2)(0νββ)(136Xe)>1.6×10(25)  yr (90% C.L.), corresponding to effective Majorana masses of less than 140-380 meV, depending on the matrix element calculation.

The (2)H(e,e'p)n cross section at a momentum transfer of 3.5 (GeV/c)(2) was measured over a kinematical range that made it possible to study this reaction for a set of fixed missing momenta as a function of the neutron recoil angle θ(nq) and to extract missing momentum distributions for fixed values of θ(nq) up to 0.55  GeV/c. In the region of 35°≤θ(nq)≤45° recent calculations, which predict that final-state interactions are small, agree reasonably well with the experimental data. Therefore, these experimental reduced cross sections provide direct access to the high momentum component of the deuteron momentum distribution in exclusive deuteron electrodisintegration.

Experimental studies that follow behavior of single probes embedded in heterogeneous systems are increasingly common. The presence of probes may perturb the system, and such perturbations may or may not affect interpretation of host behavior from the probe observables typically measured. In this study, the manifestations of potential probe-induced changes to host dynamics in supercooled liquids are investigated via molecular dynamics simulations. It is found that probe dynamics do not necessarily mirror host dynamics as they exist either in the probe-free or probe-bearing systems. In particular, for a binary supercooled liquid, we find that smooth probes larger than the host particles induce increased translational diffusion in the host system; however, the diffusion is anisotropic and enhances caging of the probe, suppressing probe translational diffusion. This in turn may lead experiments that follow probe diffusion to suggest Stokes-Einstein behavior of the system even while both the probe-free and probe-bearing systems exhibit deviations from that behavior., (© 2011 American Institute of Physics)

We report the observation of two-neutrino double-beta decay in (136)Xe with T(1/2) = 2.11 ± 0.04(stat) ± 0.21(syst) × 10(21) yr. This second-order process, predicted by the standard model, has been observed for several nuclei but not for (136)Xe. The observed decay rate provides new input to matrix element calculations and to the search for the more interesting neutrinoless double-beta decay, the most sensitive probe for the existence of Majorana particles and the measurement of the neutrino mass scale.

To mimic the extracellular matrix surrounding high grade gliomas, composite matrices composed of either acid-solubilized (AS) or pepsin-treated (PT) collagen and the glycosaminoglycans chondroitin sulfate (CS) and hyaluronic acid (HA) are prepared and characterized. The structure and mechanical properties of collagen/CS and collagen/HA gels are studied via confocal reflectance microscopy (CRM) and rheology. CRM reveals that CS induces fibril bundling and increased mesh size in AS collagen but not PT collagen networks. The presence of CS also induces more substantial changes in the storage and loss moduli of AS gels than of PT gels, in accordance with expectation based on network structural parameters. The presence of HA significantly reduces mesh size in AS collagen but has a smaller effect on PT collagen networks. However, both AS and PT collagen network viscoelasticity is strongly affected by the presence of HA. The effects of CS and HA on glioma invasion is then studied in collagen/GAG matrices with network structure both similar to (PT collagen-based gels) and disparate from (AS collagen-based gels) those of the corresponding pure collagen matrices. It is shown that CS inhibits and HA has no significant effect on glioma invasion in 1.0 mg/ml collagen matrices over 3 days. The inhibitory effect of CS on glioma invasion is more apparent in AS than in PT collagen gels, suggesting invasive behavior in these environments is affected by both biochemical and network morphological changes induced by GAGs. This study is among the few efforts to differentiate structural, mechanical and biochemical effects of changes to matrix composition on cell motility in 3D., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A magnetically driven piston pump for xenon gas recirculation is presented. The pump is designed to satisfy extreme purity and containment requirements, as is appropriate for the recirculation of isotopically enriched xenon through the purification system and large liquid xenon time projection chamber of EXO-200. The pump, using sprung polymer gaskets, is capable of pumping more than 16 standard liters per minute of xenon gas with 750 Torr differential pressure.

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.

The electric form factor of the neutron was determined from studies of the reaction 3He(e,e'n)pp in quasielastic kinematics in Hall A at Jefferson Lab. Longitudinally polarized electrons were scattered off a polarized target in which the nuclear polarization was oriented perpendicular to the momentum transfer. The scattered electrons were detected in a magnetic spectrometer in coincidence with neutrons that were registered in a large-solid-angle detector. More than doubling the Q2 range over which it is known, we find G(E)(n)=0.0236±0.0017(stat)±0.0026(syst), 0.0208±0.0024±0.0019, and 0.0147±0.0020±0.0014 for Q(2)=1.72, 2.48, and 3.41 GeV2, respectively.

We describe a source capable of producing single barium ions through nuclear recoils in radioactive decay. The source is fabricated by electroplating (148)Gd onto a silicon α-particle detector and vapor depositing a layer of BaF(2) over it. (144)Sm recoils from the alpha decay of (148)Gd are used to dislodge Ba(+) ions from the BaF(2) layer and emit them in the surrounding environment. The simultaneous detection of an α particle in the substrate detector allows for tagging of the nuclear decay and of the Ba(+) emission. The source is simple, durable, and can be manipulated and used in different environments. We discuss the fabrication process, which can be easily adapted to emit most other chemical species, and the performance of the source.

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., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

High grade gliomas are aggressive cancers that are not well addressed by current chemotherapies, in large measure because these drugs do not curtail the diffuse invasion of glioma cells into brain tissue surrounding the tumor. Here, we investigate the effects of suberoylanilide hydroxamic acid (SAHA) on glioma cells in 2D and 3D in vitro assays, as SAHA has previously been shown to significantly increase apoptosis, decrease proliferation, and interfere with migration in other cell lines. We find that SAHA has significant independent effects on proliferation, migration, and invasion. These effects are seen in both 2D and 3D culture. In 3D culture, with glioma spheroids embedded in collagen I matrices, SAHA independently limits both glioma invasion and the reorganization of the tumor surroundings that usually proceeds such invasion. The decreased matrix reorganization and invasion is not accompanied by decreased production or activity of matrix-metalloproteases but instead may be related to increased cell-cell adhesion., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)