Search

We demonstrate simultaneous spatial and spectral subtraction-based super-resolution microscopy based on photobleaching, photoblinking, or photoactivation in multichromophoric systems to detail the ...

To bridge the gap between single-molecule and bulk thin film studies of organic semiconductors and understand the influence of molecular structure on photophysical properties across scales, we prepared and characterized aggregates containing a few to more than a thousand single chains via well-controlled solvent vapor swelling of an inert polymeric matrix containing the conjugated polymer poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV). The single chains were initially immobilized in conformations with few interchain contacts, which can support exciton funneling on the single-molecule level. We show that aggregates prepared via solvent swelling and templated on such molecules result in largely isotropic aggregates. Analysis of aggregate polarization anisotropy, photoluminescence transient decay, and photoluminescence spectra as a function of aggregate size suggests that in these aggregates exciton diffusion is locally enhanced upon initial aggregation but does not reach extended length...

Polarization modulation depth (M) measurements have been widely used as a technique to report on transition dipole alignment and thus molecular conformation of single conjugated polymers and aggregates. Such extrapolation of conformation from these measurements, however, is complicated by the fact that photophysical processes (which themselves are coupled to conformation) may influence M values. Here, we show that the presence of intensity-dependent partial photoluminescence quenching can suppress M values, with this suppression more prominent in molecules with highly aligned transition dipoles. We show that these findings on M values are a direct consequence of behaviors of fluorescence intensity maxima and minima as a function of excitation polarization in poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] single molecules, as supported by a simulation that reproduces the experimental results. Our findings show that interpreting M values of molecules with complex photophysics should be done with ...

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.

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.

Lipoprotein(a), Lp(a), is a modified low-density lipoprotein particle that contains apolipoprotein(a), encoded by LPA, and is a highly heritable, causal risk factor for cardiovascular diseases that varies in concentrations across ancestries. Here, we use deep-coverage whole genome sequencing in 8392 individuals of European and African ancestry to discover and interpret both single-nucleotide variants and copy number (CN) variation associated with Lp(a). We observe that genetic determinants between Europeans and Africans have several unique determinants. The common variant rs12740374 associated with Lp(a) cholesterol is an eQTL for SORT1 and independent of LDL cholesterol. Observed associations of aggregates of rare non-coding variants are largely explained by LPA structural variation, namely the LPA kringle IV 2 (KIV2)-CN. Finally, we find that LPA risk genotypes confer greater relative risk for incident atherosclerotic cardiovascular diseases compared to directly measured Lp(a), and are significantly associated with measures of subclinical atherosclerosis in African Americans., Circulating lipoprotein(a) is an important risk factor for cardiovascular disease and shows variability between different ethnic groups. Here, Zekavat et al. perform whole-genome sequencing in individuals of European and African ancestries and find ancestry-specific genetic determinants for lipoprotein(a) levels.

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.

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 operati...

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.

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.

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 th...

The gap between graduate research and introductory undergraduate teaching laboratories is often wide, but the development of teaching activities rooted within the research environment offers an opportunity for undergraduate students to have first-hand experience with research currently being conducted and for graduate students to develop communication and pedagogical skills. We describe the development, evaluation, and redesign of a Microsoft Excel-based activity where data analysis from contemporary research was adapted into a data analysis training exercise, requiring undergraduate students to engage with the data by performing a series of analytical tasks guided by commentary from the researcher. In addition to learning common data analysis techniques, this exercise provided an opportunity for undergraduate students to practice problem solving and collaboratively work through trouble-shooting. Throughout the activity development, multiple iterations allowed the authors to explore the impact of differen...

Heterogeneity in relaxation rates is a key feature of supercooled liquids. It implies the existence of a rate-exchange process to restore ergodicity, but the experimental characterization of that exchange has been incomplete and controversial. Here, a recently proposed, ensemble-based analysis is applied to single-molecule dichroism data to extract a detailed correlation function for rotational-rate exchange. A large, late phase is $8.7\ifmmode\pm\else\textpm\fi{}0.3$ times slower than the probe rotation and 22 times the alpha-relaxation time, and it has its own exchange-rate dispersion. A small, early phase tracks the initial rotational decay. We propose that the late phase is due to molecules in the core of spatial regions of correlated rates and that the early phase is due to molecules on the boundaries. The results imply that multiple processes and spatial fields are involved in the primary relaxation in supercooled liquids.

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.

Significance Supercooled liquids are believed to exhibit spatially heterogeneous dynamics, where molecular mobility within a given spatial region may differ from that of a neighboring region, potentially by orders of magnitude. If supercooled liquids are ergodic, such that the spatial average of all regions with distinct dynamics equals the time average of a given region, these regions of distinct dynamics must interchange over time. With an appropriate probe, similar in size and mobility to the host, single-molecule measurements can provide direct access to these spatial and temporal variations. Here, such a probe is used, revealing how relaxation dynamics are distributed in time and space and directly demonstrating ergodicity of a prototypical glass former down to the glass transition temperature.

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.

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.

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.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....

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.

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.

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.

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 (τα) of the polystyrene host at all temperatures studied.

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.

The performance of a light microscope is intrinsically constrained by the Abbe diffraction limit. No matter how close to optical perfection it is, an imaging system cannot resolve two objects that are beyond this natural limit, which is dependent on the wavelength of the observed light and its angular distribution. Several methods have been devised to beat the diffraction limit, but these have generally required esoteric excitation schemes, so remain impractical. Lee et al. are working on a new way of beating the limit, using nanoscale spherical lenses that self-assemble by bottom-up integration of cup-shaped organic molecules called calixarenes. Lenses produced in this way have very short focal lengths that can generate near-field magnification beyond the diffraction limit, enabling the resolution of features of the order of 200 nm. The lenses can be placed at will on a surface and, among other things, can be used to reduce the size of deep-ultraviolet lithography features. Cup-shaped molecules of calix[4]hydroquinone self-assemble on a surface into a lens shape; these lenses are shown to generate near-field magnification beyond the diffraction limit, enabling the resolution of features of the order of 200 nanometres. Such spherical nanolenses provide new pathways for lens-based near-field focusing and high-resolution optical imaging at very low intensities, which are useful for, among other things, bio-imaging and near-field lithography. It is well known that a lens-based far-field optical microscope cannot resolve two objects beyond Abbe’s diffraction limit. Recently, it has been demonstrated that this limit can be overcome by lensing effects driven by surface-plasmon excitation1,2,3, and by fluorescence microscopy driven by molecular excitation4. However, the resolution obtained using geometrical lens-based optics without such excitation schemes remains limited by Abbe’s law even when using the immersion technique5, which enhances the resolution by increasing the refractive indices of immersion liquids. As for submicrometre-scale or nanoscale objects, standard geometrical optics fails for visible light because the interactions of such objects with light waves are described inevitably by near-field optics6. Here we report near-field high resolution by nanoscale spherical lenses that are self-assembled by bottom-up integration7 of organic molecules. These nanolenses, in contrast to geometrical optics lenses, exhibit curvilinear trajectories of light, resulting in remarkably short near-field focal lengths. This in turn results in near-field magnification that is able to resolve features beyond the diffraction limit. Such spherical nanolenses provide new pathways for lens-based near-field focusing and high-resolution optical imaging at very low intensities, which are useful for bio-imaging, near-field lithography, optical memory storage, light harvesting, spectral signal enhancing, and optical nano-sensing.

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.

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 degrees C and 32 degrees C. The following parameters are used to describe the self-assembly process in all gels: the crossover time (t(c)), the slope of the growth phase (k(g)), and the arrest time (t(a)). The first two measures are determined by rheology, and the third by CRM. A frequency-independent rheological measure of gelation, t(g), is also measured at 37 degrees C. However, this quantity cannot be straightforwardly determined for gels formed at 32 degrees C, indicating that percolation theory does not fully capture the dynamics of collagen network formation. The effects of collagen concentration and gelation temperature on k(g), t(c), and t(a) 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 degrees C and 32 degrees 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.

A straightforward technique to align thin collagen gels is presented. This technique requires only collagen solution, surface-modified magnetic beads, a small magnet, and an incubator. As such, this is the only collagen alignment technique that requires no specialized equipment. The collagen gels are imaged with confocal reflectance microscopy, and degree of alignment is quantitatively assessed using image analysis techniques that allow for identification of fiber position and angular distribution. A series of experiments shows that magnetic beads coated with streptavidin lead to the most highly aligned gels. Rheology and microscopy experiments suggest that alignment results from bead coupling to, and entrainment and entrapment in, collagen fibrils during their assembly into fibers that form a sample-spanning gel. The timescales of gelation and bead motion to the poles of the external magnet must be similar to effect good alignment over large areas with this technique. It is also demonstrated that alignment can be attained in both plain and cell-bearing gels that are several millimeters thick.

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.

Two different electronically resonant two-dimensional spectroscopies are described. The first, two-color photon echo peak shift spectroscopy, is sensitive to correlations in transition frequency between the initial and probed (final) states. It provides new insight into the mechanism of ultrafast solvation and should prove useful for characterizing dynamics in inhomogeneous systems in general. The second technique, fifth order threepulse scattering, contains two coherence periods whose durations are controlled. The entire two-dimensional surface was recorded for a dye molecule in dilute solution and a photosynthetic light-harvesting complex. The data provide insight into the short-time dynamics of solvation and exciton relaxation, respectively.

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.

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.

This paper presents an analytical Fourier deconvolution procedure for homodyne detected electronically non-resonant fifth-order signal that reveals the bare nuclear response function free from the influence of the electronic (hyperpolarizability) responses generated by the five potentially overlapping finite duration pulses used in the experiment. In developing the fifth-order deconvolution procedure, an analogous procedure for homodyne detected third-order responses is elaborated. The potential implementation problems with the homodyne deconvolution procedure are evaluated through comparison of the third-order homodyne deconvolution result with that of the well-known third-order heterodyne deconvolution. Then, the homodyne deconvolution is extended to fifth-order where it is used on several measured tensor elements of the direct fifth-order signal. Suggestions are given for improving implementation of the procedure in fifth order so that more information on the direct fifth-order nuclear response as well as the hyperpolarizability responses can be recovered via the deconvolution procedure.

This paper presents several experimentally collected tensor elements of the direct fifth-order Raman signal of room temperature CS2. All results were collected so as to reduce contamination from third-order cascade signals, and it is shown that while these cascade signals are of varying importance in different tensor elements, phase matching considerations are sufficient to make the direct fifth-order signal the dominant signal in every tensor element collected. We show, as predicted, that the different tensor elements appear to weight particular Liouville pathways of the direct fifth-order signal, and thus particular dynamics of the studied system, differently. The magic angle signals are shown to contain information about the intermolecular vibrational echo and to put a limit on the time scale of rephasing of the room temperature liquid. The Ryzzzzy tensor element, in conjunction with the Ryyzzzz tensor element, is shown to be useful in studying the pure dephasing and population dynamics of the liquid. ...

Two-photon excitation results from the near simultaneous absorption of two relatively low-energy photons by a fluorophore, causing a transition to an excited state with an energy difference close to that of the combined two photons (Figure 1 A).1 For most biologically relevant two-photon fluorophores, the excitation light used is in the near infrared (NIR) region.2 Several characteristics of two-photon microscopy make it an attractive technique for biological imaging. Two-photon absorption is strongly dependent on the intensity of the incident light, and therefore, excitation only takes place in a small volume centered at the focal plane, giving inherent sectioning and greatly reducing background fluorescence from out-of-focus excitation. Because the two-photon excitation is limited to the focal plane, photodamage and bleaching to the sample are minimized.3 Additionally, the greatly reduced absorption and scattering of NIR light allows for deeper penetration into biological samples.4 Here we report the development of a trimethoprim (TMP) chemical tag suitable for two-photon imaging in live cells; this adds a robust two-photon fluorophore to the repertoire of labels available with this technology. Figure 1 A trimethoprim (TMP)-based chemical tag for two-photon imaging. A) Energy level diagram illustrating one- and two-photon excitation. Two-photon excitation occurs from the near simultaneous absorption of two photons that are approximately half of the energy ... With the chemical tags, rather than tagging the protein of interest with a fluorescent protein (FP), the protein of interest is tagged with a polypeptide that is subsequently modified with an organic fluorophore.5–8 Thus, the chemical tags combine the selectivity of genetic encoding with a modular organic fluorophore.9–11 We have previously established that proteins tagged with E. coli dihydrofolate reductase (eDHFR) can be labeled noncovalently12–14 (and recently covalently)15 with cell-permeable trimethoprim (TMP)–fluorophore conjugates in mammalian cells. The TMP-based tag is an attractive chemical tag because eDHFR is small in size (18 kD), TMP analogues are straightforward to synthesize, and labels based on the TMP antibiotic have no apparent cross reactivity or toxicity in mammalian cells.16, 17 Currently, the TMP-tag is one of the few available chemical tags capable of specifically labeling intracellular proteins in living cells.14, 18 To our knowledge, while a variety of organic fluorophores, quantum dots,19 and lanthanide chelates17 have been adapted for the chemical tag technology, currently there are no reported chemical tags optimized for two-photon imaging. Key to the design of a TMP-tag for two-photon microscopy was selection of a two-photon fluorophore for conjugation to TMP that would have the desired photophysical properties yet also be cell permeable. The fluorophore must have a high two-photon action (TPA) cross section, which is the product of the two-photon absorption cross section and the fluorescence quantum yield. Second, when linked to TMP, the two-photon fluorophore must behave well in the cell; it must be both cell-permeable and not partition into lipid vesicles.14 In this study, we employed a 2H-benzo[h]chromene-2-one derivative, which we refer to as BC575. This fluorophore was recently reported by Kim and co-workers to have a high TPA cross-section (67 Goeppert–Mayer, or GM [1 GM=10−50 cm4 s per photon]), and to be cell permeable.20, 21 The retrosynthetic analysis of the TMP-BC575 conjugate is shown in Scheme 1. As previously reported,14 the synthesis began with the selective hydrolysis of the 4′-methoxy group of TMP in 48 % HBr; this resulted in a phenol, which was alkylated with ethyl 5-bromovalerate. Saponification of the ester produced a carboxylic acid, which was coupled to mono-Boc protected 1,13-diamino-4,7,10-trioxatridecane and then deprotected to generate a free amine. The synthesis of BC575 was completed in four steps.20 The amino group of 6-aminotetralone was methylated by iodomethane to yield the corresponding dimethylaniline, which was formylated by treatment with ethyl formate. The ring was aromatized and condensed with dimethyl malonate, giving BC575. The methyl ester was then hydrolyzed, and the resulting carboxylic acid was coupled to the free amine of the TMP portion by using standard peptide-coupling conditions. Thus, the TMP-BC575 conjugate was synthesized from two components in six linear steps in 0.3 % overall yield from 6-amino-3,4-dihydronapthalen-1(2 H)-one, the longest linear route. Scheme 1 >Retrosynthetic analysis of the TMP-BC575 conjugate. To verify the performance of BC575, its two-photon excitation and emission spectra were first measured in vitro. Rectangular glass capillaries (300×50 microns) were filled with a 100 μm solution of BC575 dissolved in DMF. The capillaries were sealed and fixed on a coverslip immersed in a drop of water. The two-photon fluorescence data were acquired by using a custom-made two-photon laser scanning microscope based on the Olympus FV-300 system (FV-300 side-mounted to a BX50WI microscope with a 60×, 1.1 numerical aperture, water immersion objective) and a Ti:sapphire laser (Chameleon Ultra II, Coherent).22 Fluorescence of BC575 was evaluated following two-photon excitation by using wavelengths from 750–1050 nm, which are typically used for two-photon biological imaging applications. Additionally, we measured the fluorescence intensity of a 100 μm sample of rhodamine B in H2O by using the same microscope setting; this allowed for the direct comparison of BC575 to rhodamine B, which is known to be a bright two-photon dye.23 The total signal intensities were then determined by using the program ImageJ24 and were used to calculate the normalized fluorescence intensities for the dyes (Figure 2 A).23, 25 The results show that BC575 has appreciable two-photon excited fluorescence from 750–950 nm and confirms the utility of BC575 for two-photon microscopy. Figure 2 Characterization of the two-photon fluorescent chemical tag TMP-BC575. A) In vitro characterization of two-photon excitation of BC575. To evaluate the utility of the fluorophore for two-photon imaging, the two-photon excited fluorescence intensity (in ... Having demonstrated in vitro that BC575 is a good two-photon fluorophore, we then evaluated the utility of the TMP-BC575 conjugate for in vivo imaging. Human embryonic kidney (HEK) 293 cells were seeded on coverslips and transiently transfected with vector DNA encoding nucleus-targeted eDHFR.13 The cells were then incubated in media containing 1 μm TMP-BC575 for 10 min at 37 °C, washed and then imaged by oblique illumination and by two-photon microscopy by using excitation at 940 nm (Figure 2 B). Notably, the transfected cells showed distinct nuclear labeling without any significant staining of the cytoplasm or untransfected cells. Comparison of the fluorescence signal intensities from two-photon excitation of both transfected and untransfected cells incubated with TMP-BC575 verified that the conjugate can be used to label proteins of interest with high signal-to-noise (Figure S5 in the Supporting Information). These results establish that, significantly, the TMP-BC575 conjugate has the combination of cell permeability and two-photon brightness necessary for two-photon live cell imaging. Thus, TMP-BC575 is an immediately viable tool for imaging proteins in live cells by using two-photon microscopy. This two-photon fluorophore expands the TMP-tag tool kit, adding to the value of this modular-labeling technology. A protein of interest can be tagged with eDHFR, and different labels can then be swapped in, allowing the protein to be readily analyzed by multiple techniques. While cell permeability and lipid partitioning appear to be tag dependent, these experiments suggest BC575 might also be compatible with other chemical tags. Given its broad excitation maxima and distinct emission wavelength, TMP-BC575 offers an alternative to other FPs for multicolor two-photon imaging with enhanced green fluorescent protein (EGFP; see Figures S2 and S3 in the Supporting Information),26–28 Alternatively, an orthogonal chemical tag with a nonoverlapping two-photon fluorophore could be developed for multicolor two-photon microscopy. Furthermore, TMP-BC575 might be advantageous in applications in which the larger FP fusion interferes with biological function or where the reversibility of the fluorophore labeling can be exploited. The next step is to challenge TMP-BC575 to label a variety of intracellular proteins. We are also exploring the utility of TMP-BC575 for two-photon imaging of tissue sections and live animals. This work opens the door for two-photon imaging with chemical tags and further illustrates how the modular organic label of chemical tags can be harnessed for state-of-the-art biological imaging.

In this paper we report the measurement of the direct fifth-order electronically nonresonant intermolecular response from liquid CS2 at room temperature. We recently demonstrated that all previous attempts to measure the direct fifth-order response were dominated by third-order cascaded signals [J. Chem Phys. 111, 3105 (1999)]. Here we show that phase matching considerations can be used to provide substantial discrimination against the cascaded third-order signals, and the direct fifth-order response can be measured. The measured responses indicate that the intermolecular spectrum of liquid CS2 at room temperature is well described as homogenous. Comparisons of the data to simulations are discussed.

We have demonstrated that fifth-order stimulated Raman spectra of the intermolecular modes in CS2 are dominated by cascading third-order processes. Previous studies have successfully discriminated against a sequential cascading process, but did not account for parallel third-order cascades. All of our measured spectra were successfully simulated considering only cascades built directly from our measured third-order spectra. Using an appropriately chosen phase matching geometry we also measured the sequential cascade, which should exist with equal probability to the parallel cascade. When employing a phase matching geometry that provided substantial discrimination against all of the third-order cascades we were not able to measure any signal. We assign an upper limit for the true fifth-order signal of 2% of the cascaded signal.

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.

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.

Breast cancer is dangerous not for its activity at the initial site but due to the ability of cells within the primary tumor to breach the surrounding basement membrane, invade dense collagen I-rich breast tissue, and ultimately reach and form secondary tumors in distant organs with critical functions. Previous studies on breast cancer cell lines have shown correlations between measures of invasion and static properties including cell aggregate morphology. Specifically, it has been shown that cells demonstrating stellate aggregate morphology typically outperform cell lines demonstrating other aggregate morphologies in the Transwell invasion assay. To further assess whether morphology can be used as a predictor of invasive capacity, six cell lines that have previously been identified as forming putatively aggressive stellate aggregates or less aggressive grape-like aggregates were evaluated for their ability to invade in various contexts- from a simple two dimensional gap migration assay to a more complex three dimensional spheroid invasion assay that recapitulates cell-cell and cell-environment contacts as they exist in vivo in the context of the primary breast tumor. We found that morphology alone was insufficient to predict how well cells performed in each of these assays. Migratory ability on a two dimensional substrate, contractility of the three dimensional environment, and presence of proper integrins for binding to the extracellular matrix also failed to fully predict invasive capacity in a three dimensional environment. Correlations between performance in the three dimensional spheroid invasion assay and gene expression profiles suggest this assay can be used to identify invasive breast cancer cells independent of their morphology.

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.

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.

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.