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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.

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.

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.

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.