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Multiple particle tracking microrheological characterization: Fundamentals, emerging techniques and applications
Journal of Applied Physics
Abstract
Multiple particle tracking microrheology (MPT) is a passive microrheological technique that measures the Brownian motion of probe particles embedded in a sample to characterize material rheological properties. MPT is a powerful tool that quantifies material rheology in the low moduli range while requiring only small sample volumes and relatively simple data acquisition using video microscopy. MPT quantitatively characterizes spatiotemporal rheological properties and is particularly well suited for the investigation of evolving materials with complex microenvironments. MPT has expanded the study of a variety of materials including biofilms, colloidal gels, hydrogels, stimuli-responsive materials, and cell-laden biomaterials. The aim of this Tutorial is to summarize the fundamentals, illustrate the versatility, and highlight recent advances in MPT. In each application, we will highlight how MPT is uniquely positioned to gather rheological properties, which would be difficult, if not impossible, to attain with other rheological characterization techniques and highlight how MPT can be used to supplement other measurement techniques. This Tutorial should provide researchers with the fundamental basis and skills needed to use MPT and develop new MPT techniques to characterize materials for their unique applications.
... This allows simultaneous measurements of material properties at different length scales. 38,39 In this study, we characterize the rheological properties and microstructure during gelation of OMFC in differently charged surfactant solutions using bi-disperse MPT, MPT, and bulk rheological characterization. This mimics the manufacturing of products that encapsulate surfactants for delivery during consumer use. ...
... Therefore, by selecting the right exposure time and frame rate, these two types of errors can be balanced. 37,39,50 Our experimental setup is calibrated by measuring varying concentrations of glycerin diluted in water, a Newtonian fluid, with 1 μm particles. Each concentration is measured at different frame rates and exposure times until the errors are balanced, and we measure the expected viscosity of the fluid. ...
... In this study, we use a frame rate of 30 frames per second for a total of 800 frames and an exposure time of 1000 μs to minimize static and dynamic particle tracking errors. 37,39,50 Additionally, the static error for all particle sizes is provided in the Supporting Information in Figure S3. ...
Rheological modifiers are used to tune rheology or induce phase transitions of products. Microfibrillated cellulose (MFC), a renewable material, has the potential to be used for rheological modification. However, the lack of studies on the evolution in rheological properties and structure during its phase transitions has prevented MFC from being added to consumer, fabric, and home care products. In this work, we characterize surface-oxidized MFC (OMFC), a negatively charged colloidal rod suspension. We measure the rheological properties and structure of OMFC during sol-gel phase transitions induced by either anionic or cationic surfactant using multiple particle tracking microrheology (MPT). MPT tracks the Brownian motion of fluorescent probe particles embedded in a sample, which is related to the sample's rheological properties. Using MPT, we measure that OMFC gelation evolution is dependent on the charge of the surfactant that induces the phase transition. OMFC gelation is gradual in anionic surfactant. In cationic surfactant, gelation is rapid followed by length scale-dependent colloidal fiber rearrangement. Initial OMFC concentration is directly related to how tightly associated the network is at the phase transition, with an increase in concentration resulting in a more tightly associated network with smaller pores. Bulk rheology measures that OMFC forms a stiffer structure but yields at lower strains in cationic surfactant than in anionic surfactant. This study characterizes the role of surfactant in inducing phase transitions, which can be used as a guide for designing future products.
... Passive microrheology has been particularly useful to explore evolving and aging materials with minimal perturbation, such as polymer sol-gel transition and degradation (Gomez-Solano et al., 2013;Xing et al., 2018;McGlynn et al., 2020). Gelation should be slow enough to satisfy the GSER assumption that the material is at quasi-equilibrium (Furst and Squires, 2017). ...
... Synthetic particles, intracellular organelles and proteins have been used as probes in bio-microrheology. Synthetic probes are usually washed and sonicated prior to mixing with samples McGlynn et al., 2020). For intracellular measurements, cells are typically allowed to adhere to the bottom surface of a petri dish or well plate (Wirtz, 2009). ...
... Intracellular MPT and TPM experiment method and principles is discussed in these reports (Lau et al., 2003;Hoffman et al., 2006;Crocker and Hoffman, 2007). For comprehensive experiment method and principles in cells and biomaterials, including sample preparation and data interpretation, we direct the readers to these excellent reviews (Kasza et al., 2007;Wirtz, 2009;Waigh, 2016;Joyner et al., 2020;McGlynn et al., 2020). ...
Microrheology encompasses a range of methods to measure the mechanical properties of soft materials. By characterizing the motion of embedded microscopic particles, microrheology extends the probing length scale and frequency range of conventional bulk rheology. Microrheology can be characterized into either passive or active methods based on the driving force exerted on probe particles. Tracer particles are driven by thermal energy in passive methods, applying minimal deformation to the assessed medium. In active techniques, particles are manipulated by an external force, most commonly produced through optical and magnetic fields. Small-scale rheology holds significant advantages over conventional bulk rheology, such as eliminating the need for large sample sizes, the ability to probe fragile materials non-destructively, and a wider probing frequency range. More importantly, some microrheological techniques can obtain spatiotemporal information of local microenvironments and accurately describe the heterogeneity of structurally complex fluids. Recently, there has been significant growth in using these minimally invasive techniques to investigate a wide range of biomedical systems both in vitro and in vivo. Here, we review the latest applications and advancements of microrheology in mammalian cells, tissues, and biofluids and discuss the current challenges and potential future advances on the horizon.
... In addition, as droplets shrink, the confinement effect of the oil/water interface on the particle movement needs to be considered. Based on Faxen's law, the extension of hindrance on particle mobility depends on the particle radius,a , and the distance from the particles to the interface,l , as shown in Eq. (4) [37,38], where b 0 and b l represent the free and hindered mobility, respectively. From Eq. (4), larger particles are more strongly affected by confinement, the following discussion is based on particles with radius of 0.50 m . ...
... the probes is suggested to be less than 0.01 to avoid brightness saturation during MPT [38]. The initial concentration of 0.5 m and 0.26 m probes are 0.072% and 0.036%, respectively, therefore the droplet is not recommended to shrink more than 15 and 30 times correspondingly, which is the same as the fold change of solute concentration. ...
... Second, the concentration of the probes needs to be chosen carefully to provide good statistics for the MSD calculation. Meanwhile, the probe particles concentration needs to be maintained lower than 0.01 during droplet shrinking to prevent brightness saturation [38], which also affects the extent of droplet shrinking and the increase in solute concentration. Additionally, proteins are surface active, to avoid significant depletion effect, the loading concentration cannot be too low otherwise the assumed initial concentration in the droplet will be incorrect. ...
Purpose
Measurement of the viscosity of concentrated protein solutions is vital for the manufacture and delivery of protein therapeutics. Conventional methods for viscosity measurements require large solution volumes, creating a severe limitation during the early stage of protein development. The goal of this work is to develop a robust technique that requires minimal sample.
Methods
In this work, a droplet-based microfluidic device is developed to quantify the viscosity of protein solutions while concentrating in micrometer-scale droplets. The technique requires only microliters of sample. The corresponding viscosity is characterized by multiple particle tracking microrheology (MPT).
Results
We show that the viscosities quantified in the microfluidic device are consistent with macroscopic results measured by a conventional rheometer for poly(ethylene) glycol (PEG) solutions. The technique was further applied to quantify viscosities of well-studied lysozyme and bovine serum albumin (BSA) solutions. Comparison to both macroscopic measurements and models (Krieger-Dougherty model) demonstrate the validity of the approach.
Conclusion
The droplet-based microfluidic device provides accurate quantitative values of viscosity over a range of concentrations for protein solutions with small sample volumes (~ μL) and high compositional resolution. This device will be extended to study the effect of different excipients and other additives on the viscosity of protein solutions.
Graphical abstract
... 22,23 Particle tracking micro-rheology (PTM) is another passive approach that involves injecting fluorescently labeled sub-micron beads into the cytoplasm of live cells. 24,25 High-frame rate fluorescent video microscopy is used to track the motion of particles. Statistical averaging of particle trajectories yields the mean-square displacement (MSD) hΔr 2 ðtÞi. ...
... Statistical averaging of particle trajectories yields the mean-square displacement (MSD) hΔr 2 ðtÞi. 24 Replacing the MSD in the generalized Stokes-Einstein relation (GSER) returns the complex frequency-dependent G Ã of the cytoplasm over the frequency range of 0.1 to 10 s −1 . 26 The upper frequency is limited to the rate at which the particle position is recorded, i.e., the frame rate of the camera. ...
... In comparison, other passive techniques such as PTM resolve only considerably larger displacements via video microscopy and exhibit much smaller accessible range of G 0 in the order of a few Pa. 24,26 These distinct features of LSM permit extending the passive micro-rheology to the context of stiffer biological tissues, in which intrinsic scattering particles exhibit arbitrary concentrations and size distributions. ...
Significance:
The ability to measure the micro-mechanical properties of biological tissues and biomaterials is crucial for numerous fields of cancer research, including tumor mechanobiology, tumor-targeting drug delivery, and therapeutic development.
Aim:
Our goal is to provide a renewed perspective on the mainstream techniques used for micro-mechanical evaluation of biological tissues and biomimetic scaffoldings. We specifically focus on portraying the outlook of laser speckle micro-rheology (LSM), a technology that quantifies the mechanical properties of biomaterials and tissues in a rapid, non-contact manner.
Approach:
First, we briefly explain the motivation and significance of evaluating the tissue micro-mechanics in various fields of basic and translational cancer research and introduce the key concepts and quantitative metrics used to explain the mechanical properties of tissue. This is followed by reviewing the general active and passive themes of measuring micro-mechanics. Next, we focus on LSM and elaborate on the theoretical grounds and working principles of this technique. Then, the perspective for measuring the micro-mechanical properties via LSM is outlined. Finally, we draw an overview picture of LSM in cancer mechanobiology research.
Results:
With the continued emergence of new approaches for measuring the mechanical attributes of biological tissues, the field of micro-mechanical imaging is at its boom. As one of these competent innovations, LSM presents a tremendous potential for both technical maturation and prospective applications in cancer biomechanics and mechanobiology research.
Conclusion:
By elaborating the current viewpoint of LSM, we expect to accelerate the expansion of this approach to new territories in both technological domains and applied fields. This renewed perspective on LSM may also serve as a road map for other micro-mechanical measurement concepts to be applied for answering mechanobiological questions.
... Time-cure or time-concentration superposition analysis of microrheology experiments was introduced by Larsen and Furst [70]. To date, the tried-andtrue method to determine gelling time from microrheology data is mostly done through shifting the MSD curves by hand [71,75,76]. ...
... Exploring particle dynamics opens up exciting avenues for mechanical testing, as highlighted in previous studies [1,76]. AIUQ methods prove valuable in this context by generating smooth MSD, facilitating the connection of MSD data to frequency-dependent viscoelastic moduli using the Generalized Stokes-Einstein Relation (GSER) [93]. ...
Estimating parameters from data is a fundamental problem in physics, customarily done by minimizing a loss function between a model and observed statistics. In scattering-based analysis, researchers often employ their domain expertise to select a specific range of wavevectors for analysis, a choice that can vary depending on the specific case. We introduce another paradigm that defines a probabilistic generative model from the beginning of data processing and propagates the uncertainty for parameter estimation, termed ab initio uncertainty quantification (AIUQ). As an illustrative example, we demonstrate this approach with differential dynamic microscopy (DDM) that extracts dynamical information through Fourier analysis at a selected range of wavevectors. We first show that DDM is equivalent to fitting a temporal variogram in the reciprocal space using a latent factor model as the generative model. Then we derive the maximum marginal likelihood estimator, which optimally weighs information at all wavevectors, therefore eliminating the need to select the range of wavevectors. Furthermore, we substantially reduce the computational cost by utilizing the generalized Schur algorithm for Toeplitz covariances without approximation. Simulated studies validate that AIUQ significantly improves estimation accuracy and enables model selection with automated analysis. The utility of AIUQ is also demonstrated by three distinct sets of experiments: first in an isotropic Newtonian fluid, pushing limits of optically dense systems compared to multiple particle tracking; next in a system undergoing a sol-gel transition, automating the determination of gelling points and critical exponent; and lastly, in discerning anisotropic diffusive behavior of colloids in a liquid crystal. These outcomes collectively underscore AIUQ's versatility to capture system dynamics in an efficient and automated manner.
... If the MSD is proportional to the decorrelation time, it indicates that the droplets undergo Brownian motion in the emulsion, and the emulsion system is dominated by viscosity. If MSD is not proportional to the decorrelation time, it indicates that the free movement of the droplets in the organogels is blocked, and the emulsion gel system is dominated by elasticity [63]. Fresh organogels were tested for four hours to obtain the full MSD plot line. ...
... Conversely, the droplets under acidic pHs (≤5.0) and high ionic strengths (≥0.2 M) gave a scaled linearly MSD curve with clear plateau regions in the early stage, reflecting the viscoelastic characteristics of samples [63]. Samples at pH 5.0 and 0.2 M displayed congregated MSD plot lines, indicating the lack of interaction between droplets within the gel system under these conditions over time. ...
Organogels have emerged as potential alternatives to trans and saturated fats. We developed semi-solid stable organogels using egg yolk granules-chitosan complex (EYGs-CS). We investigated the impacts of pH and NaCl ionic strength on the structural, rheological, and stability characteristics of the organogels. We found that stable organogels formed under acidic pHs (≤ 5.0) and high ionic strengths (≥ 0.2 M), displaying small droplet sizes, excellent elasticity behavior, poor oil migration, and high stability. The efficient adsorption of EYGs-CS at the oil surfaces formed stable droplets with compact walls. A colloidal 3D-network was formed within the aqueous phase, resulting in elastic and firm organogels. The droplets under acidic conditions showed higher surface charges, which blocked their coalescence through the electrostatic repulsion. The electrostatic shielding at high ionic strengths caused high steric repulsion, resulting in higher stability. This work provides insight into the replacement of harmful fats with healthier alternatives.
... The principles and applications of particle-tracking microrheology of living cells followed here are detailed in an extensive review and tutorial (Wirtz, 2009;McGlynn et al, 2020). ...
... Tracking of bead movements was performed using TrackMate with the following parameters: 1 μm diameter for spot detection, threshold value of 1.0 to eliminate spurious spots, 3 μm max distance and 3 μm gap-closing max distance. Beads that were outof-focus, aggregated or did not complete an entire recording duration of 20 s were excluded for analysis due to violations to assumptions involved in inferring viscosity from particle movement (McGlynn et al, 2020). To obtain statistical averaging, > 200 beads were tracked to calculate the ensemble-averaged MSD as a function of lag time: MSD ¼ r 2 ¼ 4Dτ: ...
Microtubules typically promote nuclear centring during early embryonic divisions in centrosome-containing vertebrates. In acentrosomal mouse zygotes, microtubules also centre male and female pronuclei prior to the first mitosis, this time in concert with actin. How nuclear centring is brought about in subsequent acentrosomal embryonic divisions has not been studied. Here, using time-lapse imaging in mouse embryos, we find that although nuclei are delivered to the cell centre upon completion of the first mitotic anaphase, the majority do not remain stationary and instead travel all the way to the cortex in a microtubule-dependent manner. High cytoplasmic viscosity in 2-cell embryos is associated with non-diffusive mechanisms involving actin for subsequent nuclear centring when microtubules again exert a negative influence. Thus, following the first mitotic division, pro-centring actin-dependent mechanisms work against microtubule-dependent de-centring forces. Disrupting the equilibrium of this tug-of-war compromises nuclear centring and symmetry of the subsequent division potentially risking embryonic development. This circuitous centring process exposes an embryonic vulnerability imposed by microtubule-dependent de-centring forces.
... The following variables were obtained or derived when possible: anomalous diffusion exponent (α), particle effective surface charge (ζ), mucus pH, mucus salt concentration, and mucin concentration. The anomalous exponent, also known as the logarithmic slope of the mean-squared displacement in the microrheology community [26], was obtained from the subdiffusion equation: ...
... It is puzzling, however, that only a third of the experiments measured the anomalous exponent. One possible explanation is the fact that the anomalous exponent is a well-known emerging property, but the relationship between this exponent and the underlying molecular factors determining its value is not that established in the field yet [26]. Below, we argue that investigating the molecular basis of the anomalous exponent is the key to characterizing and controlling particle diffusion in mucus and other polymeric fluids at relevant biological timescales. ...
Mucus is a complex fluid that coats multiple organs in animals. Various physicochemical properties can alter the diffusion of microscopic particles in mucus, impacting drug delivery, virus infection, and disease development. The simultaneous effect of these physicochemical properties in particle diffusion, however, remains elusive. Here, we analyzed 106 published experiments to identify the most dominant factors controlling particle diffusion in mucus. The effective diffusion—defined using a one-second sampling time window across experiments—spanned seven orders of magnitude, from 10 –5 to 10 ² μm ² /s. Univariate and multivariate statistical analyses identified the anomalous exponent (the logarithmic slope of the mean-squared displacement) as the strongest predictor of effective diffusion, revealing an exponential relationship that explained 89 % of the variance. A theoretical scaling analysis revealed that a stronger correlation of the anomalous exponent over the generalized diffusion constant occurs for sampling times two orders of magnitude larger than the characteristic molecular (or local) displacement time. This result predicts that at these timescales, the molecular properties controlling the anomalous exponent, like particle–mucus unbinding times or the particle to mesh size ratio, would be the most relevant physicochemical factors involved in passive microrheology of particles in mucus. Our findings contrast with the fact that only one-third of the studies measured the anomalous exponent, and most experiments did not report the associated molecular properties predicted to dominate the motion of particles in mucus. The theoretical foundation of our work can be extrapolated to other systems, providing a guide to identify dominant molecular mechanisms regulating the mobility of particles in mucus and other polymeric fluids.
... MPT is a passive microrheological technique which is sensitive in the low viscosity and elastic moduli range. [23][24][25][26][27][28][29] In MPT, fluorescent probe particles are embedded in the sample and their Brownian motion is recorded using video microscopy. Particle motion is tracked with classic tracking algorithms and particle trajectories throughout the video are determined. ...
... where N A is Avogadro's number. 16 29 To make the sample chamber, we cut a coverslip into two 22 Â 7 mm pieces. ...
Rheological modifiers are added to formulations to tune rheology, enable function and drive phase changes requiring an understanding of material structure and properties. We characterize two colloidal rod systems during phase transitions using multiple particle tracking microrheology, which measures the Brownian motion of probes embedded in a sample. These systems include a colloid (monodisperse polyamide or polydisperse hydrogenated castor oil), surfactant (linear alkylbenzene sulfonate [LAS]), and nonabsorbing polymer (polyethylene oxide [PEO]) which drives gelation by depletion interactions. Phase transitions are characterized at all concentrations using time‐cure superposition. We determine that rheological evolution depends on LAS:colloid. The critical PEO concentration required to form a gel, cc/c*, is independent of LAS:colloid, critical relaxation exponent, n, is dependent on LAS:colloid, and both are independent of colloid polydispersity. n indicates the material structure at the phase transition. At LAS:colloid > 16, the scaffold is a tightly associated network and at LAS:colloid = 16 a loosely associated network.
... The MSD power law exponent, α, is a derivative quantitative parameter based on the EAMSD. It describes the diffusive mode of tracked particles and is calculated as the logarithmic gradient of the MSD-curve 27 : ...
Kaposi's sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus that has a tropism for endothelial cells and leads to the development of Kaposi's sarcoma, especially in people living with HIV. KSHV induces spindling in endothelial cells. The present study aimed to quantify morphological and mechanical changes in endothelial cells after infection with KSHV to assess their potential as diagnostic and therapeutic markers. Vascular (HuARLT2) and lymphatic endothelial cells (LEC) were infected with recombinant KSHV (rKSHV) by spinoculation, establishing stable infections (HuARLT2-rKSHV and LEC-rKSHV). Cellular changes were assessed using mitochondria-tracking microrheology and morphometric analysis. rKSHV infection increased cellular deformability, indicated by higher mitochondrial mean squared displacement (MSD) for short lag times. Specifically, MSD at τ = 0.19 s was 49.4% and 42.2% higher in HuARLT2-rKSHV and LEC-rKSHV, respectively, compared to uninfected controls. There were 23.9% and 36.7% decreases in the MSD power law exponents for HuARLT2-rKSHV and LEC-rKSHV, respectively, indicating increased cytosolic viscosity associated with rKSHV infection. Infected cells displayed a marked spindloid phenotype with an increase in aspect ratio (29.7%) and decreases in roundness (26.1%) and circularity (25.7%) in HuARLT2-rKSHV, with similar changes observed in LEC-rKSHV. The quantification of distinct KSHV-induced morpho-mechanical changes in endothelial cells demonstrates the potential of these changes as diagnostic markers and therapeutic targets. These findings offer novel avenues to describe how the cellular changes may contribute to disease progression and to investigate the broader implications of KSHV pathology.
... In this case, the thermal motion of tracers (if detectable) shall reflect the local elasticity according to microrheology. Specifically, the plateau MSD is related to elastic modulus G 0 by [25] ...
Molecular dynamics and mass transportation in porous structures provide a basis for us to understand catalysis, energy storage and generation, and biological processes in porous confinements. While conventional methods extract macroscopic information in an ensemble-averaged manner, we intend to follow the journey of individual particles and molecules in porous structures relevant to cigarette filters by tracking the single-object dynamics in real space and real time. Nanoparticles of various sizes are embedded in fibrous frameworks of agarose where small particles (50 nm) can explore pores and their connections, locally mapping out the porous structure, middle-sized particles (100 nm) are trapped in single pores to fluctuate within, and large particles (500 nm) are fully immobilized by surrounding fibers. This model system is relevant to the retention and filtration of tar particles or other kinds of particulate matters by fibrous cellulose frequently used in cigarette filters. A molecular tracer is loaded to zeolite-based porous structures, where the majority are fixated in space by adsorption or micropore trapping, exhibiting localized trajectories within a 10-nm radius, and the minority are mobile to scout macropores. This molecular system may elucidate on how aromatic molecules like PAHs are adsorbed and transported in a matrix of mixed micro-, meso-, and macropores.
... The static error is observed as a time-independent average error in each of the particles' displacements. 40,41 Thus, small static errors were observed in high viscosity, high shutter speed samples where tracer particles move little and they were subtracted by calculating the linear intercept at low s. A drift error is attributed to convective particle movement between individual frames. ...
The viscoelasticity of monoclonal antibodies (mAbs) is important during their production, formulation, and drug delivery. High concentration mAbs can provide higher efficacy therapeutics (e.g., during immunotherapy) and improved efficiency during their production (economy of scale during processing). Two humanized mAbs were studied (mAb-1 and mAb-2) with differing isoelectric points. Using high speed particle tracking microrheology, we demonstrated that the mAb solutions have significant viscoelasticities above concentrations of 40 mg/ml. Power law viscoelasticity was observed over the range of time scales ( 10 − 4–1 s) probed for the high concentration mAb suspensions. The terminal viscosity demonstrated an exponential dependence on mAb concentration (a modified Mooney relationship) as expected for charged stabilized Brownian colloids. Gelation of the mAbs was explored by lowering the pH of the buffer and a power law scaling of the gelation transition was observed, i.e., the exponent of the anomalous diffusion of the probe particles scaled inversely with the gelation time.
... The surface of the tracer particles is coated with a PEG brush, which is a common strategy to impede interactions of the particles with the cytoskeleton. [63][64][65] Prior to imaging, cells were washed twice with PBS to remove beads that have not been incorporated into cells. Subsequently the cells are incubated in KRBH buffer containing a low concentration of glucose (2.8 mM), to reach basal insulin secretion conditions, for 1 h at 37 • C. Live cell microscopy was then conducted using an inverted light microscope (Nikon Ti-2 A) equipped with a Hamamatsu ImageEM-X2 CCD camera, with a frame rate of 1 s −1 and an exposure time of 100 ms in an environmental chamber (Okolab) at 37 • C and 5% CO 2 atmosphere. ...
Pancreatic β-cells regulate glucose homeostasis through glucose-stimulated insulin secretion, which is hindered in type-2 diabetes. Transport of the insulin vesicles is expected to be affected by changes in the viscoelastic and transport properties of the cytoplasm. These are evaluated in-situ through particle-tracking measurements using a rat insulinoma β-cell line. The use of inert probes assists in decoupling the material properties of the cytoplasm from the active transport through cellular processes. The effect of glucose-stimulated insulin secretion is examined, and the subsequent remodeling of the cytoskeleton, at constant effects of cell activity, is shown to result in reduced mobility of the tracer particles. Induction of diabetic-like conditions is identified to alter the mean-squared displacement of the passive particles in the cytoplasm and diminish its reaction to glucose stimulation.
... Interestingly, at lower viscosity values, it is only the 2.8 ps reference pulse duration after the filter (our effective IRF) that is limiting the viscosity measurement to a value as low as 0.0065 mPa ⋅ s, which is more than two orders of magnitude lower than that of water (which is η~1mPa ⋅ s). Moreover, FL-HOM returns a resolution of <1% and is thus competitive to more established microrheology measurement techniques such as multiple particle tracking or optical tweezers 47,48 . At relatively high viscosity values, the noise in the decay function measurement at long times is the limiting factor affecting the measurements, yet the method still returns a~2−4% error in viscosity measurements. ...
Fluorescence Lifetime Imaging Microscopy in the time domain is typically performed by recording the arrival time of photons either by using electronic time tagging or a gated detector. As such the temporal resolution is limited by the performance of the electronics to 100’s of picoseconds. Here, we demonstrate a fluorescence lifetime measurement technique based on photon-bunching statistics with a resolution that is only dependent on the duration of the reference photon or laser pulse, which can readily reach the 1–0.1 picosecond timescale. A range of fluorescent dyes having lifetimes spanning from 1.6 to 7 picoseconds have been here measured with only ~1 s measurement duration. We corroborate the effectiveness of the technique by measuring the Newtonian viscosity of glycerol/water mixtures by means of a molecular rotor having over an order of magnitude variability in lifetime, thus introducing a new method for contact-free nanorheology. Accessing fluorescence lifetime information at such high temporal resolution opens a doorway for a wide range of fluorescent markers to be adopted for studying yet unexplored fast biological processes, as well as fundamental interactions such as lifetime shortening in resonant plasmonic devices.
... Applied engineering systems whose performance analyses depend on fluid flow measurement span from biochemical reactors (Devasenathipathy et al. 2002;Kováts et al. 2018;Romano et al. 2021;Hofmann et al. 2022;Romano et al. 2023) and fundamental fluid dynamics (Humphrey et al. 1974;Kasagi and Nishino 1991;Kim et al. 2016) to manufacturing (Eschner et al. 2019;Fischer et al. 2022) and civil (Bautista-Capetillo et al. 2014;Fu et al. 2015;Zhao et al. 2020) industry. The imaging system must fulfill resolution requirements relative to the physical system; however, no constraint is imposed on the overall scale of the physical system, PTV being applicable to nano, (Faez et al. 2015;Matsuura et al. 2018;Wang et al. 2018;Bos et al. 2021; Satake 2022) micro, (Devasenathipathy et al. 2002;Fan et al. 2010;Choi et al. 2012;Huang and Choma 2015;Marin et al. 2017;Kawaguchi et al. 2019;Landauer et al. 2019;Dehnavi et al. 2020;McGlynn et al. 2020;Savorana et al. 2022) meso, (Humphrey et al. 1974;Kasagi and Nishino 1991;Boukany and Wang 2008;Gaudin et al. 2014;Kim et al. 2016;Lee et al. 2019) and macroscale (Bautista-Capetillo et al. 2014;Fu et al. 2015;Aksamit and Pomeroy 2016;Zhao et al. 2020) systems. Selection of tracer particles and advanced post-processing analyses can improve PTV algorithms allowing in situ velocimetry measurement in previously unsuitable, sensitive, or inaccuracy-prone systems. ...
Development of experimental methods for in situ particle tracking velocimetry (PTV) is fundamental for allowing measurement of moving systems non-tailored for velocimetry. This investigation focuses on the development of a post-processing methodology for single-camera PTV, without laser-sheet illumination, tracking native air bubbles as tracer particles within a liquid drop of human insulin in microgravity. Human insulin functioned as a sufficiently complex, non-Newtonian fluid system for testing fluid measurement methodology. The PTV scenario was facilitated by microgravity technology known as the ring-sheared drop (RSD), aboard the International Space Station, which produced an optical imaging scenario and fluid flow geometry suitable as a testbed for PTV research. The post-processing methodology performed included five steps: (i) physical system characterization and native air bubble tracer identification, (ii) image projection and single-camera calibration, (iii) depth determination and 3D particle position determination, (iv) ray tracing and refraction correction, and (v) particle history and data expansion for suboptimal particles. Overall, this post-processing methodology successfully allowed for a total of 1085 particle measurements in a scenario where none were previously possible. Such post-processing methodologies have promise for application to other in situ PTV scenarios allowing better understanding of physical systems whose flow is difficult to measure and/or where PTV-specific optical elements (such as laser light sheets and dual-camera setups) are not permissible due to physical or safety constraints.
... Most developments and applications of particle tracking in the last three decades are found in biophysics [36,42,[49][50][51], where particle tracking is used extensively for micro-rheology with the help of the mean squared displacement (MSD) of particles. The primary reason for this application is the simplicity of the initial guess for linking particles -as they undergo Brownian motion, their expected subsequent location in a given timestep is identical to their initial location. ...
Background
Obtaining accurate displacement measurements for large material deformation and/or rotation presents a distinct challenge to digital image correlation (DIC) due to cumulative and decorrelation errors, particularly near material boundaries.
Objective
We aim to accurately measure the deformation gradient tensor near boundary discontinuities in situations of large deformation and large deformation gradients.
Methods
To achieve this goal, the locations of randomly distributed particles are tracked using an open-source particle-tracking software, Trackpy. A least-squares estimate of the deformation gradient tensor field uses nearest-neighbor material vectors and a first-order Finite Difference (FD) approximation, circumventing common errors in other methods. The error caused by FD approximation and that incurred by measurement are derived and tested with exhaustive numerical simulations. Furthermore, a uniaxial tensile test and mode-I fracture experiment are conducted with particle-embedded hydrogels to validate the method.
Results
Numerical results corroborate a theoretical expression of measurement error. They show that the FD error increases while the measurement error decreases for a growing estimating radius. Moreover, measurement error is linearly correlated to displacement noise. A benchmark uniaxial tensile test validates the accuracy of the proposed estimator, and the near-crack-tip measurements in a tensile fracture experiment demonstrate the estimator’s capabilities near a free surface, when a material undergoes large deformation and rotation. The results of the displacement and strain data are benchmarked against kinematic data obtained using an open-source DIC software, Ncorr. Computation time for both methods is compared.
Conclusions
A deformation gradient tensor estimator is developed based on a particle tracking technique and a least squares routine. Theoretical error bounds on the estimator are verified by numerical simulations, and the method’s capability is confirmed by physical experiments in evaluating large deformation and rotation near a free boundary. The proposed estimator is expected to open a door towards future material tests and experimental mechanics studies, especially in large deformation and large rotation scenarios.
... Among the image-based techniques, conventional MPT is used to analyze passive microrheology data to extract particle trajectories to calculate the mean square displacement (MSD) [34,[49][50][51]. MSD found from the Brownian motion of the probing particles reveals the frequency-dependent complex viscosity of the material [36,[52][53][54]. ...
We present a novel approach to perform passive microrheology. A method to measure the rheological properties of fluids from the Brownian motion of suspended particles. Rheological properties are found from the particles' mean square displacements (MSDs) as a function of measurement time lag. Current state-of-the-art approaches find the MSD by tracking multiple particles' trajectories. However, particle tracking approaches face many limitations, including low accuracy and high computational cost, and they are only applicable to low particle seeding densities. Here, we present a novel method, termed particle image rheometry (PIR), for estimating the particle ensemble MSD from the temporal evolution of the probability density function of the displacement as a function of measurement time lag. First, the probability density function (PDF) of the particle displacements for each time lag is found using a generalized ensemble image cross-correlation approach that eliminates the need for particle tracking. Then, PDFs are used to calculate the MSD from which the complex viscosity of the solution is measured. We evaluate the performance of PIR using synthetic datasets and show that it can achieve an error of less than 1% in passive microrheology measurements, which corresponds to a twofold lower error than existing methods. Finally, we compare the measured complex viscosity from PIR with bulk rheometry for a polymeric solution and show agreement between the two measurements.
... Mislocalizations in the trajectories were corrected using the surrounding axial estimates 35 . Sample drift was corrected by calculating the collective motion of all particles tracked in the frame and subtracting it from each of the individual trajectories 38 . ...
Single particle tracking in three dimensions is an indispensable tool for studying dynamic processes in various disciplines, including material sciences, physics, and biology, but often shows anisotropic three-dimensional spatial localization precision, which restricts the tracking precision, and/or a limited number of particles that can be tracked simultaneously over extended volumes. Here we developed an interferometric, three-dimensional fluorescence single particle tracking method based on conventional widefield excitation and temporal phase-shift interference of the emitted, high-aperture-angle, fluorescence wavefronts in a greatly simplified, free-running, triangle interferometer that enables tracking of multiple particles at the same time with <10-nm spatial localization precision in all three dimensions over extended volumes (~35 × 35 × 2 μm3) at video rate (25 Hz). We applied our method to characterize the microenvironment of living cells and up to ~40 μm deep in soft materials. Conventional widefield excitation and temporal phase-shift fluorescence interference in a basic 4π-interferometer enable video rate, multiple particle tracking with isotropic, 3D precision over large volumes in cells and deep in soft materials.
... Recent efforts have used microfluidic approaches to improve throughput and control the microenvironment to observe phase changes through changes in pH or temperature. 108 A high-throughput approach developed by Schultz and Furst used a T-junction drop generator to adjust drop composition through changes in relative flow rates of the fluid components. 109 The flow was stopped and particles included in the fluids were tracked to make microrheology measurements. ...
The use of microfluidics to make mechanical property measurements is increasingly common. Fabrication of microfluidic devices has enabled various types of flow control and sensor integration at micrometer length scales to interrogate biological materials. For rheological measurements of biofluids, the small length scales are well suited to reach high rates, and measurements can be made on droplet-sized samples. The control of flow fields, constrictions, and external fields can be used in microfluidics to make mechanical measurements of individual bioparticle properties, often at high sampling rates for high-throughput measurements. Microfluidics also enables the measurement of bio-surfaces, such as the elasticity and permeability properties of layers of cells cultured in microfluidic devices. Recent progress on these topics is reviewed, and future directions are discussed.
... Measurements of the velocity field is fundamental in understanding the behavior of flowing systems both natural, ranging from molecular biology (Bos et al., 2021;Faez et al., 2015;Huang and Choma, 2015;Wang et al., 2018) and cellular biology (Marin et al., 2017;Savorana et al., 2022;Wang et al., 2020) to physiology (Oeler et al., 2021;Sampath et al., 2018;Zhang et al., 2022) and the environment, (Aksamit and Pomeroy, 2016;Gaudin et al., 2014;Mujtaba and de Lima, 2018;Tagliavini et al., 2022;Tauro et al., 2019) and engineered, spanning biochemical reactors (Devasenathipathy et al., 2002;Hofmann et al., 2022;Kováts et al., 2018;Romano et al., 2023Romano et al., , 2021 and fundamental fluid dynamics (Humphrey et al., 1974;Kasagi and Nishino, 1991;Kim et al., 2016) to manufacturing (Eschner et al., 2019;Fischer et al., 2022) and civil (Bautista-Capetillo et al., 2014;Fu et al., 2015;Zhao et al., 2020) industry. The imaging system must fulfill resolution requirements relative to the physical system, however, no constraint is imposed on the overall scale of the physical system, PTV being applicable to nano, (Bos et al., 2021;Faez et al., 2015;Matsuura et al., 2018;Satake, 2022;Wang et al., 2018) micro, (Choi et al., 2012Dehnavi et al., 2020;Devasenathipathy et al., 2002;Fan et al., 2010;Huang and Choma, 2015;Kawaguchi et al., 2019;Landauer et al., 2019;Marin et al., 2017;McGlynn et al., 2020;Savorana et al., 2022;Wang et al., 2020;Wang.W et al., 2020) meso, (Boukany and Wang, 2008;Gaudin et al., 2014;Humphrey et al., 1974;Kasagi and Nishino, 1991;Kim et al., 2016;Lee et al., 2019) and macroscale (Aksamit and Pomeroy, 2016;Bautista-Capetillo et al., 2014;Fu et al., 2015;Zhao et al., 2020) systems. Selection of tracer particles and advanced post-processing analyses can improve PTV algorithms allowing in situ velocimetry measurement in previously unsuitable, sensitive, or inaccuracy-prone systems. ...
Development of experimental methods for in situ particle tracking velocimetry (PTV) is fundamental for allowing measurement of moving systems non-tailored for velocimetry. This investigation focuses on the development of a post-processing methodology for single-camera PTV, without laser-sheet illumination, tracking native air-bubbles as tracer particles within a liquid drop of human insulin in microgravity. This PTV scenario was facilitated by microgravity technology known as the ring-sheared drop (RSD), aboard the International Space Station, which produced an optical imaging scenario and fluid flow geometry suitable as a testbed for PTV research. The post processing methodology performed included five steps: (i) physical system characterization and native air-bubble tracer identification, (ii) image projection and single-camera calibration, (iii) depth determination and 3D particle position determination, (iv) ray tracing and refraction correction, and (v) particle history and data expansion for suboptimal particles. Overall, this post processing methodology successfully allowed for a total of 1,085 particle measurements in a scenario where none were previously possible. Such post processing methodologies have promise for application to other in situ PTV scenarios allowing better understanding of physical systems whose flow is difficult to measure and or where PTV-specific optical elements (such as laser lights sheets and dual camera setups) are not permissible due to physical or safety constraints.
... A microplate rheometer may be used for the study of cell cultures such as human tissues [22]. Particle-tracking microrheology in cells may be used without the need to perturb marker beads [23,24]. Here, the diffusion of a particle through the internal cellular landscape is observed through optical microscopy. ...
In eukaryotes, intracellular physicochemical properties like macromolecular crowding and cytoplasmic viscoelasticity influence key processes such as metabolic activities, molecular diffusion, and protein folding. However, mapping crowding and viscoelasticity in living cells remains challenging. One approach uses passive rheology in which diffusion of exogenous fluorescent particles internalised in cells is tracked and physicochemical properties inferred from derived mean square displacement relations. Recently, the crGE2.3 Fӧrster Resonance Energy Transfer (FRET) biosensor was developed to quantify crowding in cells, though it is unclear how this readout depends on viscoelasticity and the molecular weight of the crowder.
Here, we present correlative, multidimensional data to explore diffusion and molecular crowding characteristics of molecular crowding agents using super-resolved fluorescence microscopy and ensemble time-resolved spectroscopy. We firstly characterise in vitro and then apply these insights to live cells of budding yeast Saccharomyces cerevisiae. It is to our knowledge the first time this has been attempted. We demonstrate that these are usable both in vitro and in the case of endogenously expressed sensors in live cells. Finally, we present a method to internalise fluorescent beads as in situ viscoelasticity markers in the cytoplasm of live yeast cells, and discuss limitations of this approach including impairment of cellular function.
... This explains the use of a passive probing technique based on the motion of single particles, rather than pairs (the latter is particularly useful for probing heterogeneous media) [42]. Alternative methods of quantifying heterogeneity do exist, the most common being the van Hove analysis of the probability density functions (PDFs) of particle displacements at selected lag times-which should be Gaussian if the medium is homogeneous [41,43], corresponding to classical random walk statistics (measured in Cartesian coordinates) [44]. ...
Blood plasma (BP) is a borderline non-Newtonian fluid. Few studies have characterized the rheology of BP and even less focused on understanding its subtle viscoelastic traits, which were only somewhat recently acknowledged. We use passive microrheology to measure the bulk response of human plasma samples under shear at body and ambient temperatures. Evidence of subdiffusive behavior in the mean-squared displacement is observed at the highest frequencies probed, which we attribute to the stress relaxation of protein molecules or chains. Jeffreys-like complex shear moduli were computed thereof. The microenvironments of albumin, fibrinogen, and gamma-globulin solutions (key plasma proteins) were probed as well. Single proteins in an aqueous buffer showed no signs of viscoelasticity within experimental resolution. Conversely, mixed together, they appear to promote the same kind of short-term elastic behavior seen in plasma. All in all, a fresh look at the shear rheology of BP is presented.
... where τ stands for the lag time, k B T denotes the thermal energy, and a indicates the radius of the particle. The GSER is applicable in cases where several conditions are fulfilled: firstly, the length of the probe particle is much longer than the characteristic length span of the material, secondly, effects of inertia on the sensor probe and the fluid are ignorable, and thirdly, the length compression factor of the fluid is ignorable (Squires and Mason, 2010;McGlynn et al., 2020). AFM techniques evidenced that the elastic modulus of the lower invasive epithelial bladder cancer cells RT112 displayed a plateau modulus at the slower frequencies, which is not the case for the two other stronger invasive epithelial bladder cancer cells T24 and J82, implying that the invasiveness renders the cells to be less elastic (Abidine et al., 2021). ...
Viscoelasticity and its alteration in time and space has turned out to act as a key element in fundamental biological processes in living systems, such as morphogenesis and motility. Based on experimental and theoretical findings it can be proposed that viscoelasticity of cells, spheroids and tissues seems to be a collective characteristic that demands macromolecular, intracellular component and intercellular interactions. A major challenge is to couple the alterations in the macroscopic structural or material characteristics of cells, spheroids and tissues, such as cell and tissue phase transitions, to the microscopic interferences of their elements. Therefore, the biophysical technologies need to be improved, advanced and connected to classical biological assays. In this review, the viscoelastic nature of cytoskeletal, extracellular and cellular networks is presented and discussed. Viscoelasticity is conceptualized as a major contributor to cell migration and invasion and it is discussed whether it can serve as a biomarker for the cells’ migratory capacity in several biological contexts. It can be hypothesized that the statistical mechanics of intra- and extracellular networks may be applied in the future as a powerful tool to explore quantitatively the biomechanical foundation of viscoelasticity over a broad range of time and length scales. Finally, the importance of the cellular viscoelasticity is illustrated in identifying and characterizing multiple disorders, such as cancer, tissue injuries, acute or chronic inflammations or fibrotic diseases.
... The variance of the image difference is then computed to give σ 2 0 . In MPT, a "noise floor" is commonly quantified [12] and frequently subtracted from all data to give a more realistic estimate of the MSD [28][29][30]; however, such characterization is not currently routine for DDM. ...
Differential dynamic microscopy (DDM) is a form of video image analysis that combines the sensitivity of scattering and the direct visualization benefits of microscopy. DDM is broadly useful in determining dynamical properties including the intermediate scattering function for many spatiotemporally correlated systems. Despite its straightforward analysis, DDM has not been fully adopted as a routine characterization tool, largely due to computational cost and lack of algorithmic robustness. We present a comprehensive statistical framework that aims at quantifying error, reducing the computational order and enhancing the robustness of DDM analysis. We quantify the error, and propagate an independent noise term to derive a closed-form expression of the expected value and variance of the observed image structure function. Significantly, we propose an unbiased estimator of the mean of the noise in the observed image structure function, which can be determined experimentally and significantly improves the accuracy of applications of DDM. Furthermore, through use of Gaussian Process Regression (GPR), we find that predictive samples of the image structure function require only around 1% of the Fourier Transforms of the observed quantities. This vastly reduces computational cost, while preserving information of the quantities of interest, such as quantiles of the image scattering function, for subsequent analysis. The approach, which we call DDM with Uncertainty Quantification (DDM-UQ), is validated using both simulations and experiments with respect to accuracy and computational efficiency, as compared with conventional DDM and multiple particle tracking. Overall, we propose that DDM-UQ lays the foundation for important new applications of DDM, as well as to high-throughput characterization.
... Aside from cryopreservation so as to halt confounding mechanotranductive effects, traditional AFM was replaced for the assessment of hMSCs mechanical characteristics. To ideally evaluate spatiotemporal rheological properties of hMSCs, the passive microrheology technique, VPTM [16,17], was firstly employed to study Young's modulus during osteogenesis. Soft tissue was especially feasible and already ubiquitous for VPTM measurement. ...
Human mesenchymal stem cells (hMSCs) possess potential of bone formation and were proposed as ideal material against osteoporosis. Although interrogation of directing effect on lineage specification by physical cues has been proposed, how mechanical stimulation impacts intracellular viscoelasticity during osteogenesis remained enigmatic. Cyto-friendly 3D matrix was prepared with polyacrylamide and conjugated fibronectin. The hMSCs were injected with fluorescent beads and chemically-induced toward osteogenesis. The mechanical properties were assessed using video particle tracking microrheology. Inverted epifluorescence microscope was exploited to capture the Brownian trajectory of hMSCs. Mean square displacement was calculated and transformed into intracellular viscoelasticity. Two different stiffness of microspheres (12 kPa, 1 kPa) were established. A total of 45 cells were assessed. hMSCs possessed equivalent mechanical traits initially in the first week, while cells cultured in rigid matrix displayed significant elevation over elastic (G′) and viscous moduli (G″) on day 7 (p < 0.01) and 14 (p < 0.01). However, after two weeks, soft niches no longer stiffened hMSCs, whereas the effect by rigid substrates was consistently during the entire differentiation course. Stiffness of matrix impacted the viscoelasticity of hMSCs. Detailed recognition of how microenvironment impacts mechanical properties and differentiation of hMSCs will facilitate the advancement of tissue engineering and regenerative medicine.
... Aside from cryopreservation so as to halt confounding mechanotranductive effects, traditional AFM was replaced for the assessment of hMSCs mechanical characteristics. To ideally evaluate spatiotemporal rheological properties of hMSCs, the passive microrheology technique, VPTM [16,17], was rstly employed to study Young's modulus during osteogenesis. Soft tissue was especially feasible and already ubiquitous for VPTM measurement. ...
Background: Human mesenchymal stem cells (hMSCs) possess potential of bone formation and were proposed as ideal material for tissue regeneration against osteoporosis. Although a plethora of elucidation regarding the directing effect on lineage specification by physical cues has been proposed, how mechanical stimulations impact the intracellular viscoelasticity during osteo-lineage commitment remained enigmatic.
Methods: Cyto-friendly 3D matrix was prepared on soft-lithographed device. The substrate was basically polyacrylamide and conjugated with fibronectin. Stiffness of the scaffolds was determined by the mixing ratio of monomer and crosslinker. The hMSCs were injected with fluorescent beads by gene gun before being cultured in the matrix and chemically induced toward osteogenesis. The mechanical properties were assessed using video particle tracking microrheology. On post-induction day 0, 7, 14, 21, inverted epifluorescence microscope with charge-coupled camera was exploited to capture the projected Brownian trajectory of indwelling hMSCs. Mean square displacement thereof was calculated and transformed into intracellular viscoelasticity by generalized Stokes-Einstein equation.
Results: Two different stiffness of 3D culture microspheres (12 kPa, 1 kPa) were established. A total of 45 cells were assessed. Initially, hMSCs possessed equivalent mechanical traits in the first week. Nonetheless, cells cultured in rigid matrix displayed statistically significant elevation over elastic (G’) and viscous moduli (G”) on day 7 (G’: 192±8 vs. 114±8, p<0.01; G” 204±9 vs. 169±10 Pa, p<0.01) and day 14 (G’: 190±6 vs. 129±7, p<0.01; G” 222±10 vs. 161±9 Pa, p<0.01). To appreciate the trend of fluctuation, subsequent measurements were compared with the respective modulus at day 0. The soft niches no longer facilitated stiffening hMSCs, whereas the effect by rigid substrates was consistently during the entire differentiation course.
Conclusions: Stiffness of the matrix impacted the viscoelasticity of hMSCs. Detailed elucidation on how microenvironment impacts mechanical properties and differentiation of hMSCs will facilitate the advancement of tissue engineering and regenerative medicine.
The SARS-CoV-2 Nucleocapsid protein (N) performs several functions during the viral lifecycle, including transcription regulation and viral genome encapsulation. We hypothesized that N toggles between these functions via phosphorylation-induced conformational change, thereby altering N interactions with membranes and RNA. We found that phosphorylation changes how biomolecular condensates composed of N and RNA interact with membranes: phosphorylated N (pN) condensates form thin films, while condensates with unmodified N are engulfed. This partly results from changes in material properties, with pN forming less viscous and elastic condensates. The weakening of protein-RNA interaction in condensates upon phosphorylation is driven by a decrease in binding between pN and unstructured RNA. We show that phosphorylation induces a conformational change in the serine/arginine-rich region of N that increases interaction between pN monomers and decreases nonspecific interaction with RNA. These findings connect the conformation, material properties, and membrane-associated states of N, with potential implications for COVID-19 treatment.
Estimating parameters from data is a fundamental problem in physics, customarily done by minimizing a loss function between a model and observed statistics. In scattering-based analysis, it is common to work in the reciprocal space. Researchers often employ their domain expertise to select a specific range of wave vectors for analysis, a choice that can vary depending on the specific case. We introduce another paradigm that defines a probabilistic generative model from the beginning of data processing and propagates the uncertainty for parameter estimation, termed the ab initio uncertainty quantification (AIUQ). As an illustrative example, we demonstrate this approach with differential dynamic microscopy (DDM) that extracts dynamical information through minimizing a loss function for the squared differences of the Fourier-transformed intensities, at a selected range of wave vectors. We first show that the conventional way of estimation in DDM is equivalent to fitting a temporal variogram in the reciprocal space using a latent factor model as the generative model. Then we derive the maximum marginal likelihood estimator, which optimally weighs the information at all wave vectors, therefore eliminating the need to select the range of wave vectors. Furthermore, we substantially reduce the computational cost of computing the likelihood function without approximation, by utilizing the generalized Schur algorithm for Toeplitz covariances. Simulated studies of a wide range of dynamical systems validate that the AIUQ method improves estimation accuracy and enables model selection with automated analysis. The utility of AIUQ is also demonstrated by three distinct sets of experiments: first in an isotropic Newtonian fluid, pushing limits of optically dense systems compared to multiple particle tracking; next in a system undergoing a sol-gel transition, automating the determination of gelling points and critical exponent; and lastly, in discerning anisotropic diffusive behavior of colloids in a liquid crystal. These studies demonstrate that the new approach does not require manually selecting the wave vector range and enables automated analysis.
Inflammatory Bowel Disease (IBD) is a chronic inflammatory condition affecting the gastrointestinal tract (GIT). Glucagon‐like peptide‐2 (GLP‐2) analogs possess high potential in the treatment of IBD by enhancing intestinal repair and attenuating inflammation. Due to the enzymatic degradation and poor intestinal absorption, GLP‐2 analogs are administered parenterally, which leads to poor patient compliance. This work aims to develop IBD‐targeted nanoparticles (NPs) for the oral delivery of the GLP‐2 analog, Teduglutide (TED). Leveraging the overproduction of Reactive Oxygen Species (ROS) in the IBD environment, ROS‐sensitive NPs are developed to target the intestinal epithelium, bypassing the mucus barrier. PEGylation of NPs facilitates mucus transposition, but subsequent PEG removal is crucial for cellular internalization. This de‐PEGylation is possible by including a ROS‐sensitive thioketal linker within the system. ROS‐sensitive NPs are established, with the ability to fully de‐PEGylate via ROS‐mediated cleavage. Encapsulation of TED into NPs resulted in the absence of absorption in 3D in vitro models, potentially promoting a localized action, and avoiding adverse effects due to systemic absorption. Upon oral administration to colitis‐induced mice, ROS‐sensitive NPs are located in the colon, displaying healing capacity and reducing inflammation. Cleavable PEGylated NPs demonstrate effective potential in managing IBD symptoms and modulating the disease's progression.
Mucus is a dynamic biological hydrogel, composed primarily of the glycoprotein mucin, exhibits unique biophysical properties and forms a barrier protecting cells against a broad‐spectrum of viruses. Here, this work develops a polyglycerol sulfate‐based dendronized mucin‐inspired copolymer (MICP‐1) with ≈10% repeating units of activated disulfide as cross‐linking sites. Cryo‐electron microscopy (Cryo‐EM) analysis of MICP‐1 reveals an elongated single‐chain fiber morphology. MICP‐1 shows potential inhibitory activity against many viruses such as herpes simplex virus 1 (HSV‐1) and SARS‐CoV‐2 (including variants such as Delta and Omicron). MICP‐1 produces hydrogels with viscoelastic properties similar to healthy human sputum and with tuneable microstructures using linear and branched polyethylene glycol‐thiol (PEG‐thiol) as cross‐linkers. Single particle tracking microrheology, electron paramagnetic resonance (EPR) and cryo‐scanning electron microscopy (Cryo‐SEM) are used to characterize the network structures. The synthesized hydrogels exhibit self‐healing properties, along with viscoelastic properties that are tuneable through reduction. A transwell assay is used to investigate the hydrogel's protective properties against viral infection against HSV‐1. Live‐cell microscopy confirms that these hydrogels can protect underlying cells from infection by trapping the virus, due to both network morphology and anionic multivalent effects. Overall, this novel mucin‐inspired copolymer generates mucus‐mimetic hydrogels on a multi‐gram scale. These hydrogels can be used as models for disulfide‐rich airway mucus research, and as biomaterials.
Introduction:
Intranasal administration is an effective drug delivery routes in modern pharmaceutics. However, unlike other in vivo biological barriers, the nasal mucosal barrier is characterized by high turnover and selective permeability, hindering the diffusion of both particulate drug delivery systems and drug molecules. The in vivo fate of administrated nanomedicines is often significantly affected by nano-biointeractions.
Areas covered:
The biological barriers that nanomedicines encounter when administered intranasally are introduced, with a discussion on the factors influencing the interaction between nanomedicines and the mucus layer/mucosal barriers. General design strategies for nanomedicines administered via the nasal route are further proposed. Furthermore, the most common methods to investigate the characteristics and the interactions of nanomedicines when in presence of the mucus layer/mucosal barrier are briefly summarized.
Expert opinion:
Detailed investigation of nanomedicine-mucus/mucosal interactions and exploration of their mechanisms provide solutions for designing better intranasal nanomedicines. Designing and applying nanomedicines with mucus interaction properties or non-mucosal interactions should be customized according to the therapeutic need, considering the target of the drug, i.e. brain, lung or nose. Then how to improve the precise targeting efficiency of nanomedicines becomes a difficult task for further research.
Despite the critical role of hydrogels in material science and biotechnology, current methods for analyzing their formation lack real‐time monitoring and require complex sample preparation and instrumentation. In this work, an innovative methodology is introduced for the real‐time analysis of enzymatically catalyzed hydrogelation. Electrochemical impedance spectroscopy (EIS) coupled with interdigitated electrodes (IDEs) to sense and transduce the gelation reaction of model precursor carboxymethyl cellulose‐tyramine (CMC‐TA) conjugates that undergoes enzymatic cross‐linking by horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). Real‐time monitoring involves single‐frequency analyses at 3 × 10⁵ Hz, where the measured impedance consists solely of a resistive component, and the admittance equates to solution conductance. The gelation trajectories for all tested enzymatically cross‐linked hydrogel component combinations are determined by substituting the conductance data in the modified Michaelis–Menten kinetic model. Specifically, for CMC‐TA cross‐linked by HRP, the authors calculate apparent KM and kcat values of 82.1 µM and 95.5 s⁻¹, respectively. These findings are further validated through rheological characterization, including oscillatory shear measurements and microrheology. Overall, this research paves the way for a streamlined, accurate, and cost‐effective approach to controllable enzymatically initiated hydrogel synthesis, enhancing their successful application in various fields ranging from material science to biotechnology.
Monitoring the kinetic evolution of the molecular weight of a growing polymer is critical to understand and optimize polymerization reactions for materials development and discovery. In this work, we propose...
Covalent adaptable networks are designed for applications including cell and drug delivery and tissue regeneration. These applications require network degradation at physiological conditions and on a physiological timescale with microstructures...
Particle tracking (PT) microrheology is a passive microrheological approach that characterizes material properties of soft matter. Multicomponent materials with the ability to create extensive crosslinking, such as supra-assemblies, may exhibit a complex interplay of viscous and elastic properties with a substantial contribution of liquid phase still diffusing through the system. Microrheology analyzes the motion of microscopic beads immersed in a sample, making it possible to evaluate the rheological properties of biological supra-assemblies. This method requires only a small volume of the sample and a relatively simple, inexpensive experimental setup. The objective of this chapter is to describe the experimental procedures for the observation of particle motion, calibration of an optical setup for particle tracking, preparation of imaging chambers, and the use of image analysis software for particle tracking in viscoelastic nucleic acid-based supra-assemblies.Key wordsNucleic acidsSupra-assembliesViscoelastic propertiesMicrorheologyParticle tracking
Viscosity control is essential for the manufacturing and delivery of concentrated therapeutic proteins. Limited availability of the precious protein-based drugs hinders the characterization and screening of the formulation conditions with new types or different combinations of excipients. In this work, a droplet-based microfluidic device with incorporated multiple particle tracking microrheology (MPT) is developed to quantify the effects of two excipients, arginine hydrochloride (ArgHCl) and caffeine, on the viscosity of concentrated bovine gamma globulin (BGG) dispersions at two different values of pH. The effectiveness of both ArgHCl and caffeine show dependence on the BGG concentration and solution pH. The data set with high compositional resolution provides useful information to guide formulation with multiple viscosity-reducing excipients and quantification appropriate to start elucidating the connection to protein-protein interaction mechanisms. Overall, this work has demonstrated that the developed microfluidic approach has the potential to effectively assess the impact of multiple excipients on the viscosity and provide data for computational methods to predict viscosity for high concentration protein formulations.
Lymph nodes (LNs) are highly structured lymphoid organs that compartmentalize B and T cells in the outer cortex and inner paracortex, respectively, and are supported by a collagen-rich reticular network. Tissue material properties like viscoelasticity and diffusion of materials within extracellular spaces and their implications on cellular behavior and therapeutic delivery have been a recent topic of investigation. Here, we developed a nanoparticle system to investigate the rheological properties, including pore size and viscoelasticity, through multiple particle tracking (MPT) combined with LN slice cultures. Dense coatings with polyethylene glycol (PEG) allow nanoparticles to diffuse within the LN extracellular spaces. Despite differences in function in B and T cell zones, we found that extracellular tissue properties and mesh spacing do not change significantly in the cortex and paracortex, though nanoparticle diffusion was slightly reduced in B cell zones. Interestingly, our data suggest that LN pore sizes are smaller than the previously predicted 10-20 μm, with pore sizes ranging from 500 nm-1.5 μm. Our studies also confirm that LNs exhibit viscoelastic properties, with an initial solid-like response followed by stress-relaxation at higher frequencies. Finally, we found that nanoparticle diffusion is dependent on LN location, with nanoparticles in skin draining LNs exhibiting a higher diffusion coefficient and pore size compared to mesenteric LNs. Our data shed new light onto LN interstitial tissue properties, pore size, and define surface chemistry parameters required for nanoparticles to diffuse within LN interstitium. Our studies also provide both a tool for studying LN interstitium and developing design criteria for nanoparticles targeting LN interstitial spaces.
In early breast cancer progression, cancer cells invade through a nanoporous basement membrane (BM) as a first key step toward metastasis. This invasion is thought to be mediated by a combination of proteases, which biochemically degrade BM matrix, and physical forces, which mechanically open up holes in the matrix. To date, techniques that quantify cellular forces of BM invasion in 3D culture have been unavailable. Here, we developed cellular-force measurements for breast cancer cell invasion in 3D culture that combine multiple-particle tracking of force-induced BM-matrix displacements at the nanoscale, and magnetic microrheometry of localized matrix mechanics. We find that cancer-cell protrusions exert forces from picoNewtons up to nanoNewtons during invasion. Strikingly, the protrusions extension involves stepwise increases in force, in steps of 0.2 to 0.5 nN exerted from every 30 s to 6 min. Thus, this technique reveals previously unreported dynamics of force generation by invasive protrusions in cancer cells.
Human mesenchymal stem cells (hMSCs) are instrumental in the wound healing process. They migrate to wounds from their native niche in response to chemical signals released during the inflammatory phase of healing. At the wound, hMSCs downregulate inflammation and regulate tissue regeneration. Delivering additional hMSCs to wounds using cell-laden implantable hydrogels has the potential to improve healing outcomes and restart healing in chronic wounds. For these materials to be effective, cells must migrate from the scaffold into the native tissue. This requires cells to traverse a step-change in material properties at the implant-tissue interface. Migration of cells in material with highly varying properties is not well characterized. We measure 3D encapsulated hMSC migration and remodeling in a well-characterized hydrogel with a step-change in stiffness. This cell-degradable hydrogel is composed of 4-arm poly(ethylene glycol)-norbornene cross-linked with an enzymatically-degradable peptide. The scaffold is made with two halves of different stiffnesses separated by an interface where stiffness changes rapidly. We characterize changes in structure and rheology of the pericellular region using multiple particle tracking microrheology (MPT). MPT measures Brownian motion of embedded particles and relates it to material rheology. We measure more remodeling in the soft region of the hydrogel than the stiff region on day 1 post-encapsulation and similar remodeling everywhere on day 6. In the interface region, we measure hMSC-mediated remodeling along the interface and migration towards the stiff side of the scaffold. These results can improve materials designed for cell delivery from implants to a wound to enhance healing.
The viscoelastic properties of a cell cytoskeleton contain abundant information about the state of a cell. Cells show a response to a specific environment or an administered drug through changes in their viscoelastic properties. Studies of single cells have shown that chemical agents that interact with the cytoskeleton can alter mechanical cell properties and suppress mitosis. This envisions using rheological measurements as a non-specific tool for drug development, the pharmacological screening of new drug agents, and to optimize dosage. Although there exists a number of sophisticated methods for studying mechanical properties of single cells, studying concentration dependencies is difficult and cumbersome with these methods: large cell-to-cell variations demand high repetition rates to obtain statistically significant data. Furthermore, method-induced changes in the cell mechanics cannot be excluded when working in a nonlinear viscoelastic range. To address these issues, we not only compared narrow-gap rheometry with commonly used single cell techniques, such as atomic force microscopy and microfluidic-based approaches, but we also compared existing cell monolayer studies used to estimate cell mechanical properties. This review provides insight for whether and how narrow-gap rheometer could be used as an efficient drug screening tool, which could further improve our current understanding of the mechanical issues present in the treatment of human diseases.
Microrheology based on video microscopy of embedded tracer particles has the potential to be used for high-throughput protein-based materials characterization. This potential is due to a number of characteristics of the techniques, including the suitability for measurement of low sample volumes, noninvasive and noncontact measurements, and the ability to set up a large number of samples for facile, sequential measurement. In addition to characterization of the bulk rheological properties of proteins in solution, for example, viscosity, microrheology can provide insight into the dynamics and self-assembly of protein-based materials as well as heterogeneities in the microenvironment being probed. Specifically, passive microrheology in the form of multiple particle tracking and differential dynamic microscopy holds promise for applications in high-throughput characterization because of the lack of user interaction required while making measurements. Herein, recent developments in the use of multiple particle tracking and differential dynamic microscopy are reviewed for protein characterization and their potential to be applied in a high-throughput, automatable setting.
Among the new molecular tools available to scientists and engineers, some of the most useful include fluorescently tagged biomolecules. Tools, such as green fluorescence protein (GFP), have been applied to perform semi-quantitative studies on biological signal transduction and cellular structural dynamics involved in the physiology of healthy and disease states. Such studies focus on drug pharmacokinetics, receptor-mediated endocytosis, nuclear mechanobiology, viral infections, and cancer metastasis. In 1976, fluorescence recovery after photobleaching (FRAP), which involves the monitoring of fluorescence emission recovery within a photobleached spot, was developed. FRAP allowed investigators to probe two-dimensional (2D) diffusion of fluorescently-labelled biomolecules. Since then, FRAP has been refined through the advancements of optics, charged-coupled-device (CCD) cameras, confocal microscopes, and molecular probes. FRAP is now a highly quantitative tool used for transport and kinetic studies in the cytosol, organelles, and membrane of a cell. In this work, the authors intend to provide a review of recent advances in FRAP. The authors include epifluorescence spot FRAP, total internal reflection (TIR)/FRAP, and confocal microscope-based FRAP. The underlying mathematical models are also described. Finally, our understanding of coupled transport and kinetics as determined by FRAP will be discussed and the potential for future advances suggested.
Rheological modifiers tune product rheology with a small amount of material. To effectively use rheological modifiers, characterizing the rheology of the system at different compositions is crucial. Two colloidal rod system, hydrogenated castor oil and polyamide, are characterized in a formulation that includes a surfactant (linear alkylbenzene sulfonate) and a depletant (polyethylene oxide). We characterize both rod systems using multiple particle tracking microrheology (MPT) and bulk rheology and build phase diagrams over a large component composition space. In MPT, fluorescent particles are embedded in the sample and their Brownian motion is measured and related to rheological properties. From MPT, we determine that in both systems: (1) microstructure is not changed with increasing colloid concentration, (2) materials undergo a sol-gel transition as depletant concentration increases and (3) the microstructure changes but does not undergo a phase transition as surfactant concentration increases in the absence of depletant. When comparing MPT and bulk rheology results different trends are measured. Using bulk rheology we observe: (1) elasticity of both systems increase as colloid concentration increases and (2) the storage modulus does not change when PEO or LAS concentration is increased. The differences measured with MPT and bulk rheology are likely due to differences in sensitivity and measurement method. This work shows the utility of using both techniques together to fully characterize rheological properties over a large composition space. These gelation phase diagrams will provide a guide to determine the composition needed for desired rheological properties and eliminate trial-and-error experiments during product formulation.
Differential dynamic microscopy (DDM) is a form of video image analysis that combines the sensitivity of scattering and the direct visualization benefits of microscopy. DDM is broadly useful in determining dynamical properties including the intermediate scattering function for many spatiotemporally correlated systems. Despite its straightforward analysis, DDM has not been fully adopted as a routine characterization tool, largely due to computational cost and lack of algorithmic robustness. We present statistical analysis that quantifies the noise, reduces the computational order, and enhances the robustness of DDM analysis. We propagate the image noise through the Fourier analysis, which allows us to comprehensively study the bias in different estimators of model parameters, and we derive a different way to detect whether the bias is negligible. Furthermore, through use of Gaussian process regression (GPR), we find that predictive samples of the image structure function require only around 0.5%–5% of the Fourier transforms of the observed quantities. This vastly reduces computational cost, while preserving information of the quantities of interest, such as quantiles of the image scattering function, for subsequent analysis. The approach, which we call DDM with uncertainty quantification (DDM-UQ), is validated using both simulations and experiments with respect to accuracy and computational efficiency, as compared with conventional DDM and multiple particle tracking. Overall, we propose that DDM-UQ lays the foundation for important new applications of DDM, as well as to high-throughput characterization.
Interfacial instabilities present challenges in common industrial processes involving multiphase flows. One such phenomenon—the Saffman-Taylor instability—involves the formation of fingerlike protrusions when a less viscous fluid displaces a more viscous fluid. Recent studies have shown that complex surfactant-laden interfaces with surface rheological stresses resist interfacial deformation and alter the fluid dynamics of the system. Despite current progress, the impact of surface rheological stresses on the Saffman-Taylor instability is yet unknown. In this work, we demonstrate the stabilizing effect of surface rheology in radial viscous fingering using linear stability analysis. We quantify the growth rates of perturbations to show that surface viscosity slows the growth of the instability and results in thicker fingers. Finally, we highlight the quantitative changes that are predicted to occur when a typical surface viscous surfactant is present.
Living cells organize their internal space into dynamic condensates through liquid-liquid phase separation of multivalent proteins in association with cellular nucleic acids. Here we study how variations in nucleic acid (NA)-to-protein stoichiometry modulate the condensed phase organization and fluid dynamics in a model system of multi-component heterotypic condensates. Employing a multi-parametric approach of particle tracking video microscopy and optical-tweezer induced droplet fusion, we show that the interfacial tension, but not viscosity, of protein-NA condensates is controlled by the NA-to-protein ratio across the two-phase regime. In parallel, we utilize fluorescence correlation spectroscopy (FCS) to quantify protein and nucleic acid diffusion in the condensed phase. FCS measurements reveal that the diffusion of the component protein and NA within the condensate core is governed by the viscosity, and hence, also remains insensitive to the changes in NA-to-protein stoichiometry. Collectively, our results provide insights into the regulation of multi-component heterotypic liquid condensates, reflecting how the bulk mixture composition affects their core vs. surface organization and dynamical properties.
Cross-linked polymeric gels are an important class of materials with applications that broadly range from synthetic wound healing scaffolds to materials used in enhanced oil recovery. To effectively design these materials for each unique applications a deeper understanding of the structure and rheological properties as a function of polymeric interactions is required. Increasing the concentration of polymer in each scaffold increases physical interactions between the molecules that can be reflected in the material structure. To characterize the structure and material properties, we use multiple particle tracking microrheology (MPT) to measure scaffolds during gelation. In MPT, fluorescently labeled probe particles are embedded in the material and the Brownian motion of these particles is captured using video microscopy. Particle motion is related to rheological properties using the Generalized Stokes-Einstein Relation. In this work, we characterize gelation of a photopolymerized scaffold composed of a poly(ethylene glycol) (PEG)-acrylate backbone and a PEG-dithiol cross-linker. Scaffolds with backbone concentrations below and above the overlap concentration, concentration where polymer pervaded volume begins to overlap, are characterized. Using time-cure superposition (TCS) we determine the critical relaxation exponent, n, of each scaffold. The critical relaxation exponent is a quantitative measure of the scaffold structure and is similar to a complex modulus, G*, which is a measure of energy storage and dissipation. Our results show that below the overlap concentration the scaffold is a tightly cross-linked network, navg = 0.40 ± 0.03, which stores energy but can also dissipate energy. As polymeric interactions increase, we measure a step change in the critical relaxation exponent above the overlap concentration to navg = 0.20 ± 0.03. After the overlap concentration the scaffold has transitioned to a more tightly cross-linked network that primarily stores energy. Additionally, continuing to increase concentration results in no change in the scaffold structure. Therefore, we determined that the properties of this scaffold can be tuned above and below the overlap concentration by changing the polymer concentration but the structure will remain the same in each concentration regime. This is advantageous for a wide range of applications that require scaffolds with varying stiffness and the same scaffold architecture.
During wound healing, human mesenchymal stem cells (hMSCs) migrate to injuries to regulate inflammation and coordinate tissue regeneration. To enable migration, hMSCs re-engineer the extracellular matrix rheology. Our work determines the correlation between cell-engineered rheology and motility. We encapsulate hMSCs in a cell-degradable peptide-polymeric hydrogel and characterize the change in rheological properties in the pericellular region using multiple particle tracking microrheology. Previous studies determined that pericellular rheology is correlated with motility. Additionally, hMSCs re-engineer their microenvironment by regulating cell-secreted enzyme, matrix metalloproteinases (MMPs), activity by also secreting their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). We independently inhibit TIMPs and measure two different degradation profiles, reaction-diffusion and reverse reaction-diffusion. These profiles are correlated with cell spreading, speed and motility type. We model scaffold degradation using Michaelis-Menten kinetics, finding a decrease in kinetics between joint and independent TIMP inhibition. hMSCs ability to regulate microenvironmental remodeling and motility could be exploited in design of new materials that deliver hMSCs to wounds to enhance healing.
Hydrogels serve as valuable tools for studying cell–extracellular matrix interactions in three-dimensional environments that recapitulate aspects of native extracellular matrix. However, the impact of early protein deposition on cell behaviour within hydrogels has largely been overlooked. Using a bio-orthogonal labelling technique, we visualized nascent proteins within a day of culture across a range of hydrogels. In two engineered hydrogels of interest in three-dimensional mechanobiology studies—proteolytically degradable covalently crosslinked hyaluronic acid and dynamic viscoelastic hyaluronic acid hydrogels—mesenchymal stromal cell spreading, YAP/TAZ nuclear translocation and osteogenic differentiation were observed with culture. However, inhibition of cellular adhesion to nascent proteins or reduction in nascent protein remodelling reduced mesenchymal stromal cell spreading and nuclear translocation of YAP/TAZ, resulting in a shift towards adipogenic differentiation. Our findings emphasize the role of nascent proteins in the cellular perception of engineered materials and have implications for in vitro cell signalling studies and application to tissue repair. The extracellular matrix surrounding cells plays a significant role in their behaviour. The spreading, mechanosensing and differentiation of mesenchymal stem cells are shown to be dependent on the early deposition and remodelling of local nascent proteins within degradable and viscoelastic hydrogels.
As mechanical properties of cell culture substrates matter, methods for mechanical characterization of scaffolds on a relevant length scale are required. We used multiple particle tracking microrheology to close the gap between elasticity determined from bulk measurements and elastic properties sensed by cells. Structure and elasticity of macroporous, three-dimensional cryogel scaffolds from mixtures of hyaluronic acid (HA) and collagen (Coll) were characterized. Both one-component gels formed homogeneous networks, whereas hybrid gels were heterogeneous in terms of elasticity. Most strikingly, local elastic moduli were significantly lower than bulk moduli presumably due to non-equilibrium chain conformations between crosslinks. This was more pronounced in Coll and hybrid gels than in pure HA gels. Local elastic moduli were similar for all gels, irrespective of their different swelling ratio and bulk moduli. Fibroblast cell culture proved the biocompatibility of all investigated compositions. Coll containing gels enabled cell migration, adhesion and proliferation inside the gels.
Complex fluid flow in porous media is ubiquitous in many natural and industrial processes. Direct visualization of the fluid structure and flow dynamics is critical for understanding and eventually manipulating these processes. However, the opacity of realistic porous media makes such visualization very challenging. Micromodels, microfluidic model porous media systems, have been developed to address this challenge. They provide a transparent interconnected porous network that enables the optical visualization of the complex fluid flow occurring inside at the pore scale. In this Review, the materials and fabrication methods to make micromodels, the main research activities that are conducted with micromodels and their applications in petroleum, geologic, and environmental engineering, as well as in the food and wood industries, are discussed. The potential applications of micromodels in other areas are also discussed and the key issues that should be addressed in the near future are proposed.
Natural extracellular matrices (ECMs) are viscoelastic and exhibit stress relaxation. However, hydrogels used as synthetic ECMs for three-dimensional (3D) culture are typically elastic. Here, we report a materials approach to tune the rate of stress relaxation of hydrogels for 3D culture, independently of the hydrogel's initial elastic modulus, degradation, and cell-adhesion-ligand density. We find that cell spreading, proliferation, and osteogenic differentiation of mesenchymal stem cells (MSCs) are all enhanced in cells cultured in gels with faster relaxation. Strikingly, MSCs form a mineralized, collagen-1-rich matrix similar to bone in rapidly relaxing hydrogels with an initial elastic modulus of 17 kPa. We also show that the effects of stress relaxation are mediated by adhesion-ligand binding, actomyosin contractility and mechanical clustering of adhesion ligands. Our findings highlight stress relaxation as a key characteristic of cell-ECM interactions and as an important design parameter of biomaterials for cell culture.
Studies focused on understanding the role of matrix biophysical signals on cells, especially those when cells are encapsulated in hydrogels that are locally remodelled, are often complicated by appropriate methods to measure differences between the bulk and local material properties. From this perspective, stress-relaxing materials that allow long-term culture of embedded cells provide an opportunity to elucidate aspects of this biophysical signalling. In particular, rheological characterization of the stress relaxation properties allows one to link a bulk material measurement to local aspects of cellular functions by quantifying the corresponding cellular forces that must be applied locally. Here, embryonic stem cell-derived motor neurons were encapsulated in a well-characterized covalently adaptable bis-aliphatic hydrazone crosslinked PEG hydrogel, and neurite outgrowth was observed over time. Using fundamental physical relationships describing classical mechanics and viscoelastic materials, we calculated the forces and energies involved in neurite extension, the results of which provide insight to the role of biophysical cues on this process.
Biofilms are densely populated communities of microbial cells protected and held together by a matrix of extracellular polymeric substances. The structure and rheological properties of the matrix at the microscale influence the retention and transport of molecules and cells in the biofilm, thereby dictating population and community behavior. Despite its importance, quantitative descriptions of the matrix microstructure and microrheology are limited. Here, particle-tracking microrheology in combination with genetic approaches was used to spatially and temporally study the rheological contributions of the major exopolysaccharides Pel and Psl in Pseudomonas aeruginosa biofilms. Psl increased the elasticity and effective cross-linking within the matrix, which strengthened its scaffold and appeared to facilitate the formation of microcolonies. Conversely, Pel reduced effective cross-linking within the matrix. Without Psl, the matrix becomes more viscous, which facilitates biofilm spreading. The wild-type biofilm decreased in effective cross-linking over time, which would be advantageous for the spreading and colonization of new surfaces. This suggests that there are regulatory mechanisms to control production of the exopolysaccharides that serve to remodel the matrix of developing biofilms. The exopolysaccharides were also found to have profound effects on the spatial organization and integration of P. aeruginosa in a mixed-species biofilm model of P. aeruginosa-Staphylococcus aureus. Pel was required for close association of the two species in mixed-species microcolonies. In contrast, Psl was important for P. aeruginosa to form single-species biofilms on top of S. aureus biofilms. Our results demonstrate that Pel and Psl have distinct physical properties and functional roles during biofilm formation.
Mesenchymal stem cells (MSCs) are attractive candidates for inclusion in cell-based therapies by virtue of their abilities to home to wound sites. However, in-depth characterization of the specific effects of MSCs on their microenvironments is needed to realize their full therapeutic potentials. Furthermore, since MSCs of varying properties can be isolated from a diverse spectrum of tissues, a strategic and rational approach in MSC sourcing for a particular application has yet to be achieved. For example, MSCs that activate their proteolytic environments may promote tissue remodeling, while those from different tissue sources may inhibit proteases and promote tissue stabilization. This study attempts to address these issues by analyzing MSCs isolated from three adult tissue sources in terms of their effects on their proteolytic microenvironments. Human bone marrow, adipose, and traumatized muscle derived MSCs were compared in their soluble and cellular-associated MMP components and activity. For all types of MSCs, MMP activity associated with the cell surface, but activity levels and MMP profiles differed with tissue source. All MSC types bound exogenous active MMPs at their surfaces. MSCs were also able to activate exogenous proMMP-2 and proMMP-13. This is in marked contrast to the MSC soluble compartment, which strongly inhibited MMPs via endogenous TIMPs. The exact TIMP used to inhibit the exogenous MMP differed with MSC type. Thus, MSCs saturate their environment with both MMPs and TIMPs. Since they bind and activate MMPs at their surfaces, the net result is a very controlled pericellular localization of MMP activities by MSCs.
We present a new method for measuring the linear viscoelastic shear moduli of complex fluids. Using photodiode detection of laser light scattered from a thermally excited colloidal probe sphere, we track its trajectory and extract the moduli using a frequency-dependent Stokes-Einstein equation. Spectra obtained for polyethylene oxide in water are in excellent agreement with those found mechanically and using diffusing wave spectroscopy. Since only minute sample volumes are required, this method is well suited for biomaterials of high purity, as we demonstrate with a concentrated DNA solution.
We suggest a very simple memory integral constitutive equation for the stress in crosslinking polymers at their transition from liquid to solid state (gel point). The equation allows for only a single (!) material parameter, the strength S[Pas 1/2 ], and it is able to describe every known viscoelastic phenomenon at the gel point. Measurements were performed on polydimethylsiloxane model networks with balanced stoichiometry for which the crosslinking reaction has been stopped at different degrees of conversion. At the gel point, the loss and storage moduli were found to be congruent and proportional to ω 1/2 over a wide range of temperature (−50°C to +180°C) and five decades of frequency ω. The hypothesis is made that this behavior is valid in the entire range 0<ω<∞. This congruence hypothesis is consistent with the Kramers‐Kronig relation and leads to a constitutive equation which shows that, for our polymer, congruent functions G′(ω)=G″(ω) are as much a rheological property at the gel point as are infinite viscosity and zero equilibrium modulus. This makes it now possible to measure exactly the instant of gelation of a crosslinking polymer without having to stop the crosslinking reaction.
Cell migration through 3D tissue depends on a physicochemical balance between cell deformability and physical tissue constraints. Migration rates are further governed by the capacity to degrade ECM by proteolytic enzymes, particularly matrix metalloproteinases (MMPs), and integrin- and actomyosin-mediated mechanocoupling. Yet, how these parameters cooperate when space is confined remains unclear. Using MMP-degradable collagen lattices or nondegradable substrates of varying porosity, we quantitatively identify the limits of cell migration by physical arrest. MMP-independent migration declined as linear function of pore size and with deformation of the nucleus, with arrest reached at 10% of the nuclear cross section (tumor cells, 7 µm(2); T cells, 4 µm(2); neutrophils, 2 µm(2)). Residual migration under space restriction strongly depended upon MMP-dependent ECM cleavage by enlarging matrix pore diameters, and integrin- and actomyosin-dependent force generation, which jointly propelled the nucleus. The limits of interstitial cell migration thus depend upon scaffold porosity and deformation of the nucleus, with pericellular collagenolysis and mechanocoupling as modulators.
This review describes the design and fabrication of microfluidic systems in poly(dimethylsiloxane) (PDMS). PDMS is a soft polymer with attractive physical and chemical properties: elasticity, optical transparency, flexible surface chemistry, low permeability to water, and low electrical conductivity. Soft lithography makes fabrication of microfluidic systems in PDMS particularly easy. Integration of components, and interfacing of devices with the user, is also convenient and simpler in PDMS than in systems made in hard materials. Fabrication of both single and multilayer microfluidic systems is straightforward in PDMS. Several components are described in detail: a passive chaotic mixer, pneumatically actuated switches and valves, a magnetic filter, functional membranes, and optical components.
The feasibility of a covalent adaptable hydrogel (CAH) as an oral delivery platform is explored using μ²rheology, microrheology in a microfluidic device. CAH degradation is initiated by physiologically relevant pHs, including incubation at a single pH and consecutively at different pHs. At a single pH, we determine CAH degradation can be tuned by changing the pH, which can be exploited for controlled release. We calculate the critical relaxation exponent, which defines the gel-sol transition and is independent of the degradation pH. We mimic the changing pH environment through part of the gastrointestinal tract (pH 4.3 to 7.4 or pH 7.4 to 4.3) in our microfluidic device. We determine that dynamic material property evolution is consistent with degradation at a single pH. However, the time scale of degradation is reduced by the history of degradation. These investigations inform the design of this material as a new vehicle for targeted delivery.
The diverse requirements of hydrogels for tissue engineering motivates development of crosslinking reactions to fabricate hydrogel networks with specific features, particularly those amenable to the activity of biological materials (e.g. cells, proteins) that do not require exposure to UV light. We describe gelation kinetics for a library of thiolated polyethylene glycol (PEG-SH) hydrogels undergoing enzymatic crosslinking via horseradish peroxidase, a catalyst-driven reaction activated by hydrogen peroxide. We report use of small-amplitude oscillatory shear (SAOS) to quantify gelation kinetics as a function of reaction conditions (hydrogen peroxide and polymer concentration). We employ a novel approach to monitor the change of viscoelastic properties of hydrogels over the course of gelation (Δtgel) via the time derivative of the storage modulus (dG’/dt). This approach, fundamentally distinct from traditional methods for defining a gel point, quantifies the time interval over which gelation events occur. We report that gelation depends on peroxide and polymer concentration as well as system temperature, where the effects of hydrogen peroxide tend to saturate over a critical concentration. Further, this crosslinking reaction can be reversed using L-cysteine for rapid cell isolation, and the rate of hydrogel dissolution can be monitored using SAOS.
The ability to behave in a fluidlike manner fundamentally separates thermoset and thermoplastic polymers. Bridging this divide, covalent adaptable networks (CANs) structurally resemble thermosets with permanent covalent crosslinks but are able to flow in a manner that resembles thermoplastic behavior only when a dynamic chemical reaction is active. As a consequence, the rheological behavior of CANs becomes intrinsically tied to the dynamic reaction kinetics and the stimuli that are used to trigger those, including temperature, light, and chemical stimuli, providing unprecedented control over viscoelastic properties. CANs represent a highly capable material that serves as a powerful tool to improve mechanical properties and processing in a wide variety of polymer applications, including composites, hydrogels, and shape-memory polymers. This review aims to highlight the enabling material properties of CANs and the applied fields where the CAN concept has been embraced. Expected final online publication date for the Annual Review of Chemical and Biomolecular Engineering Volume 10 is June 7, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Human mesenchymal stem cells (hMSCs) are motile cells that migrate from their native niche to wounded sites where they regulate inflammation during healing. New materials are being developed as hMSC delivery platforms to enhance wound healing. To act as an effective wound healing material, the hydrogel must degrade at the same rate as tissue regeneration while maintaining high cell viability. This work determines the kinetics and mechanism of cell-mediated degradation in hMSC-laden poly(ethylene glycol) (PEG) hydrogels. We use a well-established hydrogel scaffold that is composed of a backbone of four-arm star PEG functionalized with norbornene that is cross-linked with a matrix metalloproteinase (MMP) degradable peptide. This peptide sequence is cleaved by cell-secreted MMPs, which allow hMSCs to actively degrade the hydrogel during motility. Three mechanisms of degradation are characterized: hydrolytic, non-cellular enzymatic and cell-mediated degradation. We use bulk rheology to characterize hydrogel material properties and quantify degradation throughout the entire reaction. Hydrolysis and non-cellular enzymatic degradation are first characterized in hydrogels without hMSCs, and follow first-order and Michaelis-Menten kinetics, respectively. High cell viability is measured in hMSC-laden hydrogels, even after shearing on the rheometer. After confirming hMSC viability, bulk rheology characterizes cell-mediated degradation. When comparing cell-mediated degradation to non-cellular degradation mechanisms, cell-mediated degradation is dominated by enzymatic degradation. This indicates hydrogels with hMSCs are degraded primarily due to cell-secreted MMPs and very little network structure is lost due to hydrolysis. Modeling cell-mediated degradation provides an estimate of the initial concentration of MMPs secreted by hMSCs. By changing the concentration of hMSCs, we determine the initial MMP concentration increases with increasing hMSC concentration. This work characterizes the rate and mechanism of scaffold degradation, giving new insight into the design of these materials as implantable scaffolds.
We present a comprehensive overview of microrheology, emphasizing the underlying theory, practical aspects of its implementation, and current applications to rheological studies in academic and industrial laboratories. Key methods and techniques are examined, including important considerations to be made with respect to the materials most amenable to microrheological characterization and pitfalls to avoid in measurements and analysis. The fundamental principles of all microrheology experiments are presented, including the nature of colloidal probes and their movement in fluids, soft solids, and viscoelastic materials. Microrheology is divided into two general areas, depending on whether the probe is driven into motion by thermal forces (passive), or by an external force (active). We present the theory and practice of passive microrheology, including an in-depth examination of the Generalized Stokes-Einstein Relation (GSER). We carefully treat the assumptions that must be made for these techniques to work, and what happens when the underlying assumptions are violated. Experimental methods covered in detail include particle tracking microrheology, tracer particle microrheology using dynamic light scattering and diffusing wave spectroscopy, and laser tracking microrheology. Second, we discuss the theory and practice of active microrheology, focusing specifically on the potential and limitations of extending microrheology to measurements of non-linear rheological properties, like yielding and shear-thinning. Practical aspects of magnetic and optical tweezer measurements are preseted. Finally, we highlight important applications of microrheology, including measurements of gelation, degradation, high-throughput rheology, protein solution viscosities, and polymer dynamics.
Multiple particle tracking microrheology (MPT) is a powerful tool for quantitatively characterizing rheological properties of soft matter. Traditionally, MPT uses a single particle size to characterize rheological properties. But in complex systems, MPT measurements with a single size particle can characterize distinct properties that are linked to the materials’ length scale dependent structure. By varying the size of probes, MPT can measure the properties associated with different length scales within a material. We develop a technique to simultaneously track a bi-disperse population of probe particles. 0.5 and 2 μm particles are embedded in the same sample and these particle populations are tracked separately using a brightness-based squared radius of gyration, R²g. Bi-disperse MPT is validated by measuring the viscosity of glycerol samples at varying concentrations. Bi-disperse MPT measurements agree well with literature values. This technique then characterizes a homogeneous poly(ethylene glycol)-acrylate:poly(ethylene glycol)-dithiol gelation. The critical relaxation exponent and critical gelation time are consistent and agree with previous measurements using a single particle. Finally, degradation of a heterogeneous hydrogenated castor oil colloidal gel is characterized. The two particle sizes measure a different value of the critical relaxation exponent, indicating that they are probing different structures. Analysis of material heterogeneity shows measured heterogeneity is dependent on probe size indicating that each particle is measuring rheological evolution of a length scale dependent structure. Overall, bi-disperse MPT increases the amount of information gained in a single measurement, enabling more complete characterization of complex systems that range from consumer care products to biological materials.
The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement during previous phase changes and applied shear. To determine the extent of these changes, we have developed a microfluidic device that enables repeated phase transitions induced by exchange of the surrounding fluid and microrheological characterization while limiting shear on the sample. This technique is µ²rheology, the combination of microfluidics and microrheology. The microfluidic device is a two-layer design with symmetric inlet streams entering a sample chamber that traps the gel sample in place during fluid exchange. Suction can be applied far away from the sample chamber to pull fluids into the sample chamber. Material rheological properties are characterized using multiple particle tracking microrheology (MPT). In MPT, fluorescent probe particles are embedded into the material and the Brownian motion of the probes is recorded using video microscopy. The movement of the particles is tracked and the mean-squared displacement (MSD) is calculated. The MSD is related to macroscopic rheological properties, using the Generalized Stokes-Einstein Relation. The phase of the material is identified by comparison to the critical relaxation exponent, determined using time-cure superposition. Measurements of a fibrous colloidal gel illustrate the utility of the technique. This gel has a delicate structure that can be irreversibly changed when shear is applied. µ²rheology data shows that the material repeatedly equilibrates to the same rheological properties after each phase transition, indicating that phase transitions do not play a role in microstructural changes. To determine the role of shear, samples can be sheared prior to injection into our microfluidic device. µ²rheology is a widely applicable technique for the characterization of soft matter enabling the determination of rheological properties of delicate microstructures in a single sample during phase transitions in response to repeated changes in the surrounding environmental conditions.
Human mesenchymal stem cells (hMSCs) dynamically remodel their microenvironment during basic processes, such as migration and differentiation. Migration requires extracellular matrix invasion, necessitating dynamic cell-material interactions. Understanding these interactions is critical to advancing materials design that harness and manipulate these processes for applications including wound healing and tissue regeneration. In this work, we encapsulate hMSCs in a cell-degradable poly(ethylene glycol)-peptide hydrogel to determine how cell-secreted enzymes, specifically matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), create unique pericellular microenvironments. Using multiple particle tracking microrheology (MPT), we characterize spatio-temporal rheological properties in the pericellular region during cell-mediated remodeling. In MPT, the thermal motion of probes embedded in the network is measured. A newly designed sample chamber that limits probe drift during degradation and minimizes high value antibody volumes required for cell treatments enables MPT characterization. Previous MPT measurements around hMSCs show that directly around the cell the scaffold remains intact with the cross-link density decreasing as distance from the cell increases. This degradation profile suggests that hMSCs are simultaneously secreting TIMPs, which are inactivating MMPs through MMP−TIMP complexes. By neutralizing TIMPs using antibodies, we characterize the changes in matrix degradation. TIMP inhibited hMSCs create a reaction-diffusion type degradation profile where MMPs are actively degrading the matrix immediately after secretion. In this profile, the cross-link density increases with increasing distance from the cell. This change in material properties also increases the speed of migration. This simple treatment could increase delivery of hMSCs to injuries to aid wound healing and tissue regeneration.
Rheological modifiers are subject to processing steps, including mixing and dilution, that can have permanent structural effects. This work investigates rheological changes of a fibrous colloid, hydrogenated castor oil (HCO), when sheared during sample preparation. HCO is a polydisperse system that undergoes phase transitions in response to osmotic pressure gradients. Two HCO materials are characterized during phase transitions, a nonsheared 4 wt. % gel and a presheared 0.125 wt. % solution. Material properties are measured using multiple particle tracking (MPT) microrheology, μ²rheology, the combination of microfluidics and MPT, and bulk rheology. MPT quantitatively determines the critical relaxation exponent, n, which is constant for a material. MPT determines n is dependent on the starting HCO material, indicating that preshear has changed the structure. μ²rheology identifies consistent equilibrium states during consecutive phase transitions. Bulk rheology determines that the nonsheared gel does not completely degrade into a sol, indicated by no G′ and G″ crossover. The presheared material has a crossover indicating a sol-gel transition. The phases of HCO are identified by comparison of rheological data to previous work by Wilkins et al. [Langmuir 25, 8951–8959 (2009)], who determined the structure of a similar colloidal fiber system using confocal microscopy. The equilibrium moduli at the completion of both experiments are similar and indicate that the scaffold is in a transitional phase. These three techniques give consistent measurements of the rheological properties, and indicate the structure of the scaffold by comparison to previous works. During degradation, nonsheared HCO gels change from entangled networks to a transitional state with fiber entanglement. During gelation, presheared HCO solutions transition from bundles in solution to an associated network of bundles with few entanglements. These measurements confirm that shear history can permanently change rheological properties, affecting the scaffolds applications.
Human mesenchymal stem cells (hMSCs) are encapsulated in synthetic matrix metalloproteinase (MMP) degradable poly(ethylene glycol)-peptide hydrogels to characterize cell-mediated degradation of the pericellular region using multiple particle tracking microrheology. The hydrogel scaffold is degraded by cell-secreted enzymes and cytoskeletal tension. We determine that cell-secreted enzymatic degradation can be the main contributor to changes in the pericellular region, with cytoskeletal tension playing a minimal role. Measured degradation profiles for untreated and myosin II inhibited hMSCs have with the highest cross-link density around the cell. We hypothesize that cells are also secreting tissue inhibitors of metalloproteinases (TIMPs) to inhibit MMPs, which allow cell spreading and attachment prior to motility. We develop a Michaelis-Menten competitive enzymatic inhibition model which accurately describes the degradation profile due to MMP-TIMP unbinding.
Cross-linked polymeric gels are widely used in applications ranging from biomaterial scaffolds to additives in enhanced oil recovery. Despite this, fundamental understanding of the effect of polymer concentration and reaction mechanism on the scaffold structure is lacking. We measure scaffold properties and structure during gelation using multiple particle tracking microrheology. To determine the effect of concentration, we measure gelation as polymer interactions are increased in the backbone precursor solution: below, at and above the overlap concentration, c*. To determine structural changes due to the gelation mechanism, we measure gelation between the same polymers undergoing both step- and chain-growth reactions using self-assembling maleimide:thiol and photo-initiated acrylate:thiol chemistries, respectively. We determine the critical relaxation exponent, n, a measure of structure. n decreases with increasing concentration, indicating a change from a percolated (c < c*) to a tightly cross-linked network (c* < c). The gelation mechanism does not have a measurable effect on the scaffold structure. This article is protected by copyright. All rights reserved.
We perform multiple particle tracking (MPT) on a thermally-gelling oil-in-water nanoemulsion system. Carboxylated and plain polystyrene probes are used to investigate the role of colloidal probe size and surface chemistry on MPT in the nanoemulsion system. As temperature increases, hydrophobic groups of PEG-based gelators (PEGDA) partition into the oil/water interface and bridge droplets. This intercolloidal attraction generates a wide variety of microstructures consisting of droplet-rich and droplet-poor phases. By tailoring the MPT colloidal probe surface chemistry, we can control the residence of probes in each domain, thus allowing us to independently probe each phase. Our results show stark differences in probe dynamics in each domain. For certain conditions, the mean squared displacement (MSD) can differ by over four orders of magnitude for the same probe size but different surface chemistry. Carboxylated probe surface chemistries result in "slippery" probes while plain polystyrene probes appear to tether to the nanoemulsion gel network. We also observe probe hopping between pores in the gel for carboxylated probes. Our approach demonstrates that probes with different surface chemistries are useful in probing the local regions of a colloidal gel and allows the measurement of local properties within structurally heterogeneous hydrogels.
Covalent adaptable hydrogels (CAHs) dynamically evolve when pushed out of equilibrium by force or change in environmental conditions. Adapting these materials for advanced biological applications, including 3D cell culture and drug delivery platforms, requires in-depth knowledge of the evolution of scaffold microstructure and rheological properties. We use multiple particle tracking microrheology to measure the changes in a poly(ethylene glycol)–hydrazone CAH structure and properties when pushed out of equilibrium by a single change in pH. We determine the CAH degrades rapidly at acidic pH with multiple cycles of almost complete degradation and gelation. At pH 7.1, the scaffold degrades and re-forms cross-links over approximately 1.5 weeks with small oscillations between degradation and gelation. These degradation cycles are well described with first- and second-order reaction kinetics. MPT is sensitive enough to measure the phase transitions in these materials giving new insight into how CAHs evolve and their potential uses in biological applications.
Microfluidic devices are finding increasing application as analytical systems, biomedical devices, tools for chemistry and biochemistry, and systems for fundamental research. Conventional methods of fabricating microfluidic devices have centered on etching in glass and silicon. Fabrication of microfluidic devices in poly(dimethylsiloxane) (PDMS) by soft lithography provides faster, less expensive routes than these conventional methods to devices that handle aqueous solutions. These soft-lithographic methods are based on rapid prototyping and replica molding and are more accessible to chemists and biologists working under benchtop conditions than are the microelectronics-derived methods because, in soft lithography, devices do not need to be fabricated in a cleanroom. This paper describes devices fabricated in PDMS for separations, patterning of biological and nonbiological material, and components for integrated systems.
A microfluidic device is designed to measure repeated phase transitions, gelation and degradation, on a single sample by exchanging the surrounding fluid while minimizing shear stress. This device enables quantitative microrheological characterization of material properties over multiple phase transitions, determining whether the material returns to the same equilibrium state. Fluid exchange is accomplished by using a two layer design, the sample is trapped in the first layer and the second layer is a well for the exchanging fluid. Fluid enters the sample chamber symmetrically creating equal pressure around the sample, trapping it in place. Multiple particle tracking (MPT) microrheology, a passive microrheological technique, measures the dynamic rheological properties during each phase transition. Combining rheological characterization and sample manipulation using microfluidics is termed μ²rheology. The utility of this technique is demonstrated by characterizing several phase transitions of a fibrous colloidal gel, hydrogenated castor oil. Gelation and degradation is induced by an osmotic pressure gradient created by contact with a glycerine based gelling agent and water, respectively. Several transitions are measured using a single sample. Nine transitions, five gel-sol and four sol-gel, are the maximum number of transitions characterized in a single sample. This microfluidic device and measurement technique is widely applicable and can be easily adapted to any system where solvent exchange is used to induce a change in material properties.
Employing information extracted from their translational dynamics, colloidal particles have served extensively as micro-sized probes in in situ analytical techniques such as particle image velocimetry and microrheology. Janus particles, partially shadowed by metal caps appear directional when imaged with an optical microscope, and hence detectable rotational dynamics are introduced into an otherwise spherically symmetric system. Current tracking methods require high-resolution imaging, are time consuming, or focus on bulk light intensity variations. Here, we introduce two image analysis methods that identify the actual body center of gold-capped Janus particles (AuJPs), as well as distinguish the particle orientation within the space. Method I tracks the optical centroid, determines the particle’s in-plane tilting angle, and locates its actual body center (i.e., the Optical Centroid-Tilting Angle-Geometry, OCTAG procedure). Method: II tracks the diffraction ring center and the moon-phase center revealing in-plane orientation (i.e., the Ring-Phase Dual Optical Centroids, RPDOC procedure). With both methods we are able to successfully identify actual body centers and orientations of AuJPs with a resolution of 0.5 pixels and an angular uncertainty of sub−10°. It is thus possible to probe the local medium response on sub-micrometer length scales. The data analysis is applied to track dilute suspensions of AuJPs undergoing Brownian motion in a viscous glycerol-water mixture of volume ratio 1 or 2. Mean-square displacement (MSD) and angular mean-square displacement (AMSD) extracted from the experimental measurements are related to medium viscosities according to the Stokes-Einstein equation and the Perrin rotational diffusion, respectively. The viscosities calculated based on the diffusion coefficients determined from analysis of the translational and rotational dynamics of the microprobes differ by less than 6%. The 6% deviation observed is attributed to wall and gravitational effects. Results indicate that Janus particles can serve as novel probes in microrheology, and their asymmetric surface properties may potentially be used to investigate particle-medium interactions.
Rheological modifiers are essential ingredients in commercial materials that exploit facile and repeatable phase transitions. Although rheological modifiers are used to change flow behavior or quiescent stability, the complex properties of particulate gels during dilution is not well studied. We characterize a dynamically evolving colloidal gel, hydrogenated castor oil (HCO), a naturally sourced material, used in consumer products. This HCO scaffold consists of fibrous colloids, a surfactant (linear alkylbenzene sulfonate) and water. The gel undergoes critical transitions, degradation and formation, in response to an osmotic pressure gradient. Multiple particle tracking microrheology (MPT) measures the evolving material properties. In MPT, fluorescent probe particles are embedded into the sample and Brownian motion is measured. MPT data are analyzed using time-cure superposition, identifying critical transition times and critical relaxation exponents for degradation and formation where tc,deg = 102.5 min, ndeg = 0.77 ± 0.09, tc,for = 31.9 min, and nfor = 0.94 ± 0.11, respectively. During degradation and formation HCO gels evolve heterogeneously, this heterogeneity is characterized spatially and temporally. Heterogeneity of the gel is quantified by comparing variances of single particle van Hove correlation functions using an F-test with a 95% confidence interval. HCO transitions have rheological heterogeneous microenvironments that are homogeneously distributed throughout the field of view. Although HCO gels do evolve heterogeneously, this work determines that these heterogeneities do not significantly change traditional MPT measurements but the analysis techniques developed provide additional information on the unique heterogeneous scaffold microenvironments. This creates a toolbox that can be widely applied to other scaffolds during dynamic transitions.
Video-based particle tracking microrheology that requires ∼2 μl per sample is used to measure the viscosity of proteinsolutions of monoclonal antibodies. Direct imaging provides an immediate assessment of probe stability and the validity of the microrheology measurement. Precise measurements are made by choosing a displacement lag time that is a balance between minimizing tracking error while maximizing the number of sampled particle displacements. The excess kurtosis α2 of the probe displacement probability distribution and its test statistic Zα2 are used to set the optimal lag time. The viscosity is calculated by fitting a Gaussian distribution to the sampled displacements. Microrheology viscosities for two monoclonal antibody solutions are in good agreement with bulk rheology. Using a similar comparison of the microrheology of sucrose solutions with a correlation relating viscosity and concentration, an analysis of covariance (p = 0.941) demonstrates the high accuracy of small volume microrheology measurements. Based on the relative error between measured and tabulated viscosities, the uncertainty of viscosities derived from particle tracking is less than 2% of the true value.
Multiple particle tracking microrheology is used to characterize the viscoelastic properties of biomaterial and synthetic polymer gels near the liquid-solid transition. Probe particles are dispersed in the gel precursors, and their dynamics are measured as a function of the extent of reaction during gel formation. We interpret the dynamics using the generalized Stokes-Einstein relationship (GSER), using a form of the GSER that emphasizes the relationship between the probe particle mean-squared displacement and the material creep compliance. We show that long-standing concepts in gel bulk rheology are applicable to microrheological data, including time-cure superposition to identify the gel point and critical scaling exponents, and the power-law behavior of incipient network's viscoelastic response. These experiments provide valuable insight into the rheology, structure, and kinetics of gelling materials, and are especially powerful for studying the weak incipient networks of dilute gelators, as well as scarce materials, due to the small sample size requirements and rapid data acquisition.