Multiscale elasticity mapping of biological samples in 3D at optical resolution.

The mechanical properties of biological tissues have emerged as an integral determinant of tissue function in health and disease. Nonetheless, characterizing the elasticity of biological samples in 3D and at high resolution remains challenging. Here, we present a µElastography platform: a scalable elastography system that maps the elastic properties of tissues from cellular to organ scales. The platform leverages the use of a biocompatible, thermo-responsive hydrogel to deliver compressive stress to a biological sample and track its resulting deformation. By surrounding the specimen with a reference hydrogel of known Young's modulus, we are able to map the absolute values of elastic properties in biological samples. We validate the experimental and computational components of the platform using a hydrogel phantom and verify the system's ability to detect internal mechanical heterogeneities. We then apply the platform to map the elasticity of multicellular spheroids and the murine lymph node. With these applications, we demonstrate the platform's ability to map tissue elasticity at internal planes of interest, as well as capture mechanical heterogeneities neglected by most macroscale characterization techniques. The µElastography platform, designed to be implementable in any biology lab with access to 3D microscopy (e.g., confocal, multiphoton, or optical coherence microscopy), will provide the capability to characterize the mechanical properties of biological samples to labs across the large community of biological sciences by eliminating the need of specialized instruments such as atomic force microscopy. STATEMENT OF SIGNIFICANCE: Understanding the elasticity of biological tissues is of great importance, but characterizing these properties typically requires highly specialized equipment. Utilizing stimulus-responsive hydrogels, we present a scalable, hydrogel-based elastography method that uses readily available reagents and imaging modalities to generate resolved maps of internal elasticity within biomaterials and biological samples at optical resolution. This new approach is capable of detecting internal stiffness heterogeneities within the 3D bulk of samples and is highly scalable across both imaging modalities and biological length scales. Thus, it will have significant impact on the measurement capabilities of labs studying engineered biomaterials, mechanobiology, disease progression, and tissue engineering and development.

Crystal ribcage: a platform for probing real-time lung function at cellular resolution.

Understanding the dynamic pathogenesis and treatment response in pulmonary diseases requires probing the lung at cellular resolution in real time. Despite advances in intravital imaging, optical imaging of the lung during active respiration and circulation has remained challenging. Here, we introduce the crystal ribcage: a transparent ribcage that allows multiscale optical imaging of the functioning lung from whole-organ to single-cell level. It enables the modulation of lung biophysics and immunity through intravascular, intrapulmonary, intraparenchymal and optogenetic interventions, and it preserves the three-dimensional architecture, air-liquid interface, cellular diversity and respiratory-circulatory functions of the lung. Utilizing these capabilities on murine models of pulmonary pathologies we probed remodeling of respiratory-circulatory functions at the single-alveolus and capillary levels during disease progression. The crystal ribcage and its broad applications presented here will facilitate further studies of nearly any pulmonary disease as well as lead to the identification of new targets for treatment strategies.

Intravital measurements of solid stresses in tumours reveal length-scale and microenvironmentally dependent force transmission.

In cancer, solid stresses impede the delivery of therapeutics to tumours and the trafficking and tumour infiltration of immune cells. Understanding such consequences and the origin of solid stresses requires their probing in vivo at the cellular scale. Here we report a method for performing volumetric and longitudinal measurements of solid stresses in vivo, and findings from its applicability to tumours. We used multimodal intravital microscopy of fluorescently labelled polyacrylamide beads injected in breast tumours in mice as well as mathematical modelling to compare solid stresses at the single-cell and tissue scales, in primary and metastatic tumours, in vitro and in mice, and in live mice and post-mortem tissue. We found that solid-stress transmission is scale dependent, with tumour cells experiencing lower stresses than their embedding tissue, and that tumour cells in lung metastases experience substantially higher solid stresses than those in the primary tumours. The dependence of solid stresses on length scale and the microenvironment may inform the development of therapeutics that sensitize cancer cells to such mechanical forces.

The peritumor microenvironment: physics and immunity.

Cancer initiation and progression drastically alter the microenvironment at the interface between healthy and malignant tissue. This site, termed the peritumor, bears unique physical and immune attributes that together further promote tumor progression through interconnected mechanical signaling and immune activity. In this review, we describe the distinct physical features of the peritumoral microenvironment and link their relationship to immune responses. The peritumor is a region rich in biomarkers and therapeutic targets and thus is a key focus for future cancer research as well as clinical outlooks, particularly to understand and overcome novel mechanisms of immunotherapy resistance.

Correction: Anomalous and heterogeneous DNA transport in biomimetic cytoskeleton networks.

Correction for 'Anomalous and heterogeneous DNA transport in biomimetic cytoskeleton networks' by Jonathan Garamella et al., Soft Matter, 2020, DOI: 10.1039/d0sm00544d.

Anomalous and heterogeneous DNA transport in biomimetic cytoskeleton networks.

The cytoskeleton, a complex network of protein filaments and crosslinking proteins, dictates diverse cellular processes ranging from division to cargo transport. Yet, the role the cytoskeleton plays in the intracellular transport of DNA and other macromolecules remains poorly understood. Here, using single-molecule conformational tracking, we measure the transport and conformational dynamics of linear and relaxed circular (ring) DNA in composite networks of actin and microtubules with variable types of crosslinking. While both linear and ring DNA undergo anomalous, non-Gaussian, and non-ergodic subdiffusion, the detailed dynamics are controlled by both DNA topology (linear vs. ring) and crosslinking motif. Ring DNA swells, exhibiting heterogeneous subdiffusion controlled via threading by cytoskeleton filaments, while linear DNA compacts, exhibiting transport via caging and hopping. Importantly, while the crosslinking motif has little effect on ring DNA, linear DNA in networks with actin-microtubule crosslinking is significantly less ergodic and shows more heterogeneous transport than with actin-actin or microtubule-microtubule crosslinking.

Topology-dependent anomalous dynamics of ring and linear DNA are sensitive to cytoskeleton crosslinking.

Cytoskeletal crowding plays a key role in the diffusion of DNA molecules through the cell, acting as a barrier to effective intracellular transport and conformational stability required for processes such as transfection, viral infection, and gene therapy. Here, we elucidate the transport properties and conformational dynamics of linear and ring DNA molecules diffusing through entangled and crosslinked composite networks of actin and microtubules. We couple single-molecule conformational tracking with differential dynamic microscopy to reveal that ring and linear DNA exhibit unexpectedly distinct transport properties that are influenced differently by cytoskeleton crosslinking. Ring DNA coils are swollen and undergo heterogeneous and biphasic subdiffusion that is hindered by crosslinking. Conversely, crosslinking actually facilitates the single-mode subdiffusion that compacted linear chains exhibit. Our collective results demonstrate that transient threading by cytoskeleton filaments plays a key role in the dynamics of ring DNA, whereas the mobility of the cytoskeleton dictates transport of linear DNA.

Optical Tweezers Microrheology Maps the Dynamics of Strain-Induced Local Inhomogeneities in Entangled Polymers.

Optical tweezers microrheology (OTM) offers a powerful approach to probe the nonlinear response of complex soft matter systems, such as networks of entangled polymers, over wide-ranging spatiotemporal scales. OTM can also uniquely characterize the microstructural dynamics that lead to the intriguing nonlinear rheological properties that these systems exhibit. However, the strain in OTM measurements, applied by optically forcing a microprobe through the material, induces network inhomogeneities in and around the strain path, and the resultant flow field complicates the measured response of the system. Through a robust set of custom-designed OTM protocols, coupled with modeling and analytical calculations, we characterize the time-varying inhomogeneity fields induced by OTM measurements. We show that homogenization following strain does not interfere with the intrinsic stress relaxation dynamics of the system, rather it manifests as an independent component in the stress decay, even in highly nonlinear regimes such as with the microrheological large-amplitude-oscillatory-shear (MLAOS) protocols we introduce. Our specific results show that Rouse-like elastic retraction, rather than disentanglement and disengagement, dominates the nonlinear stress relaxation of entangled polymers at micro- and mesoscales. Thus, our study opens up possibilities of performing precision nonlinear microrheological measurements, such as MLAOS, on a wide range of complex macromolecular systems.

Effect of molecular architecture on ring polymer dynamics in semidilute linear polymer solutions.

Understanding the dynamics of ring polymers is a particularly challenging yet interesting problem in soft materials. Despite recent progress, a complete understanding of the nonequilibrium behavior of ring polymers has not yet been achieved. In this work, we directly observe the flow dynamics of DNA-based rings in semidilute linear polymer solutions using single molecule techniques. Our results reveal strikingly large conformational fluctuations of rings in extensional flow long after the initial transient stretching process has terminated, which is observed even at extremely low concentrations (0.025 c*) of linear polymers in the background solution. The magnitudes and characteristic timescales of ring conformational fluctuations are determined as functions of flow strength and polymer concentration. Our results suggest that ring conformational fluctuations arise due to transient threading of linear polymers through open ring chains stretching in flow.

Bridging the spatiotemporal scales of macromolecular transport in crowded biomimetic systems.

Crowding plays a key role in the transport and conformations of biological macromolecules. Gene therapy, viral infection, and transfection require DNA to traverse the crowded cytoplasm, including the cytoskeletal network of filamentous proteins. Given the complexity of cellular crowding, the dynamics of biological molecules can be highly dependent on the spatiotemporal scale probed. We present a powerful platform that spans molecular and cellular scales by coupling single-molecule conformational tracking (SMCT) and selective-plane illumination differential dynamic microscopy (SPIDDM). We elucidate the transport and conformational properties of large DNA, crowded by custom-designed networks of actin and microtubules, to link single-molecule conformations with ensemble DNA transport and cytoskeleton structure. We show that actin crowding leads to DNA compaction and suppression of fluctuations, combined with subdiffusion and heterogeneous transport, whereas microtubules have much more subdued impact across all scales. In composite networks of both filaments, scale-dependent effects emerge such that actin dictates ensemble DNA transport while microtubules influence single-molecule dynamics. We show that these intriguing results arise from a complex interplay between network rigidity, mesh size, filament concentration, and DNA size.

Crowding Induces Entropically-Driven Changes to DNA Dynamics That Depend on Crowder Structure and Ionic Conditions.

Macromolecular crowding plays a principal role in a wide range of biological processes including gene expression, chromosomal compaction, and viral infection. However, the impact that crowding has on the dynamics of nucleic acids remains a topic of debate. To address this problem, we use single-molecule fluorescence microscopy and custom particle-tracking algorithms to investigate the impact of varying macromolecular crowding conditions on the transport and conformational dynamics of large DNA molecules. Specifically, we measure the mean-squared center-of-mass displacements, as well as the conformational size, shape, and fluctuations, of individual 115 kbp DNA molecules diffusing through various in vitro solutions of crowding polymers. We determine the role of crowder structure and concentration, as well as ionic conditions, on the diffusion and configurational dynamics of DNA. We find that branched, compact crowders (10 kDa PEG, 420 kDa Ficoll) drive DNA to compact, whereas linear, flexible crowders (10, 500 kDa dextran) cause DNA to elongate. Interestingly, the extent to which DNA mobility is reduced by increasing crowder concentrations appears largely insensitive to crowder structure (branched vs. linear), despite the highly different configurations DNA assumes in each case. We also characterize the role of ionic conditions on crowding-induced DNA dynamics. We show that both DNA diffusion and conformational size exhibit an emergent non-monotonic dependence on salt concentration that is not seen in the absence of crowders.

Light-sheet microscopy with digital Fourier analysis measures transport properties over large field-of-view.

Using light-sheet microscopy combined with digital Fourier methods we probe the dynamics of colloidal samples and DNA molecules. This combination, referred to as selective-plane illumination differential dynamic microscopy (SPIDDM), has the benefit of optical sectioning to study, with minimal photobleaching, thick samples allowing us to measure the diffusivity of colloidal particles at high volume fractions. Further, SPIDDM exploits the inherent spatially-varying thickness of Gaussian light-sheets. Where the excitation sheet is most focused, we capture high spatial frequency dynamics as the signal-to-background is high. In thicker regions, we capture the slower dynamics as diffusion out of the sheet takes longer.

Particulates from hydrophilic-coated guiding sheaths embolise to the brain.

AIMS: We sought to evaluate the incidence of embolic material in porcine brains following vascular interventions using hydrophilic-coated sheaths. METHODS AND RESULTS: A new self-expanding stent and delivery system (SDS) was deployed through a hydrophilic-coated (Flexor Ansel; Cook Medical, Bloomington, IN, USA) guiding sheath into the iliac and/or carotid arteries of 23 anaesthetised Yucatan mini swine. The animals were euthanised at three, 30, 90 and 180 days and their brains were removed for histological analysis. In an additional single control animal, the guiding sheath was advanced but no SDS was deployed. Advancement of the coated guiding sheath with or without the SDS was associated with frequent foreign material in the arterioles of the brain. The embolic material was amorphous, non-refractile, non-crystalline, non-birefringent and typically lightly basophilic with a slightly stippled appearance on haematoxylin and eosin (H&E) stain. Material was observed at all time points involving 54% of all study animals (i.e., test and control) and in vitro after incubation in 0.9% saline. CONCLUSIONS: The hydrophilic coating on a clinically used guiding sheath readily avulses and embolises to the brain during deployment in a porcine model. Further documentation of this effect and monitoring in clinical scenarios are warranted.

DNA as a Model for Probing Polymer Entanglements: Circular Polymers and Non-Classical Dynamics.

Double-stranded DNA offers a robust platform for investigating fundamental questions regarding the dynamics of entangled polymer solutions. The exceptional monodispersity and multiple naturally occurring topologies of DNA, as well as a wide range of tunable lengths and concentrations that encompass the entanglement regime, enable direct testing of molecular-level entanglement theories and corresponding scaling laws. DNA is also amenable to a wide range of techniques from passive to nonlinear measurements and from single-molecule to bulk macroscopic experiments. Over the past two decades, researchers have developed methods to directly visualize and manipulate single entangled DNA molecules in steady-state and stressed conditions using fluorescence microscopy, particle tracking and optical tweezers. Developments in microfluidics, microrheology and bulk rheology have also enabled characterization of the viscoelastic response of entangled DNA from molecular levels to macroscopic scales and over timescales that span from linear to nonlinear regimes. Experiments using DNA have uniquely elucidated the debated entanglement properties of circular polymers and blends of linear and circular polymers. Experiments have also revealed important lengthscale and timescale dependent entanglement dynamics not predicted by classical tube models, both validating and refuting new proposed extensions and alternatives to tube theory and motivating further theoretical work to describe the rich dynamics exhibited in entangled polymer systems.