Highlights d Deformation microscopy is developed by combining imaging and advanced mechanics d Modulation of nuclear LINC proteins or lamin A/C reveals altered intranuclear strain d Abnormal mechanical environments cause abnormal strain in high-density chromatin d Hyperosmotic conditions lead to nuclear strain asymmetry mediated by the cytoskeleton

The ability to control the position of micron-size particles with high precision using tools such as optical tweezers has led to major advances in fields such as biology, physics and material science. In this paper, we present a novel optical strategy to confine particles in solution with high spatial control using feedback-controlled thermoviscous flows. We show that this technique allows micron-size particles to be positioned and confined with subdiffraction precision (24 nm), effectively suppressing their diffusion. Due to its physical characteristics, our approach might be particular attractive where laser exposure is of concern or materials are inherently incompatible with optical tweezing since it does not rely on contrast in the refractive index.

Biological tissues and biomaterials are often defined by unique spatial gradients in physical properties that impart specialized function over hierarchical scales. The structure and organization of these materials forms continuous transitional gradients and discrete local microenvironments between adjacent (or within) tissues, and across matrix-cell boundaries, which can be difficult to replicate with common scaffold systems. Here, we studied the matrix densification of collagen leading to gradients in density, mechanical properties, and fibril morphology. High-density regions formed via a fluid pore pressure and flow-driven mechanism, with increased relative fibril density (10×), mechanical properties (20×, to 94.40±18.74kPa), and maximum fibril thickness (1.9×, to >1μm) compared to low-density regions, while maintaining porosity and fluid/mass transport to support viability of encapsulated cells. Similar to the organization of the articular cartilage zonal structure, we found that high-density collagen regions induced cell and nuclear alignment of primary chondrocytes. Chondrocyte gene expression was maintained in collagen matrices, and no phenotypic changes were observed as a result of densification. Densification of collagen matrices provides a unique, tunable platform for the creation of gradient systems to study complex cell-matrix interactions. These methods are easily generalized to compression and boundary condition modalities useful to mimic a broad range of tissues.

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The use of optical tweezers to measure forces acting upon microscopic particles has revolutionised fields from material science to cell biology. However, despite optical control capabilities, this technology is highly constrained by the material properties of the probe, and its use may be limited due to concerns about the effect on biological processes. Here we present a novel, optically controlled trapping method based on light-induced hydrodynamic flows. Specifically, we leverage optical control capabilities to convert a translationally invariant topological defect of a flow field into an attractor for colloids in an effectively one-dimensional harmonic, yet freely rotatable system. Circumventing the need to stabilise particle dynamics along an unstable axis, this novel trap closely resembles the isotropic dynamics of optical tweezers. Using magnetic beads, we explicitly show the existence of a linear force-extension relationship that can be used to detect femtoNewton-range forces with sensitivity close to the thermal limit. Our force measurements remove the need for laser-particle contact, while also lifting material constraints, which renders them a particularly interesting tool for the life sciences and engineering.

28Structural heterogeneity is a hallmark of living cells and nuclei that drives local mechanical 29 properties and dynamic cellular responses, including adhesion, gene expression, and 30 differentiation. However, robust quantification of intracellular or intranuclear mechanics are 31 lacking from conventional methods. Here, we describe new development of deformation 32 microscopy that leverages conventional imaging and an automated hyperelastic warping 33 algorithm to investigate strain history, deformation dynamics, and changes in structural 34 heterogeneity within the interior of cells and nuclei. Using deformation microscopy, we found 35 that tensile loading modes dominated intranuclear architectural dynamics in cardiomyocytes in 36 vitro or myocytes in vivo, which was compromised by disruption of LINC complex molecule 37 nesprin-3 or Lamin A/C, respectively. We also found that cells cultured on stiff substrates or in 38 hyperosmotic conditions displayed abnormal strain burden and asymmetries compared to controls 39 at interchromatin regions where active translation was expected. Deformation microscopy 40 represents a foundational approach toward intracellular elastography, with potential utility to 41 provide new mechanistic and quantitative insights in diverse mechanobiological applications. 42 43 44 45 46 47 48 49 50 Science Advances Manuscript Template Page 2 of 34 MAIN TEXT 51 52 93 registration (21). Over the course of the analysis, a penalty factor (21) is used to enforce the 94 registration. Additionally, regularization can be achieved using a hyperelastic material model. If 95 known, the associated material properties can be assigned to improve the strain estimates in 96 regions where image intensity differences may be lacking. Spatial averaging in the form of 97 normal or Gaussian blurring are used in the image analysis to avoid local minima which would 98 stop the global image registration prematurely resulting in false, unreliable deformation data (22). 99 Consequently, defining a suitable method to map deformation, or a correct set of parameters to 100 Science Advances Manuscript Template Page 3 of 34 obtain an optimal deformation map in a manageable time frame, is challenging and largely 101 lacking. 102 103 We introduce a technique, deformation microscopy, which utilizes an automated sweep over a 104 wide range of registration parameters, to quantify precise, high-resolution, and reliable spatial 105 patterns of intracellular displacements and strain. We demonstrated and validated the efficacy of 106 the technique across several biological scales, including examples of extracellular matrix, cell, 107 and nucleus deformation in vitro and in vivo. Next, we focused on applying the technique to 108 understand the spatiotemporal mechanics of nucleus in several normal and pathological 109 conditions. We altered the integrity of nuclear envelope by modulating the KASH domain, 110 nesprin-3, and Lamin A/C to understand their structural role in nuclear mechanics both in vitro 111 (cultured cells) and ...

Graphical Abstract Decellularized cartilage microparticles, and all associated native signals, are delivered to hMSC populations in a dense, type I collagen matrix. Hybrid usage of native tissue signals and the engineering control of collagen matrices show the ability to induce local infiltration and differentiation of hMSCs. Additionally, the solid cartilage microparticles inhibit bulk cell-mediated contraction of the composite.

Mechanisms of cellular and nuclear mechanosensation are unclear, partially because of a lack of methods that can reveal dynamic processes. Here, we present a new concept for a low-cost, three-dimensionally printed device that enables high-magnification imaging of cells during stretch. We observed that nuclei of mouse embryonic skin fibroblasts underwent rapid (within minutes) and divergent responses, characterized by nuclear area expansion during 5% strain but nuclear area shrinkage during 20% strain. Only responses to low strain were dependent on calcium signaling, whereas actin inhibition abrogated all nuclear responses and increased nuclear strain transfer and DNA damage. Imaging of actin dynamics during stretch revealed similar divergent trends, with F-actin shifting away from (5% strain) or toward (20% strain) the nuclear periphery. Our findings emphasize the importance of simultaneous stimulation and data acquisition to capture mechanosensitive responses and suggest that mechanical confinement of nuclei through actin may be a protective mechanism during high mechanical stretch or loading.