Deformation microscopy for dynamic intracellular and intranuclear mapping of mechanics with high spatiotemporal resolution

Abstract

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 ...

Intra-Nuclear Tensile Strain Mediates Reorganization of Epigenetically Marked Chromatin During Cardiac Development and Disease

“…6c), To investigate the link between nuclear strain and chromatin reorganization in CMs, we performed an in-depth analysis of the strain occupancy for different chromatin types during CM contraction. For that we utilized a recently published method, termed deformation microscopy 31,32 , which generates high-resolution spatial strain maps from an undeformed template and a deformed target image ( Fig. 7a).…”

“…After two days on soft PDMS, image series (7.2 fps) of nuclear deformation during CM contractions were recorded on a Nikon A1R confocal microscope using a 60× oil immersion objective (0.14 µm/pix) via the expression of H2b-eGFP. Nuclear frames during diastolic rest and during peak contraction were used as template and target, respectively, to generate highresolution intranuclear strain maps via deformation microscopy developed previously 31,32 . The location of each cell was recorded through the use of gridded imaging dishes (µ-Dish 35 mm high Grid-500, ibidi).…”

“…Reduction of nuclear strains in stiff environments or disruption of LINC complexes inhibited the formation of the CM nuclear architecture and lead to the rearrangement of H3K27me3-modified chromatin instead. To find direct evidence for a link between nuclear deformation and chromatin reorganization in CMs, we used a recently developed method called deformation microscopy 31,32 to generate high resolution intra-nuclear strain maps from microscopy-based image series recorded during CM contractions and devised a workflow to map these strains back to chromatin regions with different epigenetic modifications.…”

Image-based elastography of heterochromatin and euchromatin domains in the deforming cell nucleus

“…Traditionally, the bulk nuclear deformation quantification and bulk mechanical properties of nuclei such as elastic modulus have been primary probed by research groups 19,20 without delving into detailed intranuclear mechanical characterization -mostly due to technical limitations. Recently, new technologies 21 have enabled us to calculate spatiotemporal displacements and generate high-resolution deformation maps in the nucleus from optical microscopy image data which enables our ability to ask and answer in depth questions regarding nuclear mechanobiology. However, as of now, no study exists that attempted to quantify intranuclear mechanical properties such as elastic modulus distribution although having this information will expand the predictive ability to quantify cell deformation by analytical and computational models.…”

TENSCell: A 3D-Printed Device for High-Magnification Imaging of Stretch-Activated Cells Reveals Divergent Nuclear Behavior to Different Levels of Strain

“…Cyclic stretch has been shown to change nuclear morphology (16)(17)(18), induce chromatin condensation (17)(18)(19), increase mechanical resistance of nuclei (16,18,20) and alter gene expression (21,22). Disruption of LINC complexes have shown to inhibit stretchinduced changes in chromatin remodeling (23)(24)(25) and gene expression (21,22), suggesting that strain transfer from the cytoskeleton plays an important role for nuclear mechanosensation. The actin skeleton, in particular, has been shown to be crucial for nuclear responses to dynamic mechanical stimulation (18,21,23,26).…”