Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture.

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Emergent structures and dynamics of cell colonies by contact inhibition of locomotion.

Cells in tissues can organize into a broad spectrum of structures according to their function. Drastic changes of organization, such as epithelial-mesenchymal transitions or the formation of spheroidal aggregates, are often associated either to tissue morphogenesis or to cancer progression. Here, we study the organization of cell colonies by means of simulations of self-propelled particles with generic cell-like interactions. The interplay between cell softness, cell-cell adhesion, and contact inhibition of locomotion (CIL) yields structures and collective dynamics observed in several existing tissue phenotypes. These include regular distributions of cells, dynamic cell clusters, gel-like networks, collectively migrating monolayers, and 3D aggregates. We give analytical predictions for transitions between noncohesive, cohesive, and 3D cell arrangements. We explicitly show how CIL yields an effective repulsion that promotes cell dispersal, thereby hindering the formation of cohesive tissues. Yet, in continuous monolayers, CIL leads to collective cell motion, ensures tensile intercellular stresses, and opposes cell extrusion. Thus, our work highlights the prominent role of CIL in determining the emergent structures and dynamics of cell colonies.

Active Tensile Modulus of an Epithelial Monolayer.

A general trait of cell monolayers is their ability to exert contractile stresses on their surroundings. The scaling laws that link such contractile stresses with the size and geometry of constituent cells remain largely unknown. In this Letter, we show that the active tension of an epithelial monolayer scales linearly with the size of the constituent cells, a surprisingly simple relationship. The slope of this relationship defines an active tensile modulus, which depends on the concentration of myosin and spans more than 2 orders of magnitude across cell types and molecular perturbations.

Compressed sensing traction force microscopy.

Adherent cells exert traction forces on their substrate, and these forces play important roles in biological functions such as mechanosensing, cell differentiation and cancer invasion. The method of choice to assess these active forces is traction force microscopy (TFM). Despite recent advances, TFM remains highly sensitive to measurement noise and exhibits limited spatial resolution. To improve the resolution and noise robustness of TFM, here we adapt techniques from compressed sensing (CS) to the reconstruction of the traction field from the substrate displacement field. CS enables the recovery of sparse signals at higher resolution from lower resolution data. Focal adhesions (FAs) of adherent cells are spatially sparse implying that traction fields are also sparse. Here we show, by simulation and by experiment, that the CS approach enables circumventing the Nyquist-Shannon sampling theorem to faithfully reconstruct the traction field at a higher resolution than that of the displacement field. This allows reaching state-of-the-art resolution using only a medium magnification objective. We also find that CS improves reconstruction quality in the presence of noise. STATEMENT OF SIGNIFICANCE: A great scientific advance of the past decade is the recognition that physical forces determine an increasing list of biological processes. Traction force microscopy which measures the forces that cells exert on their surroundings has seen significant recent improvements, however the technique remains sensitive to measurement noise and severely limited in spatial resolution. We exploit the fact that the force fields are sparse to boost the spatial resolution and noise robustness by applying ideas from compressed sensing. The novel method allows high resolution on a larger field of view. This may in turn allow better understanding of the cell forces at the multicellular level, which are known to be important in wound healing and cancer invasion.

Hydraulic fracture during epithelial stretching.

The origin of fracture in epithelial cell sheets subject to stretch is commonly attributed to excess tension in the cells' cytoskeleton, in the plasma membrane, or in cell-cell contacts. Here, we demonstrate that for a variety of synthetic and physiological hydrogel substrates the formation of epithelial cracks is caused by tissue stretching independently of epithelial tension. We show that the origin of the cracks is hydraulic; they result from a transient pressure build-up in the substrate during stretch and compression manoeuvres. After pressure equilibration, cracks heal readily through actomyosin-dependent mechanisms. The observed phenomenology is captured by the theory of poroelasticity, which predicts the size and healing dynamics of epithelial cracks as a function of the stiffness, geometry and composition of the hydrogel substrate. Our findings demonstrate that epithelial integrity is determined in a tension-independent manner by the coupling between tissue stretching and matrix hydraulics.

Structure and dynamics of soft repulsive colloidal suspensions in the vicinity of the glass transition.

We use a combination of different scattering techniques and rheology to highlight the link between structure and dynamics of dense aqueous suspensions of soft repulsive colloids in the vicinity of a glass transition. Three different latex formulations with an increasing amount of the hydrophilic component resulting in either purely electrostatically or electrosterically stabilized suspensions are investigated. From the analysis of the static structure factor measured by small-angle X-ray scattering, we derive an effective volume fraction that includes contributions from interparticle interactions. We further investigate the dynamics of the suspensions using 3D cross-correlation dynamic light scattering (3DDLS) and rheology. We analyze the data using an effective hard sphere model and in particular compare the linear viscoelasticity and flow behavior to the predictions of mode coupling theory, which accounts for a purely kinetic glass transition determined by the equilibrium structure factor. We demonstrate that seemingly very different colloidal systems exhibit the same generic behavior when the effects from interparticle interactions are incorporated using an effective volume fraction description.

Multiple particle tracking investigations of acid milk gels using tracer particles with designed surface chemistries and comparison with diffusing wave spectroscopy studies.

Multiple particle tracking (MPT) has been used in an attempt to probe the heterogeneity of acid milk gels, made with and without added pectin, by following the distribution of the displacements of added tracer beads during and after gelation using the Van Hove distribution. Furthermore, the surface chemistry of the latex probe particles was modified in an attempt to control their location in the system and probe the microrheological properties of the protein network and aqueous-phase voids independently. In addition, the mean square displacement (MSD) of the casein micelles/casein aggregates themselves, obtained by diffusing wave spectroscopy (DWS), has been compared to the ensemble-averaged MSD calculated from the data obtained by tracking the movement of the added tracers, with and without a kappa-casein coating. For the kappa-casein-coated tracer particles, upon acidification and subsequent gel formation, the MSDs obtained by MPT superimpose remarkably well with the MSDs obtained by DWS, despite the fact that one is obtained by tracking the movement of the particle network elements themselves and the other is obtained from directly tracking added tracers. This result has important implications: (i) it demonstrates that, although the DWS measurement is intrinsically ensemble-averaged, it really gives insight into the dynamics of the colloidal gel network; (ii) it confirms that the kappa-casein-coated probes used in this MPT experiment are well incorporated into the gel network; and hence (iii) that at least in gelled systems kappa-casein-coated latex probes are interesting probes that reveal the dynamics of the casein network.

Microrheological investigations give insights into the microstructure and functionality of pectin gels.

Many of the functional attributes of pectin, whether in the plant cell wall or in engineered food materials, are linked to its gelling properties and in particular to its ability to assemble in the presence of calcium. Pectin's fine structure and local concentration relative to that of its cross-linking ion play a major role in determining resultant gel micro-structures, and consequently the mechanical and transport properties of pectin matrices. Recent studies have sought to probe the basic properties of such calcium-induced matrices, using a light scattering technique called diffusing wave spectroscopy (DWS). In addition to the low frequency mechanical behaviour, which provides information about the nature and density of cross-links, microrheological measurements carried out with DWS are able to determine the high frequency behaviour, which is closely linked to the response of the basic strands of the network. By using these microrheological measurements, two distinct regimes have been identified into which pectin gels appear to fall: one corresponding to the presence of semi-flexible networks, a generally accepted paradigm in biological gels, and another where flexible networks dominate. In order to explain the origin of these dramatically different networks, distinct assembly pathways have been proposed in which the relative importance of the free energy gained by association and the frictional barrier to polymeric re-arrangement during network formation can differ significantly. By manipulating the local environment in the plant cell wall it is possible that Nature makes full use of both of these network types for fulfilling different tasks; such as providing strain-hardening, maximizing local elastic properties or controlling macromolecular transport.