Keratocytes migrate against flow with a roly-poly-like mechanism.

While cell migration can be directed by various mechanical cues such as force, deformation, stiffness, or flow, the associated mechanisms and functions may remain elusive. Single cell migration against flow, repeatedly reported with leukocytes, is arguably considered as active and mediated by integrin mechanotransduction, or passive and determined by a mechanical bias. Here, we reveal a phenotype of flow mechanotaxis with fish epithelial keratocytes that orient upstream or downstream at shear stresses around tens of dyn cm.

Leukocyte transmigration and longitudinal forward-thrusting force in a microfluidic Transwell device.

Transmigration of leukocytes across blood vessels walls is a critical step of the immune response. Transwell assays examine transmigration properties in vitro by counting cells passages through a membrane; however, the difficulty of in situ imaging hampers a clear disentanglement of the roles of adhesion, chemokinesis, and chemotaxis. We used here microfluidic Transwells to image the cells' transition from 2D migration on a surface to 3D migration in a confining microchannel and measure cells longitudinal forward-thrusting force in microchannels.

ARHGAP45 controls naïve T- and B-cell entry into lymph nodes and T-cell progenitor thymus seeding.

T and B cells continually recirculate between blood and secondary lymphoid organs. To promote their trans-endothelial migration (TEM), chemokine receptors control the activity of RHO family small GTPases in part via GTPase-activating proteins (GAPs). T and B cells express several RHO-GAPs, the function of most of which remains unknown.

Human neutrophils swim and phagocytise bacteria.

Background Information: Leukocytes migrate in an amoeboid fashion while patrolling our organism in the search for infection or tissue damage. Their capacity to migrate has been proven integrin independent, however, non-specific adhesion or confinement remain a requisite in current models of cell migration. This idea has been challenged twice within the last decade with human neutrophils and effector T lymphocytes, which were shown to migrate in free suspension, a phenomenon termed swimming.

Amoeboid Swimming Is Propelled by Molecular Paddling in Lymphocytes.

Mammalian cells developed two main migration modes. The slow mesenchymatous mode, like crawling of fibroblasts, relies on maturation of adhesion complexes and actin fiber traction, whereas the fast amoeboid mode, observed exclusively for leukocytes and cancer cells, is characterized by weak adhesion, highly dynamic cell shapes, and ubiquitous motility on two-dimensional and in three-dimensional solid matrix. In both cases, interactions with the substrate by adhesion or friction are widely accepted as a prerequisite for mammalian cell motility, which precludes swimming.

Lymphocytes perform reverse adhesive haptotaxis mediated by LFA-1 integrins.

Cell guidance by anchored molecules, or haptotaxis, is crucial in development, immunology and cancer. Adhesive haptotaxis, or guidance by adhesion molecules, is well established for mesenchymal cells such as fibroblasts, whereas its existence remains unreported for amoeboid cells that require less or no adhesion in order to migrate. We show that, , amoeboid human T lymphocytes develop adhesive haptotaxis mediated by densities of integrin ligands expressed by high endothelial venules.

Three-dimensional imaging with reflection synthetic confocal microscopy.

Biomedical imaging lacks label-free microscopy techniques able to reconstruct the contour of biological cells in solution, in 3D and with high resolution, as required for the fast diagnosis of numerous diseases. Inspired by computational optical coherence tomography techniques, we present a tomographic diffractive microscope in reflection geometry used as a synthetic confocal microscope, compatible with this goal and validated with the 3D reconstruction of a human effector T lymphocyte.

Microfluidic device to study flow-free chemotaxis of swimming cells.

Microfluidic devices have been used in the last two decades to study in vitro cell chemotaxis, but few existing devices generate gradients in flow-free conditions. Flow can bias cell directionality of adherent cells and precludes the study of swimming cells like naïve T lymphocytes, which only migrate in a non-adherent fashion. We developed two devices that create stable, flow-free, diffusion-based gradients and are adapted for adherent and swimming cells.

A Bistable Mechanism Mediated by Integrins Controls Mechanotaxis of Leukocytes.

Recruitment of leukocytes from blood vessels to inflamed zones is guided by biochemical and mechanical stimuli, with the mechanisms only partially deciphered. Here, we studied the guidance by the flow of primary human effector T lymphocytes crawling on substrates coated with ligands of integrins lymphocyte function-associated antigen 1 (LFA-1) (αβ) and very late antigen 4 (VLA-4) (αβ). We reveal that cells segregate in two populations of opposite orientation for combined adhesion and show that decisions of orientation rely on a bistable mechanism between LFA-1-mediated upstream and VLA-4-mediated downstream phenotypes.

Live nanoscopic to mesoscopic topography reconstruction with an optical microscope for chemical and biological samples.

Macroscopic properties of physical and biological processes like friction, wetting, and adhesion or cell migration are controlled by interfacial properties at the nanoscopic scale. In an attempt to bridge simultaneously investigations at different scales, we demonstrate here how optical microscopy in Wet-Surface Ellipsometric Enhanced Contrast (Wet-SEEC) mode offers imaging and measurement of thin films at solid/liquid interfaces in the range 1-500 nm with lateral optical resolution. A live, label-free and noninvasive methodology integrated with microfluidic devices allowed here characterization of polymers and proteins patterns together with corresponding phenotypes of living cells.

Absolute 3D reconstruction of thin films topography in microfluidic channels by interference reflection microscopy.

The travel of droplets, bubbles, vesicles, capsules, living cells or small organisms in microchannels is a hallmark in microfluidics applications. A full description of the dynamics of such objects requires a quantitative understanding of the complex hydrodynamic and interfacial interactions between objects and channel walls. In this paper, we present an interferometric method that allows absolute topographic reconstruction of the interspace between an object and channel walls for objects confined in microfluidic channels.

Droplets in Microchannels: Dynamical Properties of the Lubrication Film.

We study the motion of droplets in a confined, micrometric geometry, by focusing on the lubrication film between a droplet and a wall. When capillary forces dominate, the lubrication film thickness evolves nonlinearly with the capillary number due to the viscous dissipation between the meniscus and the wall. However, this film may become thin enough (tens of nanometers) that intermolecular forces come into play and affect classical scalings.

ROZA-XL, an improved FRET based biosensor with an increased dynamic range for visualizing zeta associated protein 70 kD (ZAP-70) tyrosine kinase activity in live T cells.

Genetically encoded FRET based biosensors allow one to visualize the spatial and temporal evolution of specific enzyme activities in live cells. We have previously reported the creation of a FRET based biosensor specific for Zeta-Associated Protein -70 kD (ZAP-70) (Randriamampita et al., 2008), a Syk family protein tyrosine kinase.

Lymphocytes can self-steer passively with wind vane uropods.

A wide variety of cells migrate directionally in response to chemical or mechanical cues, however the mechanisms involved in cue detection and translation into directed movement are debatable. Here we investigate a model of lymphocyte migration on the inner surface of blood vessels. Cells orient their migration against fluid flow, suggesting the existence of an adaptive mechano-tranduction mechanism.

Single cell rheometry with a microfluidic constriction: Quantitative control of friction and fluid leaks between cell and channel walls.

We report how cell rheology measurements can be performed by monitoring the deformation of a cell in a microfluidic constriction, provided that friction and fluid leaks effects between the cell and the walls of the microchannels are correctly taken into account. Indeed, the mismatch between the rounded shapes of cells and the angular cross-section of standard microfluidic channels hampers efficient obstruction of the channel by an incoming cell. Moreover, friction forces between a cell and channels walls have never been characterized.

T lymphocytes orient against the direction of fluid flow during LFA-1-mediated migration.

As they leave the blood stream and travel to lymph nodes or sites of inflammation, T lymphocytes are captured by the endothelium and migrate along the vascular wall to permissive sites of transmigration. These processes take place under the influence of hemodynamic shear stress; therefore, we investigated how migrational speed and directionality are influenced by variations in shear stress. We examined human effector T lymphocytes on intercellular adhesion molecule 1 (ICAM-1)-coated surfaces under the influence of shear stresses from 2 to 60 dyn.

Wet-surface-enhanced ellipsometric contrast microscopy identifies slime as a major adhesion factor during bacterial surface motility.

In biology, the extracellular matrix (ECM) promotes both cell adhesion and specific recognition, which is essential for central developmental processes in both eukaryotes and prokaryotes. However, live studies of the dynamic interactions between cells and the ECM, for example during motility, have been greatly impaired by imaging limitations: mostly the ability to observe the ECM at high resolution in absence of specific staining by live microscopy. To solve this problem, we developed a unique technique, wet-surface enhanced ellipsometry contrast (Wet-SEEC), which magnifies the contrast of transparent organic materials deposited on a substrate (called Wet-surf) with exquisite sensitivity.

New modeling of reflection interference contrast microscopy including polarization and numerical aperture effects: application to nanometric distance measurements and object profile reconstruction.

We have developed a new and improved optical model of reflection interference contrast microscopy (RICM) to determine with a precision of a few nanometers the absolute thickness h of thin films on a flat surface in immersed conditions. The model takes into account multiple reflections between a planar surface and a multistratified object, finite aperture illumination (INA), and, for the first time, the polarization of light. RICM intensity I is typically oscillating with h.

Influence of surface reflective properties on differential interference contrast microscopy.

We present a model describing the image formation in DIC (Differential Interference Contrast) mode microscopy, by including the actual refractive indexes and reflection coefficients of objects and substrates. We calculate the contrast of flat and level objects of nanometric thickness versus the bias retardation Gamma and the numerical aperture NA. We show that high contrasts, of the edge and of the inner object, can be achieved in DIC mode with special anti-reflective substrates and large NA values.

Surface enhanced ellipsometric contrast (SEEC) basic theory and lambda/4 multilayered solutions.

The fundamentals of a new high contrast technique for optical microscopy, named "Surface Enhanced Ellipsometric Contrast" (SEEC), are presented. The technique is based on the association of enhancing contrast surfaces as sample stages and microscope observation between cross polarizers. The surfaces are designed to become anti-reflecting when used in these conditions.

Wide-field optical imaging of surface nanostructures.

We present a new technique that increases the sensitivity of incoherent light optical microscopy to a point where it becomes possible to directly visualize ultrathin films (approximately nanometers) and isolated nano-objects. The technique is based on the use of nonreflecting substrate surfaces for cross-polarized reflected light microscopy. These surfaces generate a contrast enhancement of about 2 orders of magnitude, extending the application field of wide-field optical microscopy toward the nanoworld.

Preparation of highly stable organic steps with a fullerene-based molecule.

We report the formation of highly stable Langmuir-Blodgett (LB) organic steps made with a hexa-adduct fullerene-based amphiphile. This amphiphile forms films of excellent quality, with a very low roughness, that are structurally stable: X-ray reflectivity spectra recorded on fresh and 12-month-old samples are undiscernible. Such a behavior contrasts with that of more traditional amphiphiles, which are unfortunately well-known for their instability in time.

Reversible self-assembly and directed assembly of DNA-linked micrometer-sized colloids.

We present a technique for the directed assembly and self-assembly of micrometer-scale structures based on the control of specific DNA linkages between colloidal particles. The use of DNA links combined with polymer brushes provides an effective way to regulate the range and magnitude of addressable forces between pairs (and further combinations) of different particles. We demonstrate that the autoassembly of alternate microbeads as well as their directed assembly, by using laser tweezers, is reversible.