Marie-Pierre Valignat

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. We show that Wet-SEEC allows both the observation of unprocessed nanofilms as low as 0.2 nm thick and their accurate 3D topographic reconstructions, directly by standard light microscopy. We next used Wet-SEEC to image slime secretion, a poorly defined property of many prokaryotic and eukaryotic organisms that move across solid surfaces in absence of obvious extracellular appendages (gliding). Using combined Wet-SEEC and fluorescent-staining experiments, we observed slime deposition by gliding Myxococcus xanthus cells at unprecedented resolution. Altogether, the results revealed that in this bacterium, slime associates preferentially with the outermost components of the motility machinery and promotes its adhesion to the substrate on the ventral side of the cell. Strikingly, analogous roles have been proposed for the extracellular proteoglycans of gliding diatoms and apicomplexa, suggesting that slime deposition is a general means for gliding organisms to adhere and move over surfaces.

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. Both effects impede a quantitative determination of forces experienced by cells in a constriction. Our study is based on a new microfluidic device composed of two successive constrictions, combined with optical interference microscopy measurements to characterize the contact zone between the cell and the walls of the channel. A cell squeezed in a first constriction obstructs most of the channel cross-section, which strongly limits leaks around cells. The rheological properties of the cell are subsequently probed during its entry in a second narrower constriction. The pressure force is determined from the pressure drop across the device, the cell velocity, and the width of the gutters formed between the cell and the corners of the channel. The additional friction force, which has never been analyzed for moving and constrained cells before, is found to involve both hydrodynamic lubrication and surface forces. This friction results in the existence of a threshold for moving the cells and leads to a non-linear behavior at low velocity. The friction force can nevertheless be assessed in the linear regime. Finally, an apparent viscosity of single cells can be estimated from a numerical prediction of the viscous dissipation induced by a small step in the channel. A preliminary application of our method yields an apparent loss modulus on the order of 100 Pa s for leukocytes THP-1 cells, in agreement with the literature data.

Surface enhanced ellipsometric contrast (SEEC) basic theory and λ/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. They are defined by the simple equation rp + rs = 0 between their two Fresnel coefficients. Most often, this equation can be met by covering a solid surface with a single λ/4 layer with a well defined refractive index. A higher flexibility is obtained with multilayer stacks. Solutions with an arbitrary number of all-dielectric λ/4 layers are derived.

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. We introduce a new normalization procedure that uses the intensity extrema of the same oscillation order for both experimental and theoretical intensity values and permits us to avoid significant error in the absolute height determination, especially at high INA. We also show how the problem of solution degeneracy can be solved by taking pictures at two different INA values. The model is applied to filled polystyrene beads and giant unilamellar vesicles of radius 10-40 microm sitting on a glass substrate. The RICM profiles I(h) can be fitted for up to two to three oscillation orders, and extrema positions are correct for up to five to seven oscillation orders. The precision of the absolute distance and of the shape of objects near a substrate is about 5 nm in a range from 0 to 500 nm, even under large numerical aperture conditions. The method is especially valuable for dynamic RICM experiments and with living cells where large illumination apertures are required.

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. We find that flow detection may not require molecular mechano-sensors of shear stress, and detection of flow direction can be achieved by the orientation in the flow of the non-adherent cell rear, the uropod. Uropods act as microscopic wind vanes that can transmit detection of flow direction into cell steering via the on-going machinery of polarity maintenance, without the need for novel internal guidance signalling triggered by flow. Contrary to chemotaxis, which implies active regulation of cue-dependent signalling, upstream flow mechanotaxis of lymphocytes may only rely on a passive self-steering mechanism.

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. The calculations agree with contrast measurements on nanometric steps of silica and explain also the extreme ability to detect single molecules (stretched DNA molecules).