Submicrometre spatiotemporal characterization of the Toxoplasma adhesion strategy for gliding motility.

Toxoplasma gondii is a protozoan apicomplexan parasite that uses an adhesion-dependent mode of motility termed gliding to access host cells and disseminate into tissues. Previous studies on Apicomplexa motile morphotypes, including the T. gondii tachyzoite, have identified a cortical actin-myosin motor system that drives the rearward translocation of transmembrane adhesins, thus powering forward movement.

Competitive Specific Anchorage of Molecules onto Surfaces: Quantitative Control of Grafting Densities and Contamination by Free Anchors.

The formation of surfaces decorated with biomacromolecules such as proteins, glycans, or nucleic acids with well-controlled orientations and densities is of critical importance for the design of in vitro models, e.g., synthetic cell membranes and interaction assays.

Substrate stiffness impacts early biofilm formation by modulating twitching motility.

Surface-associated lifestyles dominate in the bacterial world. Large multicellular assemblies, called biofilms, are essential to the survival of bacteria in harsh environments and are closely linked to antibiotic resistance in pathogenic strains. Biofilms stem from the surface colonization of a wide variety of substrates encountered by bacteria, from living tissues to inert materials.

A quartz crystal microbalance method to quantify the size of hyaluronan and other glycosaminoglycans on surfaces.

Hyaluronan (HA) is a major component of peri- and extra-cellular matrices and plays important roles in many biological processes such as cell adhesion, proliferation and migration. The abundance, size distribution and presentation of HA dictate its biological effects and are also useful indicators of pathologies and disease progression. Methods to assess the molecular mass of free-floating HA and other glycosaminoglycans (GAGs) are well established.

A Method to Quantify Molecular Diffusion within Thin Solvated Polymer Films: A Case Study on Films of Natively Unfolded Nucleoporins.

We present a method to probe molecular and nanoparticle diffusion within thin, solvated polymer coatings. The device exploits the confinement with well-defined geometry that forms at the interface between a planar and a hemispherical surface (of which at least one is coated with polymers) in close contact and uses this confinement to analyze diffusion processes without interference of exchange with and diffusion in the bulk solution. With this method, which we call plane-sphere confinement microscopy (PSCM), information regarding the partitioning of molecules between the polymer coating and the bulk liquid is also obtained.

Coupling Polar Adhesion with Traction, Spring, and Torque Forces Allows High-Speed Helical Migration of the Protozoan Parasite .

Among the eukaryotic cells that navigate through fully developed metazoan tissues, protozoans from the Apicomplexa phylum have evolved motile developmental stages that move much faster than the fastest crawling cells owing to a peculiar substrate-dependent type of motility, known as gliding. Best-studied models are the sporozoite and the tachyzoite polarized cells for which motility is vital to achieve their developmental programs in the metazoan hosts. The gliding machinery is shared between the two parasites and is largely characterized.

An integrated assay to probe endothelial glycocalyx-blood cell interactions under flow in mechanically and biochemically well-defined environments.

Cell-cell and cell-glycocalyx interactions under flow are important for the behaviour of circulating cells in blood and lymphatic vessels. However, such interactions are not well understood due in part to a lack of tools to study them in defined environments. Here, we develop a versatile in vitro platform for the study of cell-glycocalyx interactions in well-defined physical and chemical settings under flow.

Third harmonic generation imaging and analysis of the effect of low gravity on the lacuno-canalicular network of mouse bone.

The lacuno-canalicular network (LCN) hosting the osteocytes in bone tissue represents a biological signature of the mechanotransduction activity in response to external biomechanical loading. Using third-harmonic generation (THG) microscopy with sub-micrometer resolution, we investigate the impact of microgravity on the 3D LCN structure in mice following space flight. A specific analytical procedure to extract the LCN characteristics from THG images is described for ex vivo studies of bone sections.

Elastohydrodynamic Lift at a Soft Wall.

We study experimentally the motion of nondeformable microbeads in a linear shear flow close to a wall bearing a thin and soft polymer layer. Combining microfluidics and 3D optical tracking, we demonstrate that the steady-state bead-to-surface distance increases with the flow strength. Moreover, such lift is shown to result from flow-induced deformations of the layer, in quantitative agreement with theoretical predictions from elastohydrodynamics.

Label-free imaging of bone multiscale porosity and interfaces using third-harmonic generation microscopy.

Interfaces provide the structural basis of essential bone functions. In the hierarchical structure of bone tissue, heterogeneities such as porosity or boundaries are found at scales ranging from nanometers to millimeters, all of which contributing to macroscopic properties. To date, however, the complexity or limitations of currently used imaging methods restrict our understanding of this functional integration.

Efficient second-harmonic imaging of collagen in histological slides using Bessel beam excitation.

Second-harmonic generation (SHG) is the most specific label-free indicator of collagen accumulation in widespread pathologies such as fibrosis, and SHG-based measurements hold important potential for biomedical analyses. However, efficient collagen SHG scoring in histological slides is hampered by the limited depth-of-field of usual nonlinear microscopes relying on focused Gaussian beam excitation. In this work we analyze theoretically and experimentally the use of Bessel beam excitation to address this issue.

The Conformation of Thermoresponsive Polymer Brushes Probed by Optical Reflectivity.

We describe a microscope-based optical setup that allows us to perform space- and time-resolved measurements of the spectral reflectance of transparent substrates coated with ultrathin films. This technique is applied to investigate the behavior in water of thermosensitive polymer brushes made of poly(N-isopropylacrylamide) grafted on glass. We show that spectral reflectance measurements yield quantitative information about the conformation and axial structure of the brushes as a function of temperature.

Mitigating phototoxicity during multiphoton microscopy of live Drosophila embryos in the 1.0-1.2 µm wavelength range.

Light-induced toxicity is a fundamental bottleneck in microscopic imaging of live embryos. In this article, after a review of photodamage mechanisms in cells and tissues, we assess photo-perturbation under illumination conditions relevant for point-scanning multiphoton imaging of live Drosophila embryos. We use third-harmonic generation (THG) imaging of developmental processes in embryos excited by pulsed near-infrared light in the 1.

Third-harmonic generation microscopy with Bessel beams: a numerical study.

We study theoretically and numerically third-harmonic generation (THG) from model geometries (interfaces, slabs, periodic media) illuminated by Bessel beams produced by focusing an annular intensity profile. Bessel beams exhibit a phase and intensity distribution near focus different from Gaussian beams, resulting in distinct THG phase matching properties and coherent scattering directions. Excitation wave vectors are controlled by adjusting the bounding aperture angles of the Bessel beam.

3D resolved mapping of optical aberrations in thick tissues.

We demonstrate a simple method for mapping optical aberrations with 3D resolution within thick samples. The method relies on the local measurement of the variation in image quality with externally applied aberrations. We discuss the accuracy of the method as a function of the signal strength and of the aberration amplitude and we derive the achievable resolution for the resulting measurements.

Multicolor two-photon tissue imaging by wavelength mixing.

We achieve simultaneous two-photon excitation of three chromophores with distinct absorption spectra using synchronized pulses from a femtosecond laser and an optical parametric oscillator. The two beams generate separate multiphoton processes, and their spatiotemporal overlap provides an additional two-photon excitation route, with submicrometer overlay of the color channels. We report volume and live multicolor imaging of 'Brainbow'-labeled tissues as well as simultaneous three-color fluorescence and third-harmonic imaging of fly embryos.

Accuracy of correction in modal sensorless adaptive optics.

We investigate theoretically and experimentally the parameters governing the accuracy of correction in modal sensorless adaptive optics for microscopy. On the example of two-photon fluorescence imaging, we show that using a suitable number of measurements, precise correction can be obtained for up to 2 radians rms aberrations without optimising the aberration modes used for correction. We also investigate the number of photons required for accurate correction when signal acquisition is shot-noise limited.

Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates.

Multiphoton microscopy is a powerful tool in neuroscience, promising to deliver important data on the spatiotemporal activity within individual neurons as well as in networks of neurons. A major limitation of current technologies is the relatively slow scan rates along the z direction compared to the kHz rates obtainable in the x and y directions. Here, we describe a custom-built microscope system based on an architecture that allows kHz scan rates over hundreds of microns in all three dimensions without introducing aberration.

Methodology for reconstructing early zebrafish development from in vivo multiphoton microscopy.

Investigating cell dynamics during early zebrafish embryogenesis requires specific image acquisition and analysis strategies. Multiharmonic microscopy, i.e.

Combined third-harmonic generation and four-wave mixing microscopy of tissues and embryos.

Nonlinear microscopy can be used to probe the intrinsic optical properties of biological tissues. Using femtosecond pulses, third-harmonic generation (THG) and four-wave mixing (FWM) signals can be efficiently produced and detected simultaneously. Both signals probe a similar parameter, i.

Advances in multiphoton microscopy for imaging embryos.

Multiphoton imaging is a promising approach for addressing current issues in systems biology and high-content investigation of embryonic development. Recent advances in multiphoton microscopy, including light-sheet illumination, optimized laser scanning, adaptive and label-free strategies, open new opportunities for embryo imaging. However, the literature is often unclear about which microscopy technique is most adapted for achieving specific experimental goals.

Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy.

Quantifying cell behaviors in animal early embryogenesis remains a challenging issue requiring in toto imaging and automated image analysis. We designed a framework for imaging and reconstructing unstained whole zebrafish embryos for their first 10 cell division cycles and report measurements along the cell lineage with micrometer spatial resolution and minute temporal accuracy. Point-scanning multiphoton excitation optimized to preferentially probe the innermost regions of the embryo provided intrinsic signals highlighting all mitotic spindles and cell boundaries.

Characterisation of the dynamic behaviour of lipid droplets in the early mouse embryo using adaptive harmonic generation microscopy.

Background: Lipid droplets (LD) are organelles with an important role in normal metabolism and disease. The lipid content of embryos has a major impact on viability and development. LD in Drosophila embryos and cultured cell lines have been shown to move and fuse in a microtubule dependent manner.