Sophie Brasselet

Translational diffusion of individual class II MHC membrane proteins in cells.

Single-molecule epifluorescence microscopy was used to observe the translational motion of GPI-linked and native I-E(k) class II MHC membrane proteins in the plasma membrane of CHO cells. The purpose of the study was to look for deviations from Brownian diffusion that might arise from barriers to this motion. Detergent extraction had suggested that these proteins may be confined to lipid microdomains in the plasma membrane. The individual I-E(k) proteins were visualized with a Cy5-labeled peptide that binds to a specific extracytoplasmic site common to both proteins. Single-molecule trajectories were used to compute a radial distribution of displacements, yielding average diffusion coefficients equal to 0.22 (GPI-linked I-E(k)) and 0.18 microm(2)/s (native I-E(k)). The relative diffusion of pairs of proteins was also studied for intermolecular separations in the range 0.3-1.0 microm, to distinguish between free diffusion of a protein molecule and diffusion of proteins restricted to a rapidly diffusing small domain. Both analyses show that motion is predominantly Brownian. This study finds no strong evidence for significant confinement of either GPI-linked or native I-E(k) in the plasma membrane of CHO cells.

In situ diagnostics of the crystalline nature of single organic nanocrystals by nonlinear microscopy.

We elucidate the crystalline nature and the three-dimensional orientation of isolated organic nanocrystals embedded in a sol-gel matrix, using a polarized nonlinear microscopy technique that combines two-photon fluorescence and second harmonic generation. This technique allows the distinction between monocrystalline structures and nanoscale polycrystalline aggregates responsible for incoherent second harmonic signals.

Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles

Animal cell cytokinesis requires a contractile ring of crosslinked actin filaments and myosin motors. How contractile rings form and are stabilized in dividing cells remains unclear. We address this problem by focusing on septins, highly conserved proteins in eukaryotes whose precise contribution to cytokinesis remains elusive. We use the cleavage of the Drosophila melanogaster embryo as a model system, where contractile actin rings drive constriction of invaginating membranes to produce an epithelium in a manner akin to cell division. In vivo functional studies show that septins are required for generating curved and tightly packed actin filament networks. In vitro reconstitution assays show that septins alone bundle actin filaments into rings, accounting for the defects in actin ring formation in septin mutants. The bundling and bending activities are conserved for human septins, and highlight unique functions of septins in the organization of contractile actomyosin rings.

Ytterbium-Based Bioprobes for Near-Infrared Two-Photon Scanning Laser Microscopy Imaging†

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Ytterbium-based bioprobes for near-infrared two-photon scanning laser microscopy imaging. Anthony D’Aléo, Adrien Bourdolle, Sophie Brustlein, Teddy Fauquier, Alexei Grichine, Alain Duperray, P. L. Baldeck, Chantal Andraud, Sophie Brasselet, Olivier Maury

Polarization-resolved nonlinear microscopy: application to structural molecular and biological imaging

In this tutorial I analyze the polarization-dependent properties of different optical contrasts widely used today in imaging, applied to biology and biomedical diagnostics. I derive the essential properties of the polarization dependence of optical processes such as two-photon fluorescence, nonlinear coherent effects in the nonresonant as well as vibrational-resonant regimes, and analyze how they can be exploited to provide information on the molecular orientational organization in a biological sample. Two examples will be detailed: the first one the measurement of lipid order in artificial and cell membranes by using fluorescent labeling, and the second one structural imaging of collagen in tissues by using second-harmonic generation.

Local second-harmonic generation enhancement on gold nanostructures probed by two-photon microscopy.

The localization of surface second-harmonic generation (S-SHG) enhancements from granular gold structures that exhibit local plasmon resonance was investigated. A two-photon microscopy technique was used to perform high spatial resolution S-SHG imaging. The magnitude and the spatial density of S-SHG enhancement confined in submicroscopic regions are strongly dependent on the morphology of the golds surface. Polarization measurements of local S-SHG responses reveal the local field anisotropy in enhancement regions and furthermore prove the incoherent and strongly depolarized nature of the emission, which is attributed to ultrafast fluctuations of the enhancement location in the focal volume.

Octupolar versus dipolar crystalline structures for nonlinear optics: a dual crystal and propagative engineering approach

A new type of crystalline structure for nonlinear optics whereby octupolar symmetry features are displayed at both molecular and crystalline levels is exemplified by the prototype 2,4,6-triaryloxy-1,3,5-triazine (TPOT) crystal and analyzed in terms of both individual molecular responses and crystal packing features. Polarized harmonic light scattering permits the full determination of the molecular β hyperpolarizability tensor and confirms the octupolar trigonal symmetry of the TPOT molecule. An oriented gas model is used to infer therefrom an estimate of the crystalline nonlinear d tensor which is predicted to be of the same order as that of the reference dipolar N-4-nitrophenyl-(L)-prolinol crystal. The concept of optimal packing toward quadratic nonlinear optics, which had been initially introduced in the realm of quasi-one-dimensional structures, is revisited and enlarged to encompass more isotropic uniaxial structures potentially amenable, in the case of octupoles, to larger optimal values than in th...

Influence of birefringence on polarization resolved nonlinear microscopy and collagen SHG structural imaging

We analyze the influence of the anisotropy of molecular and biological samples on polarization resolved nonlinear microscopy imaging. We show in particular the detrimental influence of birefringence on Second Harmonic Generation (SHG) and Two-Photon Excited Fluorescence (TPEF) polarization resolved microscopy imaging, which, if not accounted for, can lead to an erroneous determination of the sample properties and thus to a misinterpretation of the read-out information. We propose a method to measure this birefringence and account for this effect in nonlinear polarization resolved experiments.

Fluorescence Behavior of Single-Molecule pH-Sensors

The fluorescence behavior of a well-known pH-reporter molecule diluted in an aqueous gel is described at the single-molecule level. Single copies of the pH-sensitive seminaphthorhodafluor SNARF-1-dextran were immobilized in the pores of agarose gels at different pH in the range 6-9. This probe shows separate emission wavelengths for its protonated and deprotonated forms, such that the ratio between these two emission intensities is a signature of the pH of the surrounding medium. Two-color detection of the time trajectories of single chromophore emission signals allowed characterization of the behavior, molecule by molecule. In particular the distribution of the ratio between the two emission wavelengths, measured over a large number of individual molecules, shows a broadening at pH close to pKa, which cannot be explained by the experimental noise. The observed range of average ratios appears to arise from local inhomogeneities in the environment of the fluorophore which alter the response to local pH.

Defocused imaging of second harmonic generation from a single nanocrystal

We demonstrate the direct imaging of the second harmonic generation radiation from a single nonlinear nanocrystal using defocused nonlinear microscopy. This technique allows the retrieval of complete information on the 3D orientation of a nanocrystal as well as possible deviations from its purely crystalline nature, in a simple experimental implementation. The obtained images are modeled by calculation of the radiation diagram from a nonlinear dipole that accounts for the excitation beam, the crystal symmetry and the particle size. Experimental demonstrations are performed on Potassium Titanyl Phosphase (KTP) nanocrystals. The shape and structure of the radiation images show a strong dependence on both crystal orientation and field polarization state, as expected by the specific nonlinear coherent coupling between the induced dipole and the excitation field polarization state.