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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 hemi-spherical surface (of which at least one is coated with polymers) in close contact, and uses this confinement to analyse 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. Thanks to the shape of the confined geometry, diffusion and partitioning can be mapped as a function of compression and concentration of the coating in a single experiment. The method is versatile and can be integrated with conventional optical microscopes, and thus should find widespread use in the many application areas exploiting functional polymer coatings. We demonstrate the use of PSCM using brushes of natively unfolded nucleoporin domains rich in phenylalanine-glycine repeats (FG domains). A meshwork of FG domains is known to be responsible for the selective transport of nuclear transport receptors (NTR) and their macromolecular cargos across the nuclear envelope that separates the cytosol and the nucleus of living cells. We find that the selectivity of NTR uptake by FG domain films depends sensitively on FG domain concentration, and that the interaction of NTRs with FG domains obstructs NTR movement only moderately. These observations contribute important information to better understand the mechanisms of selective NTR transport., 27 pages and 6 figures of main text, 12 pages and 8 figures of supporting information

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. Here, we demonstrate experimentally that the promiscuous opportunistic pathogen Pseudomonas aeruginosa explores substrates differently based on their rigidity, leading to striking variations in biofilm structure, exopolysaccharides (EPS) distribution, strain mixing during co-colonization and phenotypic expression. Using simple kinetic models, we show that these phenotypes arise through a mechanical interaction between the elasticity of the substrate and the type IV pilus (T4P) machinery, that mediates the surface-based motility called twitching. Together, our findings reveal a new role for substrate softness in the spatial organization of bacteria in complex microenvironments, with far-reaching consequences on efficient biofilm formation.

International audience; 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 Plasmodium sporozoite and the Toxoplasma 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. Localized beneath the cell surface, it includes actin filaments, unconventional myosin motors housed within a multimember glideosome unit, and apically secreted transmembrane adhesins. In contrast, less is known about the force mechanisms powering cell movement. Pioneered biophysical studies on the sporozoite and phenotypic analysis of tachyzoite actin-related mutants have added complexity to the general view that force production for parasite forward movement directly results from the myosin-driven rearward motion of the actin-coupled adhesion sites. Here, we have interrogated how forces and substrate adhesion–de-adhesion cycles operate and coordinate to allow the typical left-handed helical gliding mode of the tachyzoite. By combining quantitative traction force and reflection interference microscopy with micropatterning and expansion microscopy, we unveil at the millisecond and nanometer scales the integration of a critical apical anchoring adhesion with specific traction and spring-like forces. We propose that the acto-myoA motor directs the traction force which allows transient energy storage by the microtubule cytoskeleton and therefore sets the thrust force required for T. gondii tachyzoite vital helical gliding capacity.

International audience; Numerous biophysical questions require the quantification of short-range interactions between (functionalized) surfaces and synthetic or biological objects such as cells. Here, we present an original, custom built setup for reflection interference contrast microscopy that can assess distances between a substrate and a flowing object at high speed with nanometric accuracy. We demonstrate its use to decipher the complex biochemical and mechanical interplay regulating blood cell homing at the vessel wall in the microcirculation using an in vitro approach. We show that in the absence of specific biochemical interactions, flowing cells are repelled from the soft layer lining the vessel wall, contributing to red blood cell repulsion in vivo. In contrast, this so-called glycocalyx stabilizes rolling of cells under flow in the presence of a specific receptor naturally present on activated leucocytes and a number of cancer cell lines.

International audience; In bone tissue, multiscale interfaces provide the structural basis of essential bone functions and contribute to itsmacroscopic mechanical properties. The lacuno-canalicular network (LCN) hosting the osteocytes in the bone matrix, inparticular, represents a biological signature of the mechanotransduction activity in response to external biomechanicalloading. We have demonstrated that label-free third-harmonic generation (THG) microscopy reveals the structure of theLCN in 3D with submicron precision over millimetric fields of view compatible with histology and can be coupled tosecond-harmonic generation (SHG) signals relating to the collagen organization in the bone matrix. Taking advantage ofthese label-free imaging methods, we investigate the impact of microgravity on the LCN structure in mice following a 1-month space flight. We show that our current lack of understanding of the extent of the LCN heterogeneity at the organlevel hinders the interpretation of such investigations based on a limited number of samples and we discuss theimplications for future biomedical studies.

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. Our approach is demonstrated with the interaction between hyaluronan (HA, a key component of the endothelial glycocalyx) and its cell receptor CD44. We generate HA brushes in situ within a microfluidic device, and demonstrate the tuning of their physical (thickness and softness) and chemical (density of CD44 binding sites) properties using characterisation with reflection interference contrast microscopy (RICM) and application of polymer theory. We highlight the interactions of HA brushes with CD44-displaying beads and cells under flow. Observations of CD44+ beads on a HA brush with RICM enabled the 3-dimensional trajectories to be generated, and revealed interactions in the form of stop and go phases with reduced rolling velocity and reduced distance between the bead and the HA brush, compared to uncoated beads. Combined RICM and bright-field microscopy of CD44+ AKR1 T-lymphocytes revealed complementary information about the dynamics of cell rolling and cell morphology, and highlighted the formation of tethers and slings, as they interacted with a HA brush under flow. This platform can readily incorporate more complex models of the glycocalyx, and should permit the study of how mechanical and biochemical factors are orchestrated to enable highly selective blood cell-vessel wall interactions under flow.

International audience; 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. The analysis conducted in different anatomical quadrants of femoral cortical bone didn't reveal any statistical differences between the control, habitat control and flight groups, suggesting that the LCN connectivity is not affected by one month space flight. However, significant variations are systematically observed within each sample. We show that our current lack of understanding of the extent of the LCN heterogeneity at the organ level hinders the interpretation of such investigations based on a limited number of samples and we discuss the implications for future biomedical studies.

International audience; We study experimentally the motion of non-deformable 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/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. This study thus provides the first experimental evidence of ``soft lubrication'' at play at small scale, in a system relevant {\it e.g.} to the physics of blood microcirculation.

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. Focused Bessel beams can provide an axially extended excitation volume for nonlinear microscopy while preserving lateral resolution. We show that shaping the focal volume has consequences on signal level and scattering directionality in the case of coherent signals (such as SHG) which significantly differ from the case of incoherent signals (two-photon excited fluorescence, 2PEF). We demonstrate extended-depth SHG-2PEF imaging of fibrotic mouse kidney histological slides. Finally, we show that Bessel beam excitation combined with spatial filtering of the harmonic light in wave vector space can be used to probe collagen accumulation more efficiently than the usual Gaussian excitation scheme. These results open the way to SHG-based histological diagnoses.

We demonstrate wavefront sensorless aberration correction in a two-photon excited fluorescence microscope. Using analysis of the image-formation process, we have developed an optimized correction scheme permitting image-quality improvement with minimal additional exposure of the sample. We show that, as a result, our correction process induces little photobleaching and significantly improves the quality of images of biological samples. In particular, increased visibility of small structures is demonstrated. Finally, we illustrate the use of this technique on various fresh and fixed biological tissues.

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. Focused Bessel beams can provide an axially extended excitation volume for nonlinear microscopy while preserving lateral resolution. We show that shaping the focal volume has consequences on signal level and scattering directionality in the case of coherent signals (such as SHG) which significantly differ from the case of incoherent signals (two-photon excited fluorescence, 2PEF). We demonstrate extended-depth SHG-2PEF imaging of fibrotic mouse kidney histological slides. Finally, we show that Bessel beam excitation combined with spatial filtering of the harmonic light in wave vector space can be used to probe collagen accumulation more efficiently than the usual Gaussian excitation scheme. These results open the way to SHG-based histological diagnoses.

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. We study how molecular parameters (grafting density, chain length) affect the hydra-tion state of a brush, and provide one of the few experimental evidence for the occurrence of vertical phase separation in the vicinity of the lower critical solution temperature of the polymer. The origin of the hysteretic behavior of poly(N-isopropylacrylamide) brushes upon cycling the temperature is also clarified. We thus demonstrate that our optical technique allows for in-depth characterization of stimuli-responsive polymer layers, which is crucial for the rational design of smart polymer coatings in actuation, gating or sensing applications., Comment: Revised version. To appear in Langmuir (in press)

We present a simple and versatile scheme for characterising amplitude and phase modulation by an active element, such as a deformable mirror, in the pupil plane of a high NA microscope. By placing a mirror in the vicinity of the focal plane of the objective and recording images of the reflected focal spot on a camera, we show that reliable measurements of the influence function of the mirror actuators in the pupil plane of the objective can be obtained using an iterative electric field retrieval algorithm. Compared to direct wavefront sensors, the proposed method allows characterisation for a variety of objectives with different NA and pupil sizes without modification of the setup, requires minimal space inside the microscope, and can be used with pulsed sources such as used for multiphoton microscopy. In order to validate our method, we compare our data to the results obtained with a Shack-Hartmann wavefront sensor, and show that comparable precision is achieved.

International audience; Third-harmonic generation (THG) microscopy provides images of unstained biological samples based on spatial variations in third-order nonlinear susceptibility, refractive index, and dispersion. In this study, we establish quantitative values for the third-order nonlinear susceptibilities of several solvents (water, ethanol, glycerol), physiological aqueous (ions, amino acids, polypeptides, bovine serum albumin, glucose) and lipid (triglycerides, cholesterol) solutions as a function of solute concentration in the 1.05-1.25 microm excitation range. We use these data in conjunction with imaging experiments to show that THG imaging with approximately 1.2 microm excitation lacks specificity and sensitivity to detect physiological ion concentration changes, and that nonaqueous structures such as lipid bodies provide a more robust source of signal. Finally, we illustrate the impact of index-matching liquids in THG images. These data provide a basis for interpreting biological THG images and for developing additional applications.

Depuis son introduction en 1990, la microscopie de fluorescence excitee a deux photons (Fluo-2P) s’est peu a peu imposee comme une methode incontournable d’imagerie de tissus intacts a l’echelle sub-cellulaire. En effet, la caracteristique la plus remarquable de la microscopie multiphotonique est de maintenir une resolution tridimensionnelle micrometrique lors de l’observation en profondeur d’un milieu optiquement diffusant. Combinee aux technologies de proteines-fusion (type GFP), cette approche est aujourd’hui utilisee dans de nombreux domaines, notamment en neurophysiologie. Un autre attrait de ce type d’imagerie reside dans l’utilisation possible d’autres phenomenes optiques non lineaires (c’est-a-dire impliquant l’interaction simultanee de plusieurs photons avec une molecule observee) comme source de contraste. Ainsi, les microscopies par generation de second harmonique (GSH) et par generation de troisieme harmonique (GTH) permettent egalement d’observer des milieux complexes et fournissent des informations complementaires par rapport a l’imagerie de fluorescence. Certaines structures cellulaires ou tissulaires fournissent, en effet, ce type de reponse optique sans necessiter de marquage exogene. La microscopie GSH permet, par exemple, de detecter le collagene fibrillaire et la microscopie GTH permet d’observer sans marquage le developpement embryonnaire de petits organismes.

We characterized the effect of femtosecond pulse trains on gastrulating Drosophila embryos using two-photonexcited fluorescence (2PEF) and third-harmonic generation (THG) microscopy. Femtosecond pulses can be used to perform controlled intravital microdissections that alter the embryo structural integrity but do not significantly perturb cytoskeleton dynamics in adjacent cells. Such targeted ablations can be used to remotely perturb cell movements in developing embryos. The transparent combination of femtosecond pulse-induced ablation with nonlinear microscopy can be used to analyze in vivo the effect of cell and tissue deformations. r 2005 Elsevier GmbH. All rights reserved.

International audience; 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 1.0–1.2 mm range. We study the influence of imaging rate, wavelength, and pulse duration on the short-term and long-term perturbation of development and define criteria for safe imaging. We show that under illumination conditions typical for multiphoton imaging, photodamage in this system arises through 2-and/or 3-photon absorption processes and in a cumulative manner. Based on this analysis, we derive general guidelines for improving the signal-to-damage ratio in two-photon (2PEF/SHG) or THG imaging by adjusting the pulse duration and/or the imaging rate. Finally, we report label-free time-lapse 3D THG imaging of gastrulating Drosophila embryos with sampling appropriate for the visualisation of morphogenetic movements in wild-type and mutant embryos, and long-term multiharmonic (THG-SHG) imaging of development until hatching. Citation: Débarre D, Olivier N, Supatto W, Beaurepaire E (2014) Mitigating Phototoxicity during Multiphoton Microscopy of Live Drosophila Embryos in the 1.0– 1.2 mm Wavelength Range. PLoS ONE 9(8): e104250.

Nonlinear optical microscopy is a biocompatible avenue for probing ordered molecular assemblies in biological tissues. As in linear optics, the nonlinear optical response from ordered systems is polarization-sensitive. This dependence can be used to identify and characterize local molecular ordering with micrometer-scale 3D resolution in a nonlinear microscope. In particular, third-harmonic generation (THG) microscopy is a nonlinear optical modality sensitive to the electronic nonlinear susceptibility χ (3) of a material. THG microscopy can be used to map χ (3) spatial variations (i.e. material interfaces), and to probe birefringence. In principle, polarization-resolved THG (P-THG) can therefore be used to probe ordered molecular arrays. However, the orientation, distribution, and nonlinear optical properties of the molecules near the beam focus all affect the detected signal. It is therefore necessary to develop a theoretical method which decouples these effects and permits the extraction of orientational information from P-THG images. In this report, we first present P-THG images of model systems (lipid droplets, multilamellar lipid vesicles) and biological tissues (human skin biopsy) which establish that P-THG is sensitive to lipid ordering and that it is maximized when excitation polarization is parallel to the ordered lipid molecules, giving impetus for the development of a thorough theoretical analysis. We then outline a multiscale model spanning the molecular (nm) and ensemble (μm) scales predicting the PTHG signal, consisting of three main steps: (i) calculation of the molecular electronic hyperpolarizability; (ii) determination of the anisotropic χ (3) for various molecular distribution parameters; and (iii) numerical calculations of the P-THG signal from lipid-water interfaces. This analysis links the measured P-THG response to lipid molecular structure and ordering.

International audience; Ordered lipid assemblies are responsible for important physiological functions including skin barrier and axon conductivity. However, techniques commonly used to probe molecular order such as X-ray scattering and nuclear magnetic resonance are not suited for in-situ tissue studies. Here, we identify and characterize a novel contrast mechanism in nonlinear optical microscopy which is sensitive to molecular ordering in multilamellar lipid vesicles (MLVs) and in samples obtained from human skin biopsy: polarized third-harmonic generation (P-THG). We develop a multiscale theoretical framework to calculate the anisotropic, nonlinear optical response of lipid arrays as a function of molecular order. This analysis reveals that conserved carbon-carbon bond and aliphatic tail directionality are the atomic- and molecular-scale sources of the observed P-THG response, respectively. Agreement between calculations and experiments on lipid droplets and MLVs validates the use of P-THG as a probe of lipid ordering. Finally, we show that P-THG can be used to map molecular ordering in the multilamellar, intercorneocyte lipid matrix of the stratum corneum of human skin. These results provide the foundation for the use of P-THG in probing molecular order and highlight a novel biomedical application of multiphoton microscopy in an optically accessible tissue relevant to monitoring lipid-related disorder.

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. In addition to extended depth-of-field imaging, this opens interesting perspectives for coherent nonlinear microscopy, such as extracting sample spatial frequencies in the lambda/8 - lambda range in the case of organized media. (C) 2012 Optical Society of America

International audience; 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.

International audience; Investigating cell dynamics during early zebrafish embryogenesis requires specific image acquisition and analysis strategies. Multiharmonic microscopy, i.e., second- and third-harmonic generations, allows imaging cell divisions and cell membranes in unstained zebrafish embryos from 1- to 1000-cell stage. This paper presents the design and implementation of a dedicated image processing pipeline (tracking and segmentation) for the reconstruction of cell dynamics during these developmental stages. This methodology allows the reconstruction of the cell lineage tree including division timings, spatial coordinates, and cell shape until the 1000-cell stage with minute temporal accuracy and micrometer spatial resolution. Data analysis of the digital embryos provides an extensive quantitative description of early zebrafish embryogenesis.

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. We further demonstrate how this high-speed 3D multiphoton imaging system can be used to study neuronal activity at millisecond resolution at the subcellular as well as the population level.

International audience; We investigate the parameters governing the accuracy of correction in modal sensorless adaptive optics for microscopy. In this paper we focus on the case of two-photon excited fluorescence. Using analytical, numerical and experimental results, we show that using a suitable number of measurements, accurate correction can be achieved for up to 2 rad rms initial aberrations even without optimisation of the correction modes. We demonstrate that this correction can be achieved using low light levels to minimise photobleaching and toxicity, and we provide examples of such optimised correction. © (2012) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

International audience; Modal sensorless adaptive optics relies on the use of an image quality metric to estimate the amplitude of aberrations, and of a well-suited set of aberration modes to describe the aberration. This set is chosen so that aberration of one mode does not influence correction in another mode. In this paper, we show how these modes can be derived experimentally, and investigate the influence of imperfect crosstalk removal on the accuracy of correction. We show that the resulting error can be mitigated using appropriate algorithms that can incorporate knowledge of the influence of the modes on the metric and, if available, partial knowledge of the aberrations. Finally, we derive from these results the minimum time required for correction in various situations. © (2012) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

International audience; Accurate control over the phase and amplitude modulation in an adaptive microscope is essential to the quality of aberration correction that can be achieved. In this paper we present a robust and compact method for characterising such amplitude and phase modulation in the pupil plane of the focussing objective. This method, based on phase diversity, permits calibrating the microscope as a whole and thus avoids errors in the alignment of the wavefront shaping device after calibration and the resulting imprecision in the induced modulation: by acquiring three 2D images of the point spread function at different distances from the focal plane, we show that the electric field distribution at the pupil plane can be retrieved using an iterative algorithm. We have applied this technique to the characterisation of the phase modulation induced by a deformable mirror when conjugated with the entrance pupil of different objectives, which permits accurate evaluation of the performance of the mirror for subsequent aberration correction. © (2012) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

International audience; 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.e. the real part of the third-order nonlinear susceptibility χ((3)). However THG and FWM images result from different phase matching conditions and provide complementary information. We analyze this complementarity using calculations, z-scan measurements on water and oils, and THG-FWM imaging of cell divisions in live zebrafish embryos. The two signals exhibit different sensitivity to sample size and clustering in the half-wavelength regime. Far from resonance, THG images reveal spatial variations |Δχ((3))(-3ω;ω,ω,ω)| with remarkable sensitivity while FWM directly reflects the distribution of χ((3))(-2ω(1) + ω(2);ω(1), -ω(2), ω(1)). We show that FWM images provide χ((3)) maps useful for proper interpretation of cellular THG signals, and that combined imaging carries additional structural information. Finally we present simultaneous imaging of intrinsic THG, FWM, second-harmonic (SHG) and two-photon-excited fluorescence (2PEF) signals in live Caenorhabditis elegans worms illustrating the information provided by multimodal nonlinear imaging of unstained tissue.

International audience; 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. In this review, we describe and discuss the key concepts of imaging speed, imaging depth, photodamage, and nonlinear contrast mechanisms in the context of recent advances in live embryo imaging. We illustrate the potentials of these new imaging approaches with a selection of recent applications in developmental biology. Cop. 2011 Elsevier Ltd.

8th IEEE International Symposium on Biomedical Imaging (ISBI) - From Nano to Macro Location: Chicago, IL MAR 30-APR 02, 2011; The reconstruction of the cell lineage tree of early zebrafish embryogenesis requires the use of in-vivo microscopy imaging and image processing strategies. Second (SHG) and third harmonic generation (THG) microscopy observations in unstained zebrafish embryos allows to detect cell divisions and cell membranes from 1-cell to 1K-cell stage. In this article, we present an ad-hoc image processing pipeline for cell tracking and cell membranes segmentation enabling the reconstruction of the early zebrafish cell lineage tree until the 1K-cell stage. This methodology has been used to obtain digital zebrafish embryos allowing to generate a quantitative description of early zebrafish embryogenesis with minute temporal accuracy and μm spatial resolution.

International audience; 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. Automated image analysis revealed the phenomenology of cell proliferation. Blastomeres continuously drift out of synchrony. After the 32-cell stage, the cell cycle lengthens according to cell radial position, leading to apparent division waves. Progressive amplification of this process is the rule, contrasting with classical descriptions of abrupt changes in the system dynamics.

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. Due to limitations in current imaging technology, little is known about the behaviour of LD in the mammalian embryo. Harmonic generation microscopy (HGM) allows one to image LD without the use of exogenous labels. Adaptive optics can be used to correct aberrations that would otherwise degrade the quality and information content of images. Results We have built a harmonic generation microscope with adaptive optics to characterise early mouse embryogenesis. At fertilization, LD are small and uniformly distributed, but in the implanting blastocyst, LD are larger and enriched in the invading giant cells of the trophectoderm. Time-lapse studies reveal that LD move continuously and collide but do not fuse, instead forming aggregates that subsequently behave as single units. Using specific inhibitors, we show that the velocity and dynamic behaviour of LD is dependent not only on microtubules as in other systems, but also on microfilaments. We explore the limits within which HGM can be used to study living embryos without compromising viability and make the counterintuitive finding that 16 J of energy delivered continuously over a period of minutes can be less deleterious than an order of magnitude lower energy delivered dis-continuously over a period of hours. Conclusions LD in pre-implantation mouse embryos show a previously unappreciated complexity of behaviour that is dependent not only on microtubules, but also microfilaments. Unlike LD in other systems, LD in the mouse embryo do not fuse but form aggregates. This study establishes HGM with adaptive optics as a powerful tool for the study of LD biology and provides insights into the photo-toxic effects of imaging embryos.

International audience; We demonstrate image-based aberration correction in a third-harmonic generation (THG) microscope. We describe a robust, mostly sample-independent correction scheme relying on prior measurement of the influence of aberration modes produced by a deformable mirror on the quality of THG images. We find that using image sharpness as an image quality metric, correction of N aberration modes is achieved using 2(2N+1) measurements in a variety of samples. We also report aberration correction in combined multiharmonic and two-photon excited fluorescence experiments. Finally, we demonstrate time-dependent adaptive THG imaging in developing embryonic tissue.

Specimen-induced aberrations are frequently encountered in high resolution microscopy, particularly when highnumerical aperture lenses are used to image deep into biological specimens. These aberrations distort the focalspot causing a reduction in resolution and, often more importantly, reduced signal level and contrast. This isparticularly problematic in multiphoton microscopy, where the non-linear nature of the signal generation processmeans that the signal level is strongly aected by changes in the focal spot intensity. We have developed anadaptive two-photon ”uorescence microscope to correct for these aberrations. Unlike a conventional adaptiveoptics system, the microscope does not include a wavefront sensor but uses an ecient sensorless optimisationscheme to obtain optimum aberration correction.Keywords: Adaptive optics, microscopy, multiphoton microscopy. 1. INTRODUCTION Adaptive optics technology has the potential to solve many of the problems of aberrations in microscopy.

Specimen-induced aberrations frequently affect microscopes, particularly when high aperture lenses image deep into specimens. We have applied adaptive optics to correct aberrations in confocal, two-photon, widefield sectioning and harmonic generation microscopes, restoring image quality.

In adaptive microscope systems, it is often desirable to dispense with the wavefront sensor and perform aberration correction through optimisation an appropriate quality metric, such as image brightness or sharpness. A sequence of trial aberrations is applied to the adaptive element and the metric values are calculated. The optimum aberration correction is derived from these measurements. An important choice in the design of these correction schemes is the modal aberration expansion. This choice may depend upon several factors, such as the deformable mirror, the optimisation metric, the aberration statistics or the image properties. We discuss these factors with particular reference to microscope imaging.

International audience; We implement wave front sensor-less adaptive optics in a structured illumination microscope. We investigate how the image formation process in this type of microscope is affected by aberrations. It is found that aberrations can be classified into two groups, those that affect imaging of the illumination pattern and those that have no influence on this pattern. We derive a set of aberration modes ideally suited to this application and use these modes as the basis for an efficient aberration correction scheme. Each mode is corrected independently through the sequential optimisation of an image quality metric. Aberration corrected imaging is demonstrated using fixed fluorescent specimens. Images are further improved using differential aberration imaging for reduction of background fluorescence.

International audience; We use a vector field model to analyze the third-harmonic generation (THG) emission patterns for isolated objects illuminated by a Gaussian beam. Simulations and experiments indicate that THG from biological (dielectric) structures is essentially forward-directed, as opposed to e.g. THG from gold particles. We then address the issue of epidetecting forward-emitted light backscattered in a turbid medium. We use Monte Carlo simulations and measurements to analyze the effect of tissue properties (absorption, scattering), and of the geometry of the collecting optics. This analysis provides guidelines for optimizing epidetection in coherent nonlinear microscopy. An erratum is attached. © (2008) COPYRIGHT SPIE--The International Society for Optical Engineering.

We present an overview of a wavefront sensor-less adaptive optics scheme for microscopy based upon the optimisation of a metric related to the spatial frequency content of images. Aberrations are expanded as a series of suitable functions that permit the independent optimisation of each aberration mode. A general scheme to derive these modes theoretically and experimentally is presented. Resulting aberration correction is demonstrated in an incoherent transmission imaging system and in a structured illumination microscope.

We present an overview of a wave front sensor-less adaptive optics scheme based upon the optimisation of the low spatial frequency content of images. Aberrations are expanded as a series of Lukosz functions as this permits the independent optimisation of each aberration mode. The scheme is demonstrated in an incoherent transmission microscope. It is shown how the method is related to so-called 'image sharpening' techniques.

International audience; We used intrinsic Second Harmonic Generation (SHG) by fibrillar collagen to visualize the three-dimensional architecture of collagen fibrosis at the micrometer scale using laser scanning nonlinear microscopy. We showed that SHG signals are highly specific to fibrillar collagen and provide a sensitive probe of the micrometer-scale structural organization of collagen in tissues. Moreover, recording simultaneously other nonlinear optical signals in a multimodal setup, we visualized the tissue morphology using Two-Photon Excited Fluorescence (2PEF) signals from endogenous chromophores such as NADH or elastin. We then compared different methods to determine accurate indexes of collagen fibrosis using nonlinear microscopy, given that most collagen fibrils are smaller than the microscope resolution and that second harmonic generation is a coherent process. In order to define a robust method to process our three-dimensional images, we either calculated the fraction of the images occupied by a significant SHG signal, or averaged SHG signal intensities. We showed that these scores provide an estimation of the extension of renal and pulmonary fibrosis in murine models, and that they clearly sort out the fibrotic mice.

International audience; The organization of collagen during fibrotic processes is poorly characterized because of the lack of appropriate methodologies. Here we show that multimodal multiphoton microscopy provides novel insights into lung fibrosis. We characterize normal and fibrotic pulmonary tissue in the bleomycin model, and show that second-harmonic generation by fibrillar collagen reveals the micrometer-scale three-dimensional spatial distribution of the fibrosis. We find that combined two-photon excited fluorescence and second-harmonic imaging of unstained lung tissue allows separating the inflammatory and fibrotic steps in this pathology, underlining characteristic features of fibroblastic foci in human Idiopathic Pulmonary Fibrosis samples. Finally, we propose phenomenological scores of lung fibrosis and we show that they unambiguously sort out control and treated mice, with a better sensitivity and reproducibility in the subpleural region. These results should be readily generalized to other organs, as an accurate method to assess extracellular matrix remodeling during fibrosis. Microsc. Res. Tech., 2007. © 2006 Wiley-Liss, Inc.

One principal advantage of multiphoton excitation microscopy is that it preserves its three-dimensional micrometer resolution when imaging inside light-scattering samples. For that reason two-photon-excited fluorescence microscopy has become an invaluable tool for cellular imaging in intact tissue, with applications in many fields of physiology. This success has driven increasing interest in other forms of nonlinear microscopy that can provide additional information on cells and tissues, such as second- (SHG) and third- (THG) harmonic generation microscopies. In recent years, significant progress has been made in understanding the contrast mechanisms of these recent methodologies, and high-resolution imaging based on intrinsic sources of signal has been demonstrated in cells and tissues. Harmonic generation exhibits structural rather than chemical specificity and can be obtained from a variety of non-fluorescent samples. SHG is observed specifically in dense, non-centrosymmetric arrangements of polarizable molecules, such as collagen fibrils, myofilaments, and polarized microtubule bundles. SHG imaging is therefore emerging as a novel approach for studying processes such as the physiopathological remodelling of the collagen matrix and myofibrillogenesis in intact tissue. THG does not require a non-centrosymmetric system ; however no signal can be obtained from a homogeneous medium. THG imaging therefore provides maps of sub-micrometer heterogeneities (interfaces, inclusions) in unstained samples, and can be used as a general purpose structural imaging tool. Recent studies showed that this technique can be used to image embryo development in small organisms and to characterize the accumulation of large lipid bodies in specialized cells. SHG and THG microscopy both rely on femtosecond laser technology and are easily combined with two-photon microscopy.

International audience; The micro- and macro- organization of collagen during fibrotic processes is usually characterized by 2D assessment using histochemical stains on selected slides such as Masson's Trichrome or Sirius Red. However, these staining methods are limited to semi quantitative evaluation on restricted surfaces. Quantitative evaluation of collagen contents, such as Hydroxyproline or Sircol assays, performed on lung homogenates requires large amounts of tissue, are time consuming and do not give the spatial distribution of collagen. Non linear multimodal, multiphoton microscopy provides novel insights into collagen deposition in tissues. In this study, the micrometerscale three-dimensional spatial distribution of fi brosis was characterized in normal mouse lung (n=5) and in the murine model of bleomycininduced lung fi brosis (n=5) by second harmonic generation (SHG) of fi brillar collagen, using C56Bl/6 mice at day 14 post intra-tracheal instillation of bleomycin, 80 µg in 50 µl of 0.9 % sterile saline.