Publications

2012

• Microscopie non linéaire de tissus biologiques : excitation multicouleur, faisceaux de Bessel, et excitation en nappe de lumière
  
  • Mahou Pierre
  , 2012. Le travail effectué au cours de cette thèse a porté sur le développement et la mise en œuvre de nouvelles stratégies en microscopie non linéaire permettant d'augmenter le nombre de signaux non linéaires simultanément observables d'une part, et la vitesse d'acquisition d'autre part. Dans un premier temps, nous avons exploré la possibilité de produire des signaux multiples au moyen de deux trains d'impulsions synchronisés de longueur d'onde centrale distincte. Nous avons montré que cette approche permet d'exciter de façon optimale et simultanée trois protéines fluorescentes respectivement bleue, jaune, et rouge. Une application de cette méthode consiste à imager de grands volumes de tissus marqués avec des transgènes Brainbow dans le but d'étudier la connectivité ou le lignage cellulaire. Plus généralement, nous avons montré que cette approche permet de combiner plusieurs signaux non linéaires tels que la fluorescence, la génération de seconde (SHG) et de troisième (THG) harmoniques, ainsi que le mélange à quatre ondes (FWM). Dans un deuxième temps, nous avons étudié la possibilité d'augmenter la vitesse d'imagerie. Pour cela, nous avons mis en œuvre plusieurs manières de produire des faisceaux de Bessel focalisés afin d'augmenter la profondeur de champ d'un microscope à balayage. Enfin, en vue d'augmenter la vitesse d'acquisition tout en préservant le sectionnement optique, nous avons construit un microscope biphotonique à nappe de lumière de profil spatial programmable. Dans cette géométrie nous avons comparé les propriétés d'imagerie de profils d'excitation de type gaussien et de Bessel pour des applications en biologie du développement.
• Microscopie optique non-linéaire quantitative utilisant un faisceau mis en forme (extension internationale du brevet français)
  
  • Schanne-Klein Marie-Claire
  • Beaurepaire Emmanuel
  • Strupler Mathias
  • Débarre Delphine
  • Olivier Nicolas
  • Mahou Pierre
  , 2012.
• Microscopie multiphoton illuminée par nappe : imagerie de fluorescence rapide et en profondeur dans les tissus vivants
  
  • Supatto Willy
  Photoniques, EDP Sciences, 2012 (62), pp.33-37. (10.1051/photon/20126233)
  DOI : 10.1051/photon/20126233
• Polarization-resolved Second Harmonic Microscopy in collagenous tissues: theoretical and experimental developments, and application to microstructural imaging during biomechanical assays in tendon.
  
  • Gusachenko Ivan
  , 2012. Collagen is a major structural protein that forms various macromolecular organizations in tissues and is responsible for the biomechanical properties of most organs. Second harmonic generation (SHG) microscopy is a valuable imaging technique to probe collagen fibrillar organization. This work is aimed at implementing and characterizing polarization-resolved SHG (P-SHG) and coupling this technique to biomechanical assays to provide multiscale structural information on collagen tissues. We first studied the linear propagation effects that affect P-SHG imaging of thick anisotropic tissues such as collagen tissues. We developed a theoretical model that accounts for birefringence, diattenuation, and polarization scrambling, and obtained an excellent agreement with our P-SHG measurements in rattail tendon. Moreover we performed numerical simulations of light propagation and harmonic generation in tendon and cornea, which confirmed the crucial role of birefringence for P-SHG signal formation. We then implemented a new experimental device that combined mechanical testing with SHG imaging. It enabled visualization of the tendon crimp morphology at the micrometer scale during macroscopic strain-stress measurements. Our results proved that continuous SHG imaging allows for elucidating the link between macroscopic response and microscopic structure of tissues. Finally, we developed a theoretical model, which relates the P-SHG signal anisotropy to the orientational order of its SHG-capable constituents at submicrometer scale. We tested our model by performing P-SHG measurements in increasingly stretched tendon, and successfully characterized variations of fibril disorder within fascicle with strain.
• Third-harmonic generation microscopy with Bessel beams: a numerical study
  
  • Olivier Nicolas
  • Débarre Delphine
  • Mahou Pierre
  • Beaurepaire Emmanuel
  Optics Express, Optical Society of America - OSA Publishing, 2012, 20 (22), pp.24886-24902. 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 λ/8 - λ range in the case of organized media. © 2012 OSA (10.1364/OE.20.024886)
  DOI : 10.1364/OE.20.024886
• In situ 3D characterization of historical coatings and wood using multimodal nonlinear optical microscopy
  
  • Latour Gaël
  • Echard Jean-Philippe
  • Didier Marie
  • Schanne-Klein Marie-Claire
  Optics Express, Optical Society of America - OSA Publishing, 2012, 20 (22), pp.24623-24635. We demonstrate multimodal nonlinear optical imaging of historical artifacts by combining Second Harmonic Generation (SHG) and Two-Photon Excited Fluorescence (2PEF) microscopies. We first identify the nonlinear optical response of materials commonly encountered in coatings of cultural heritage artifacts by analyzing one- and multi-layered model samples. We observe 2PEF signals from cochineal lake and sandarac and show that pigments and varnish films can be discriminated by exploiting their different emission spectral ranges as in luminescence linear spectroscopy. We then demonstrate SHG imaging of a filler, plaster, composed of bassanite particles which exhibit a non centrosymmetric crystal structure. We also show that SHG/2PEF imaging enables the visualization of wood microstructure through typically 60 µm-thick coatings by revealing crystalline cellulose (SHG signal) and lignin (2PEF signal) in the wood cell walls. Finally, in situ multimodal nonlinear imaging is demonstrated in a historical violin. SHG/2PEF imaging thus appears as a promising non-destructive and contactless tool for in situ 3D investigation of historical coatings and more generally for wood characterization and coating analysis at micrometer scale. © 2012 OSA (10.1364/OE.20.024623)
  DOI : 10.1364/OE.20.024623
• Procédé microfluidique de traitement et d'analyse d'une solution contenant un matériel biologique, et circuit microfluidique correspondant
  
  • Baroud Charles N.
  • Dangla Rémi
  • Türkcan Silvan
  • Abbyad Paul
  , 2012. La présente invention a pour objet un procédé microfluidique de traitement et d'analyse d'une solution contenant un matériel biologique, comprend une étape d'introduction de la solution dans des microcanaux d'un circuit microfluidique (1), une étape de formation de gouttes de cette solution, sous l'effet de modifications de la tension de surface de la solution, une étape de déplacement des gouttes vers une ou plusieurs zone de stockage de gouttes (130), sous l'effet de modifications de la tension de surface des gouttes, une étape de traitement des gouttes et une étape d'analyse des gouttes.
• Circuit microfluidique permettant la mise en contact de gouttes de plusieurs fluides, et procédé microfluidique correspondant
  
  • Fradet Etienne
  • Dangla Remi
  • Baroud Charles N.
  • Abbyad Paul
  , 2012. La présente invention a pour objet un circuit microfluidique (1), dans lequel sont définis des microcanaux pouvant contenir des fluides, et comprenant au moins un dispositif de formation de gouttes d'une solution, des moyens de guidage des gouttes (133) vers une zone de stockage (130) dans laquelle une des gouttes peut être mise en contact avec une goutte d'une autre solution, les parois de ladite portion de microcanal formant le premier dispositif de formation de gouttes s'écartent de façon à détacher des gouttes de ladite première solution sous l'effet de la tension de surface de ladite première solution ; lesdits premiers moyens de guidage (133) comprennent des portions de paroi desdits microcanaux, s'écartant de façon à déplacer lesdites gouttes sous l'effet de la tension de surface de ladite première solution.
• Device for managing pulses in pump-probe spectroscopy
  
  • Antonucci Laura
  • Bonvalet Adeline
  • Joffre Manuel
  • Solinas Xavier
  , 2012. A device for managing light pulses for measuring the reaction of a sample exposed to a first light pulse, the measurement being performed by analysis of a signal emitted by the sample subjected to a second light pulse, shifted with respect to the first pulse by a determined interval of time, the device including two optical detectors for detecting the pulses of two light beams emitted by two pulsed laser sources, respectively, each beam emitting pulses with respective repetition frequencies that are different, arbitrary and stable over a determined period in the direction of the sample; the detectors being connected to a computer for determining the interval of time between two pulses coming from the first and the second beam, respectively, and constituting the first and second pulses; the computer being connected to an analyzer for measuring the reaction of the sample having as input parameter the interval of time between the two pulses, where the computer uses an algorithm making use of the stability of the repetition frequencies for determining the interval of time.
• In situ three-dimensional monitoring of collagen fibrillogenesis using SHG microscopy.
  
  • Bancelin Stéphane
  • Aimé Carole
  • Coradin Thibaud
  • Schanne-Klein Marie-Claire
  Biomedical optics express, Optical Society of America - OSA Publishing, 2012, 3 (6), pp.1446-54. We implemented in situ time-lapse Second Harmonic Generation (SHG) microscopy to monitor the three-dimensional (3D) self-assembly of collagen in solution. As a proof of concept, we tuned the kinetics of fibril formation by varying the pH and measured the subsequent exponential increase of fibril volume density in SHG images. We obtained significantly different time constants at pH = 6.5 ± 0.3 and at pH = 7.5 ± 0.3. Moreover, we showed that we could focus on the growth of a single isolated collagen fibril because SHG microscopy is sensitive to well-organized fibrils with diameter below the optical resolution. This work illustrates the potential of SHG microscopy for the rational design and characterization of collagen-based biomaterials. (10.1364/BOE.3.001446)
  DOI : 10.1364/BOE.3.001446
• Modulation of the Pyrococcus abyssi NucS endonuclease activity by replication clamp at functional and structural levels.
  
  • Creze Christophe
  • Ligabue Alessio
  • Laurent Sébastien
  • Lestini Roxane
  • Laptenok Sergey P.
  • Khun Joelle
  • Vos Marten H.
  • Czjzek Mirjam
  • Myllykallio Hannu
  • Flament Didier
  Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2012, 287 (19), pp.15648-60. Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5' and 3' flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction. (10.1074/jbc.M112.346361)
  DOI : 10.1074/jbc.M112.346361
• Polarization-resolved second-harmonic generation in tendon upon mechanical stretching
  
  • Gusachenko Ivan
  • Tran Viet
  • Goulam Houssen Yannick
  • Allain Jean-Marc
  • Schanne-Klein Marie-Claire
  Biophysical Journal, Biophysical Society, 2012, 102 (9), pp.2220-2229. Collagen is a triple-helical protein that forms various macromolecular organizations in tissues and is responsible for the biomechanical and physical properties of most organs. Second-harmonic generation (SHG) microscopy is a valuable imaging technique to probe collagen fibrillar organization. In this article, we use a multiscale nonlinear optical formalism to bring theoretical evidence that anisotropy of polarization-resolved SHG mostly reflects the micrometer-scale disorder in the collagen fibril distribution. Our theoretical expectations are confirmed by experimental results in rat-tail tendon. To that end, we report what to our knowledge is the first experimental implementation of polarization-resolved SHG microscopy combined with mechanical assays, to simultaneously monitor the biomechanical response of rat-tail tendon at macroscopic scale and the rearrangement of collagen fibrils in this tissue at microscopic scale. These experiments bring direct evidence that tendon stretching corresponds to straightening and aligning of collagen fibrils within the fascicle. We observe a decrease in the SHG anisotropy parameter when the tendon is stretched in a physiological range, in agreement with our numerical simulations. Moreover, these experiments provide a unique measurement of the nonlinear optical response of aligned fibrils. Our data show an excellent agreement with recently published theoretical calculations of the collagen triple helix hyperpolarizability. Copyright © 2012 Biophysical Society (10.1016/j.bpj.2012.03.068)
  DOI : 10.1016/j.bpj.2012.03.068
• Methodology for reconstructing early zebrafish development from in vivo multiphoton microscopy.
  
  • Luengo-Oroz Miguel Angel
  • Rubio-Guivernau José L.
  • Faure Emmanuel
  • Savy Thierry
  • Duloquin Louise
  • Olivier Nicolas
  • Pastor-Escuredo David
  • Ledesma-Carbayo María Jesús
  • Debarre Delphine
  • Bourgine Paul
  • Beaurepaire Emmanuel
  • Peyriéras Nadine
  • Santos Andrés
  IEEE Transactions on Image Processing, Institute of Electrical and Electronics Engineers, 2012, 21 (4), pp.2335-40. 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. (10.1109/TIP.2011.2177911)
  DOI : 10.1109/TIP.2011.2177911
• Multifunctional Rare Earth Vanadate Nanoparticles: Luminescent Labels, Oxidant Sensors and Magnetic Resonance Imaging Contrast Agents
  
  • Schoeffel Markus
  , 2012. Multimodal imaging techniques are of great interest due to the wealth of information they provide. This thesis is devoted to the development and characterization of nanoparticles which can be applied as luminescent probes, for oxidant detection and as contrast agents in magnetic resonance imaging. The work is based on previous studies on Y0.6Eu0.4VO4 nanoparticles which show strong, non-blinking and stable luminescence. Time and space resolved optical oxidant detection is feasible after reversible photoreduction of Eu3+ to Eu2+ by e. g. H2O2. This oxidation is detectable by a modification of the luminescence properties. The incorporation of paramagnetic Gd3+ confers proton relaxation enhancing properties to the system. We synthesized nanoparticles of either 10 nm or 40 nm diameter and of the compositions GdVO4 and Gd0.6Eu0.4VO4 as well as core-shell systems containing a Y0.6Eu0.4VO4 core and a GdVO4 shell with 40 nm total diameter. X-ray microstructural analysis in combination with dynamic light scattering and electron microscopy enabled us to propose a model for the relationship between the shape of the nanoparticles and the crystallites contained in them. Complexometric titration indicated that rare earth leaching is negligible making this type of nanoparticles well suited for in vivo applications. We demonstrated that even after substitution of Y3+ by Gd3+, the nanoparticles retain the ability of H2O2 detection by luminescence modulation. Temperature and magnetic field dependent measurements of the magnetization of Gd0.6Eu0.4VO4 nanoparticles confirmed the paramagnetic behavior according to a Curie-Weiss law in the temperature range from 290 K down to 5 K. We found that the proton relaxivity of GdVO4 and Gd0.6Eu0.4VO4 nanoparticles of 10 nm diameter as well as of the core-shell nanoparticles is higher than that of the commercial chelate compound Dotarem®. Nuclear magnetic resonance dispersion spectroscopy showed higher proton relaxivities for nanoparticles made up from Gd0.6Eu0.4VO4 than from pure GdVO4. The present data suggest that rare earth vanadate nanoparticles containing simultaneously Gd and Eu are very promising candidates for applications as in vivo multifunctional probe. This system might also be useful as a target in gadolinium neutron capture therapy or for positron emission tomography.
• Particules d'oxyde à base de terres rares et utilisation notamment en imagerie
  
  • Schöffel Markus
  • Alexandrou Antigoni
  • Bouzigues Cedric
  • Gacoin Thierry
  • Boilot Jean-Pierre
  , 2012. La présente demande concerne des produits composites multimodaux pour l'imagerie, en particulier pour l'imagerie diagnostique, et optionnellement pour thérapie, en particulier des produits composites capables d'être utilisés comme agents de contraste en particulier en imagerie par résonance magnétique (IRM), et/ou dans des techniques d'imagerie, comme par exemple en imagerie optique, en détection optique d'oxydants, en tomographie par émission de positrons (TEP), en tomodensitométrie (TDM) et/ou en imagerie par ultrasons et optionnellement simultanément utilisables en thérapie. Ces produits sont basés sur une particule comprenant ou consistant en une partie pourvue d'une activité d'agent de contraste et/ou d'une activité paramagnétique, et une partie pourvue d'une activité luminescente et optionnellement d'une activité de détection d'oxydant.
• Ultrafast heme-ligand recombination in truncated hemoglobin HbO from Mycobacterium tuberculosis: A ligand cage
  
  • Jasaitis Audrius
  • Ouellet Hugues
  • Lambry Jean-Christophe
  • Martin Jean-Louis
  • Friedman Joel M.
  • Guertin Michel
  • Vos Marten H.
  Chemical Physics, Elsevier, 2012, 396, pp.10-16. Truncated hemoglobin HbO from Mycobacterium tuberculosis displays very slow exchange of diatomic ligands with its environment. Using femtosecond spectroscopy, we show that upon photoexcitation, ligands rebind with unusual speed and efficiency. Only ∼1% O2 can escape from the heme pocket and less than 1% NO. Most remarkably, CO rebinding occurs for 95%, predominantly in 1.2 ns. The general CO rebinding properties are unexpectedly robust against changes in the interactions with close by aromatic residues Trp88 (G8) and Tyr36 (CD1). Molecular dynamics simulations of the CO complex suggest that interactions of the ligand with structural water molecules as well as its rotational freedom play a role in the high reactivity of the ligand and the heme. The slow exchange of ligands between heme and environment may result from a combination of hindered ligand access to the heme pocket by the network of distal aromatic residues, and low escape probability from the pocket. (10.1016/j.chemphys.2011.04.003)
  DOI : 10.1016/j.chemphys.2011.04.003
• Module d'excitation multi-couleurs pour un systeme d'imagerie multi-photonique, systeme et procede associes
  
  • Beaurepaire Emmanuel
  • Mahou Pierre
  • Débarre Delphine
  • Supatto Willy
  • Martin Jean-Louis
  , 2012. La présente invention concerne un module 1 d'excitation multi-couleurs pour un système d'imagerie multi-photonique 100. Un tel module est utilisé pour imager un échantillon 7 comprenant au moins trois chromophores. Le module 1 comprend : - une première source de laser femtoseconde 2, émettant un premier faisceau d'excitation 20 ; - une deuxième source laser femtoseconde 3 émettant un deuxième faisceau d'excitation 30. Le premier faisceau d'excitation 20 comprend une partie dite « de pompage », cette partie de pompage servant de faisceau de pompe pour exciter de façon synchrone la deuxième source laser 3, et une partie dite « d'excitation ». Une ligne à retard optique 4 est agencée pour superposer spatialement et temporellement le deuxième faisceau d'excitation 30 et la partie d'excitation du premier faisceau d'excitation, de façon à exciter au moins un troisième des chromophores par absorption multi-photons, lesdits photons absorbés provenant des premier et deuxième faisceaux d'excitation 20,30. L'invention concerne également le système complet d'imagerie multi-photonique 100, ainsi qu'un procédé mis en oeuvre dans ce module 1.
• Accuracy of correction in modal sensorless adaptive optics.
  
  • Facomprez Aurélie
  • Beaurepaire Emmanuel
  • Débarre Delphine
  Optics Express, Optical Society of America - OSA Publishing, 2012, 20 (3), pp.2598-612. 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. We show that only 10(4) to 10(5) photons are required for complete correction so that the correction process can be implemented with limited extra-illumination and associated photoperturbation. Finally, we provide guidelines for implementing an optimal correction algorithm depending on the experimental conditions.
• In vivo multiphoton imaging of the cornea: Polarization-resolved second harmonic generation from stromal collagen
  
  • Latour Gaël
  • Gusachenko Ivan
  • Kowalczuk Laura
  • Lamarre Isabelle
  • Schanne-Klein Marie-Claire
  , 2012, 8226. Multiphoton microscopy provides specific and contrasted images of unstained collagenous tissues such as tendons or corneas. Polarization-resolved second harmonic generation (SHG) measurements have been implemented in a laserscanning multiphoton microscope. Distortion of the polarimetric response due to birefringence and diattenuation during propagation of the laser excitation has been shown in rat-tail tendons. A model has been developed to account for these effects and correct polarization-resolved SHG images in thick tissues. This new modality is then used in unstained human corneas to access two quantitative parameters: the fibrils orientation within the collagen lamellae and the ratio of the main second-order nonlinear tensorial components. Orientation maps obtained from polarization resolution of the trans-detected SHG images are in good agreement with the striated features observed in the raw images. Most importantly, polarization analysis of the epi-detected SHG images also enables to map the fibrils orientation within the collagen lamellae while epi-detected SHG images of corneal stroma are spatially homogenous and do not enable direct visualization of the fibrils orientation. Depth profiles of the polarimetric SHG response are also measured and compared to models accounting for orientation changes of the collagen lamellae within the focal volume. Finally, in vivo polarization-resolved SHG is performed in rat corneas and structural organization of corneal stroma is determined using epi-detected signals. © (2012) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). (10.1117/12.906603)
  DOI : 10.1117/12.906603
• Correction precision in image-based adaptive optics for nonlinear microscopy
  
  • Facomprez Aurélie
  • Beaurepaire Emmanuel
  • Débarre Delphine
  , 2012, 8227. 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). (10.1117/12.908647)
  DOI : 10.1117/12.908647
• Assessing correction accuracy in image-based adaptive optics
  
  • Débarre Delphine
  • Facomprez Aurélie
  • Beaurepaire Emmanuel
  , 2012, 8253. 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). (10.1117/12.908808)
  DOI : 10.1117/12.908808
• Calibration of an adaptive microscope using phase diversity
  
  • Débarre Delphine
  • Vieille Thibault
  • Facomprez Aurélie
  • Mahou Pierre
  • Beaurepaire Emmanuel
  , 2006, pp.poster. 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).
• Quaternary Structure Controls Ligand Dynamics in Soluble Guanylate Cyclase
  
  • Yoo Byung-Kuk
  • Lamarre Isabelle
  • Martin Jean-Louis
  • Negrerie Michel
  Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2012, 287, pp.6851-6859. Soluble guanylate cyclase (sGC) is the mammalian endogenous nitric oxide (NO) receptor. The mechanisms of activation and deactivation of this heterodimeric enzyme are unknown. For deciphering them, functional domains can be overexpressed. We have probed the dynamics of the diatomic ligands NO and CO within the isolated heme domain β1(190) of human sGC by piconanosecond absorption spectroscopy. After photo-excitation of nitrosylated sGC, only NO geminate rebinding occurs in 7.5 ps. In β1(190), both photo-dissociation of 5c-NO and photo-oxidation occur, contrary to sGC, followed by NO rebinding (7 ps) and back-reduction (230 ps and 2 ns). In full-length sGC, CO geminate rebinding to the heme does not occur. In contrast, CO geminately rebinds to β1(190) with fast multiphasic process (35, 171, and 18 ns). We measured the bimolecular association rates kon = 0.075 ± 0.01 × 106 M−1*s−1 for sGC and 0.83 ± 0.1 × 106 M−1*s−1 for β1(190). These different dynamics reflect conformational changes and less proximal constraints in the isolated heme domain with respect to the dimeric native sGC. We concluded that the α-subunit and the β1(191-619) domain exert structural strains on the heme domain. These strains are likely involved in the transmission of the energy and relaxation toward the activated state after Fe2+-His bond breaking. This also reveals the heme domain plasticity modulated by the associated domains and subunit. (10.1074/jbc.M111.299297)
  DOI : 10.1074/jbc.M111.299297
• Mechanistic and structural basis for inhibition of thymidylate synthase ThyX
  
  • Basta Tamara
  • Boum Yap
  • Briffotaux Julien
  • Becker Hubert F
  • Lamarre-Jouenne Isabelle
  • Lambry Jean-Christophe
  • Skouloubris Stephane
  • Liebl Ursula
  • Graille Marc
  • van Tilbeurgh Herman
  • Myllykallio Hannu
  Open Biology, Royal Society, 2012, 2 (10), pp.120120. Nature has established two mechanistically and structurally unrelated families of thymidylate synthases that produce de novo thymidylate or dTMP, an essential DNA precursor. Representatives of the alternative flavin-dependent thymidylate synthase family, ThyX, are found in a large number of microbial genomes, but are absent in humans. We have exploited the nucleotide binding pocket of ThyX proteins to identify non-substrate-based tight-binding ThyX inhibitors that inhibited growth of genetically modified Escherichia coli cells dependent on thyX in a manner mimicking a genetic knockout of thymidylate synthase. We also solved the crystal structure of a viral ThyX bound to 2-hydroxy-3-(4-methoxybenzyl)-1,4-naphthoquinone at a resolution of 2.6 Å. This inhibitor was found to bind within the conserved active site of the tetrameric ThyX enzyme, at the interface of two monomers, partially overlapping with the dUMP binding pocket. Our studies provide new chemical tools for investigating the ThyX reaction mechanism and establish a novel mechanistic and structural basis for inhibition of thymidylate synthesis. As essential ThyX proteins are found e.g. in Mycobacterium tuberculosis and Helicobacter pylori, our studies have also potential to pave the way towards the development of new anti-microbial compounds. (10.1098/rsob.120120)
  DOI : 10.1098/rsob.120120
• A Bayesian Inference Scheme to Extract Diffusivity and Potential Fields from Confined Single-Molecule Trajectories
  
  • Tuerkcan Silvan
  • Alexandrou Antigoni
  • Masson Jean-Baptiste
  Biophysical Journal, Biophysical Society, 2012, 102 (10), pp.2288-2298. Currently used techniques for the analysis of single-molecule trajectories only exploit a small part of the available information stored in the data. Here, we apply a Bayesian inference scheme to trajectories of confined receptors that are targeted by pore-forming toxins to extract the two-dimensional confining potential that restricts the motion of the receptor. The receptor motion is modeled by the overdamped Langevin equation of motion. The method uses most of the information stored in the trajectory and converges quickly onto inferred values, while providing the uncertainty on the determined values. The inference is performed on the polynomial development of the potential and on the diffusivities that have been discretized on a mesh. Numerical simulations are used to test the scheme and quantify the convergence toward the input values for forces, potential, and diffusivity. Furthermore, we show that the technique outperforms the classical mean-square-displacement technique when forces act on confined molecules because the typical mean-square-displacement analysis does not account for them. We also show that the inferred potential better represents input potentials than the potential extracted from the position distribution based on Boltzmann statistics that assumes statistical equilibrium. (10.1016/j.bpj.2012.01.063)
  DOI : 10.1016/j.bpj.2012.01.063