Articles

Multimodal Highlighting of Structural Abnormalities in Diabetic Rat and Human Corneas.
- Kowalczuk Laura
- Latour Gaël
- Bourges Jean-Louis
- Savoldelli Michèle
- Jeanny Jean-Claude
- Plamann Karsten
- Schanne-Klein Marie-Claire
- Behar-Cohen Françine
Translational vision science & technology, Association for Research in Vision and Ophthalmology (ARVO) , 2013, 2 (2), pp.3 . PURPOSE: This study aimed to highlight structural corneal changes in a model of type 2 diabetes, using in vivo corneal confocal microscopy (CCM). The abnormalities were also characterized by transmission electron microscopy (TEM) and second harmonic generation (SHG) microscopy in rat and human corneas. METHODS: Goto-Kakizaki (GK) rats were observed at age 12 weeks (n = 3) and 1 year (n = 6), and compared to age-matched controls. After in vivo CCM examination, TEM and SHG microscopy were used to characterize the ultrastructure and the three-dimensional organization of the abnormalities. Human corneas from diabetic (n = 3) and nondiabetic (n = 3) patients were also included in the study. RESULTS: In the basal epithelium of GK rats, CCM revealed focal hyper-reflective areas, and histology showed proliferative cells with irregular basement membrane. In the anterior stroma, extracellular matrix modifications were detected by CCM and confirmed in histology. In the Descemet's membrane periphery of all the diabetic corneas, hyper-reflective deposits were highlighted using CCM and characterized as long-spacing collagen fibrils by TEM. SHG microscopy revealed these deposits with high contrast, allowing specific detection in diabetic human and rat corneas without preparation and characterization of their three-dimensional organization. CONCLUSION: Pathologic findings were observed early in the development of diabetes in GK rats. Similar abnormalities have been found in corneas from diabetic patients. TRANSLATIONAL RELEVANCE: This multidisciplinary study highlights diabetes-induced corneal abnormalities in an animal model, but also in diabetic donors. This could constitute a potential early marker for diagnosis of hyperglycemia-induced tissue changes. (10.1167/tvst.2.2.3)
DOI : 10.1167/tvst.2.2.3
Non-invasive monitoring of cell metabolism and lipid production in 3D engineered human adipose tissues using label-free multiphoton microscopy
- Chang Tyler
- Zimmerley Maxwell
- Quinn Kyle P.
- Lamarre-Jouenne Isabelle
- Kaplan David
- Beaurepaire Emmanuel
- Georgakoudi Irene
Biomaterials, Elsevier , 2013, 34 (34), pp.8607-8616 . Non-linear optical microscopy methods can characterize over time multiple functional properties of engineered tissues during development. Here, we demonstrate how the combined use of third-harmonic generation (THG) and two-photon excited fluorescence (2PEF) imaging can provide direct quantitative biomarkers of adipogenic stem cell differentiation and metabolic state, respectively. Specifically, we imaged over nine weeks silk scaffolds embedded with human mesenchymal stem cells and exposed to either propagation (PM) or adipogenic differentiation media (AM). THG was employed to visualize the formation of lipid droplets. 2PEF was used to assess the metabolic state of the cells through the redox ratio defined based on the endogenous FAD and NADH fluorescence. The redox ratio of cells in the AM scaffold was significantly lower than that in the PM scaffold during week 5 and 9, and correlated with significant increases in lipid-to-cell volume ratio, and number and size of lipid droplets in the AM scaffold. These findings indicate that the decrease in redox ratio during adipogenic differentiation is associated with fatty acid synthesis and lipid accumulation. Our methods therefore enabled us to identify and measure dynamic correlations between lipid droplet formation and cell metabolic state, while providing insight on the spatial heterogeneity of the observed signals. (10.1016/j.biomaterials.2013.07.066)
DOI : 10.1016/j.biomaterials.2013.07.066
Probing ordered lipid assemblies with polarized third-harmonic-generation microscopy
- Zimmerley Maxwell
- Mahou Pierre
- Débarre Delphine
- Schanne-Klein Marie-Claire
- Beaurepaire Emmanuel
Physical Review X, American Physical Society , 2013, 3 (1), pp.11002 . 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. (10.1103/PhysRevX.3.011002)
DOI : 10.1103/PhysRevX.3.011002
Probing membrane protein interactions with their lipid raft environment using single-molecule tracking and Bayesian inference analysis.
- Türkcan Silvan
- Richly Maximilian U.
- Alexandrou Antigoni
- Masson Jean-Baptiste
PLoS ONE, Public Library of Science , 2013, 8 (1), pp.e53073 . The statistical properties of membrane protein random walks reveal information on the interactions between the proteins and their environments. These interactions can be included in an overdamped Langevin equation framework where they are injected in either or both the friction field and the potential field. Using a Bayesian inference scheme, both the friction and potential fields acting on the ε-toxin receptor in its lipid raft have been measured. Two types of events were used to probe these interactions. First, active events, the removal of cholesterol and sphingolipid molecules, were used to measure the time evolution of confining potentials and diffusion fields. Second, passive rare events, de-confinement of the receptors from one raft and transition to an adjacent one, were used to measure hopping energies. Lipid interactions with the ε-toxin receptor are found to be an essential source of confinement. ε-toxin receptor confinement is due to both the friction and potential field induced by cholesterol and sphingolipids. Finally, the statistics of hopping energies reveal sub-structures of potentials in the rafts, characterized by small hopping energies, and the difference of solubilization energy between the inner and outer raft area, characterized by higher hopping energies. (10.1371/journal.pone.0053073)
DOI : 10.1371/journal.pone.0053073
Arbitrary-detuning asynchronous optical sampling pump-probe spectroscopy of bacterial reaction centers
- Antonucci Laura
- Bonvalet Adeline
- Solinas Xavier
- Jones Mickael R.
- Vos Marten H.
- Joffre Manuel
Optics Letters, Optical Society of America - OSA Publishing , 2013, 38 (17), pp.3322-3324 . A recently reported variant of asynchronous optical sampling compatible with arbitrary unstabilized laser repetition rates is applied to pump-probe spectroscopy. This makes possible the use of a 5.1 MHz chirped pulse oscillator as the pump laser, thus extending the available time window to almost 200 ns with a time resolution as good as about 320 fs. The method is illustrated with the measurement in a single experiment of the complete charge transfer dynamics of the reaction center from Rhodobacter sphaeroides. © 2013 Optical Society of America (10.1364/OL.38.003322)
DOI : 10.1364/OL.38.003322
Achievement of cornea-like organizations in dense collagen I solutions: clues to the physico-chemistry of cornea morphogenesis
- de Sa Peixoto Paulo
- Deniset-Besseau Ariane
- Schmutz Marc
- Anglo Anny
- Illoul Corinne
- Schanne-Klein Marie-Claire
- Mosser Gervaise
Soft Matter, Royal Society of Chemistry , 2013, 9 (47), pp.11241-11248 . Multiphoton and electron microscopic analyses show that acido-soluble collagen I prepared in 5 mM acetic acid (pH 3.5) at concentration above 45 mg mL−1 spontaneously generates liquid crystal phases mimicking plywood organization found in cornea tissues. Those organizations extend for several hundred micrometers. Transmission electron microscopy reveals the presence of small nanofibrils organized in a complex phase, coupling overall smectic and cholesteric organizations together with local order. Those nanofibrils could be the mesogen elements giving rise to this plywood organization. These data provide clues to physico-chemical events that may take place in cornea morphogenesis in vivo. This result is invaluable for bioengineering fields, as this liquid crystal organization paves the way for the generation of collagen based bio-mimetic cornea matrices. (10.1039/c3sm52097h)
DOI : 10.1039/c3sm52097h
Picosecond binding of the his ligand to four-coordinate heme in cytochrome c ': A one-way gate for releasing proximal NO
- Yoo Byung-Kuk
- Lamarre Isabelle
- Martin Jean-Louis
- Andrew Colin R.
- Negrerie Michel
Journal of the American Chemical Society, American Chemical Society , 2013, 135 (8), pp.3248-3254 . We provide a direct demonstration of a "kinetic trap" mechanism in the proximal 5-coordinate heme-nitrosyl complex (5c-NO) of cytochrome c' from Alcaligenes xylosoxidans (AXCP) in which picosecond rebinding of the endogenous His ligand following heme-NO dissociation acts as a one-way gate for the release of proximal NO into solution. This demonstration is based upon picosecond transient absorption changes following NO photodissociation of the proximal 5c-NO AXCP complex. We have determined the absolute transient absorption spectrum of 4-coordinate ferrous heme to which NO rebinds with a time constant tNO = 7 ps (kNO = 1.4 × 1011 s-1) and shown that rebinding of the proximal histidine to the 4-coordinate heme takes place with a time constant tHis = 100 ± 10 ps (kHis = 1010 s-1) after the release of NO from the proximal heme pocket. This rapid His reattachment acts as a one-way gate for releasing proximal NO by precluding direct proximal NO rebinding once it has left the proximal heme pocket and requiring NO rebinding from solution to proceed via the distal heme face. Cop. 2013 American Chemical Society. (10.1021/ja312140f)
DOI : 10.1021/ja312140f
Crystal structure of Cex1p reveals the mechanism of tRNA trafficking between nucleus and cytoplasm
- Nozawa Kayo
- Ishitani Ryuichiro
- Yoshihisa Tohru
- Sato Mamoru
- Arisaka Fumio
- Kanamaru Shuji
- Dohmae Naoshi
- Mangroo Dev
- Senger Bruno
- Becker Hubert F.
- Nureki Osamu
Nucleic Acids Research, Oxford University Press , 2013, 41 (6), pp.3901-3914 . In all eukaryotes, transcribed precursor tRNAs are maturated by processing and modification processes in nucleus and are transported to the cytoplasm. The cytoplasmic export protein (Cex1p) captures mature tRNAs from the nuclear export receptor (Los1p) on the cytoplasmic side of the nuclear pore complex, and it delivers them to eukaryotic elongation factor 1alpha. This conserved Cex1p function is essential for the quality control of mature tRNAs to ensure accurate translation. However, the structural basis of how Cex1p recognizes tRNAs and shuttles them to the translational apparatus remains unclear. Here, we solved the 2.2 A resolution crystal structure of Saccharomyces cerevisiae Cex1p with C-terminal 197 disordered residues truncated. Cex1p adopts an elongated architecture, consisting of N-terminal kinase-like and a C-terminal alpha-helical HEAT repeat domains. Structure-based biochemical analyses suggested that Cex1p binds tRNAs on its inner side, using the positively charged HEAT repeat surface and the C-terminal disordered region. The N-terminal kinase-like domain acts as a scaffold to interact with the Ran-exportin (Los1p.Gsp1p) machinery. These results provide the structural basis of Los1p.Gsp1p.Cex1p.tRNA complex formation, thus clarifying the dynamic mechanism of tRNA shuttling from exportin to the translational apparatus. (10.1093/nar/gkt010)
DOI : 10.1093/nar/gkt010
Attenuated internal reflection terahertz imaging
- Wojdyla Antoine
- Gallot Guilhem
Optics Letters, Optical Society of America - OSA Publishing , 2013, 38 (2), pp.112-114 . We present a terahertz (THz) imaging technique based on attenuated internal reflection, which is ideally suited for the analysis of liquid and biological samples. Inserted in a THz time-domain system, and using a high-resistivity low loss silicon prism to couple the THz wave into the sample, the detection scheme is based on the relative differential spectral phase of two orthogonal polarizations. Biological sample imaging as well as subwavelength (?/16) longitudinal resolution are demonstrated. Cop. 2013 Optical Society of America.
Upregulation of Adhesion Molecules on Leukemia Targets Improves the Efficacy of Cytotoxic T Cells Transduced With Chimeric Anti-CD19 Receptor
- Laurin David
- Marin Virna
- Biagi Ettore
- Pizzitola Irene
- Agostoni Valentina
- Gallot Géraldine
- Vié Henri
- Christine Jacob Marie
- Chaperot Laurence
- Aspord Caroline
- Plumas Joël
Journal of Immunotherapy, Lippincott, Williams & Wilkins , 2013, 36 (3), pp.181-189 . (10.1097/CJI.0b013e318288f8c1)
DOI : 10.1097/CJI.0b013e318288f8c1
Parallel measurements of reaction kinetics using ultralow-volumes
- Fradet Etienne
- Abbyad Paul
- Vos Marten H.
- Baroud Charles N.
Lab on a Chip, Royal Society of Chemistry , 2013, 13 (22), pp.4326-4330 . We present a new platform for the production and manipulation of microfluidic droplets in view of measuring the evolution of a chemical reaction. Contrary to existing approaches, our device uses gradients of confinement to produce a single drop on demand and guide it to a pre-determined location. In this way, two nanoliter drops containing different reagents can be placed in contact and merged together, in order to trigger a chemical reaction. The reaction rate is extracted from an analysis of the observed reaction-diffusion front. We show that the results obtained using this platform are in excellent agreement with stopped-flow measurements, while decreasing the sample consumption 5000 fold. We also show how the device operation can be parallelized in order to react an initial sample with a range of compounds or concentrations, on a single integrated chip. This integrated chip thus further reduces sample consumption while reducing the time required for the experimental runs from hours to minutes. (10.1039/c3lc50768h)
DOI : 10.1039/c3lc50768h
Primary processes in heme-based sensor proteins
- Liebl Ursula
- Lambry Jean-Christophe
- Vos Marten H.
Biochimica et Biophysica Acta Proteins and Proteomics, Elsevier , 2013, 1834 (9), pp.1684-1692 . A wide and still rapidly increasing range of heme-based sensor proteins has been discovered over the last two decades. At the molecular level, these proteins function as bistable switches in which the catalytic activity of an enzymatic domain is altered mostly by binding or dissociation of small gaseous ligands (O2, NO or CO) to the heme in a sensor domain. The initial "signal" at the heme level is subsequently transmitted within the protein to the catalytic site, ultimately leading to adapted expression levels of specific proteins. Making use of the photolability of the heme-ligand bond that mimics thermal dissociation, early processes in this intra-protein signaling pathway can be followed using ultrafast optical spectroscopic techniques; they also occur on timescales accessible to molecular dynamics simulations. Experimental studies performed over the last decade on proteins including the sensors FixL (O2), CooA (CO) and soluble guanylate cyclase (NO) are reviewed with an emphasis on emerging general mechanisms. After heme-ligand bond breaking, the ligand can escape from the heme pocket and eventually from the protein, or rebind directly to the heme. Remarkably, in all sensor proteins the rebinding, specifically of the sensed ligand, is highly efficient. This "ligand trap" property possibly provides means to smoothen the effects of fast environmental fluctuations on the switching frequency. For 6-coordinate proteins, where exchange between an internal heme-bound residue and external gaseous ligands occurs, the study of early processes starting from the unliganded form indicates that mobility of the internal ligand may facilitate signal transfer. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. Cop. 2013 Elsevier B.V. All rights reserved. (10.1016/j.bbapap.2013.02.025)
DOI : 10.1016/j.bbapap.2013.02.025
Configurational fluctuations and flavin-substrate interactions in the flavoenzyme ThyX studied by time- and spectrally resolved fluorescence
- Laptenok Sergey P.
- Bouzhir-Sima Latifa
- Myllykallio Hannu
- Liebl Ursula
- Vos Marten H.
EPJ Web of Conferences, EDP Sciences , 2013, 41, pp.07011 . Femtosecond-resolved fluorescence of bacterial thymidilate synthase using a Kerr-gate based setup identifies a close-by tyrosine involved in flavin fluorescence quenching, shows that the substrate dUMP acts as a strong quencher itself and highlights functional configurational flexibility. © Owned by the authors, published by EDP Sciences, 2013 (10.1051/epjconf/20134107011)
DOI : 10.1051/epjconf/20134107011
Numerical simulation of polarization-resolved second harmonic microscopy in birefringent media
- Gusachenko Ivan
- Schanne-Klein Marie-Claire
Physical Review A : Atomic, molecular, and optical physics [1990-2015], American Physical Society , 2013, 88 (5), pp.053811 . Polarization-resolved second-harmonic microscopy has recently emerged as a valuable technique for in situ imaging of collagen structure in tissues. Nevertheless, collagen-rich tissues such as tendon, ligament, skin dermis, bone, cornea, or artery exhibit a heterogeneous and anisotropic architecture that results in complex optical properties. While experimental evidence of polarization distortions has been reported in various tissues, the physics of second-harmonic imaging within such tissues is not fully understood yet. In this work, we performed numerical simulations of polarization-resolved second-harmonic generation in a strongly focused regime within a birefringent tissue. We show that vectorial components due to strong focusing have a rather small effect on the measurement of the second-harmonic tensorial response, while birefringence and optical dispersion may affect these measurements dramatically. We show indeed that a difference in the focal field distribution for ordinary and extraordinary waves results in different phase-matching conditions, which strongly affects the relative efficacy of second-harmonic generation for different polarizations. These results are of great interest for extracting reliable quantitative parameters from second-harmonic images. ©2013 American Physical Society (10.1103/PhysRevA.88.053811)
DOI : 10.1103/PhysRevA.88.053811
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
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
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
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
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.
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
In vivo structural imaging of the cornea by polarization-resolved second harmonic microscopy.
- Latour Gaël
- Gusachenko Ivan
- Kowalczuk Laura
- Lamarre Isabelle
- Schanne-Klein Marie-Claire
Biomedical optics express, Optical Society of America - OSA Publishing , 2012, 3 (1), pp.1-15 . The transparency and mechanical strength of the cornea are related to the highly organized three-dimensional distribution of collagen fibrils. It is of great interest to develop specific and contrasted in vivo imaging tools to probe these collagenous structures, which is not available yet. Second Harmonic Generation (SHG) microscopy is a unique tool to reveal fibrillar collagen within unstained tissues, but backward SHG images of cornea fail to reveal any spatial features due to the nanometric diameter of stromal collagen fibrils. To overcome this limitation, we performed polarization-resolved SHG imaging, which is highly sensitive to the sub-micrometer distribution of anisotropic structures. Using advanced data processing, we successfully retrieved the orientation of the collagenous fibrils at each depth of human corneas, even in backward SHG homogenous images. Quantitative information was also obtained about the submicrometer heterogeneities of the fibrillar collagen distribution by measuring the SHG anisotropy. All these results were consistent with numerical simulation of the polarization-resolved SHG response of cornea. Finally, we performed in vivo SHG imaging of rat corneas and achieved structural imaging of corneal stroma without any labeling. Epi-detected polarization-resolved SHG imaging should extend to other organs and become a new diagnosis tool for collagen remodeling. 2011 Optical Society of America (10.1364/BOE.3.000001)
DOI : 10.1364/BOE.3.000001