Articles

Nitric oxide binding to the cardiolipin complex of ferric cytochrome
- Silkstone G.
- Kapetanaki Sofia M.
- Husu I.
- Vos Marten H.
- Wilson M.T.
Biochemistry, American Chemical Society , 2012, 51 (34), pp.6760-6766 . Cardiolipin, a phospholipid specific to the mitochondrion, interacts with the small electron transfer heme protein cytochrome c through both electrostatic and hydrophobic interactions. Once in a complex with cardiolipin, cytochrome c has been shown to undergo a conformational change that leads to the rupture of the bond between the heme iron and the intrinsic sulfur ligand of a methionine residue and to enhance the peroxidatic properties of the protein considered important to its apoptotic activity. Here we report that the ferric cytochrome c/cardiolipin complex binds nitric oxide tightly through a multistep process in which the first step is the relatively slow displacement (5 s-1) from heme coordination of an intrinsic ligand that replaces methionine in the complex. Nanosecond photolysis of the nitrosyl adduct demonstrated that a fraction of the nitric oxide escapes from the heme pocket and subsequently recombines to the heme in second-order processes (k = 1.8 × 106 and 5.5 × 105 M-1 s-1) that, under these conditions, were much faster than recombination of the intrinsic ligand with which they compete. Ultrafast (femtosecond) laser photolysis showed that the geminate recombination of nitric oxide to the heme occurred with time constants (? = 22 and 72 ps) and that ~23% of the photolyzed nitric oxide escaped into the bulk phase. This high value for the escape fraction relative to other heme proteins indicates the open nature of the heme pocket in this complex. These results are summarized in a scheme and are discussed in terms of the possible modulation of the apoptotic activity of cytochrome c by nitric oxide. Cop. 2012 American Chemical Society. (10.1021/bi300596)
DOI : 10.1021/bi300596
Asynchronous optical sampling with arbitrary detuning between laser repetition rates
- Antonucci Laura
- Solinas Xavier
- Bonvalet Adeline
- Joffre Manuel
Optics Express, Optical Society of America - OSA Publishing , 2012, 20 (16), pp.17928-17937 . A method of asynchronous optical sampling based on free-running lasers with no requirement on the repetition rates is presented. The method is based on the a posteriori determination of the delay between each pair of pulses. A resolution better than 400 fs over 13 ns total delay scan is demonstrated. In addition to the advantages of conventional asynchronous sampling techniques, this method allows a straightforward implementation on already-existing laser systems using a fiber-based setup and an appropriate acquisition procedure. (C) 2012 Optical Society of America (10.1364/OE.20.017928)
DOI : 10.1364/OE.20.017928
Glotaran: A Java-based graphical user interface for the R package TIMP
- Snellenburg Joris J.
- Laptenok Sergey
- Seger Ralf
- Mullen Katharine M.
- van Stokkum Ivo H.M.
Journal of Statistical Software, University of California, Los Angeles , 2012, 49 (3) . In this work the software application called Glotaran is introduced as a Java-based graphical user interface to the R package TIMP, a problem solving environment for fitting superposition models to multi-dimensional data. TIMP uses a command-line user interface for the interaction with data, the specification of models and viewing of analysis results. Instead, Glotaran provides a graphical user interface which features interactive and dynamic data inspection, easier -- assisted by the user interface -- model specification and interactive viewing of results. The interactivity component is especially helpful when working with large, multi-dimensional datasets as often result from time-resolved spectroscopy measurements, allowing the user to easily pre-select and manipulate data before analysis and to quickly zoom in to regions of interest in the analysis results. Glotaran has been developed on top of the NetBeans rich client platform and communicates with R through the Java-to-R interface Rserve. The background and the functionality of the application are described here. In addition, the design, development and implementation process of Glotaran is documented in a generic way.
Amplification and temporal filtering during gradient sensing by nerve growth cones probed with a microfluidic assay
- Morel Mathieu
- Shynkar Vasyl
- Galas Jean-Christophe
- Dupin Isabelle
- Bouzigues Cédric
- Studer Vincent
- Dahan Maxime
Biophysical Journal, Biophysical Society , 2012, 103 (8), pp.1648-1656 . Nerve growth cones (GCs) are chemical sensors that convert graded extracellular cues into oriented axonal motion. To ensure a sensitive and robust response to directional signals in complex and dynamic chemical landscapes, GCs are presumably able to amplify and filter external information. How these processing tasks are performed remains however poorly known. Here, we probe the signal-processing capabilities of single GCs during ?-Aminobutyric acid (GABA) directional sensing with a shear-free microfluidic assay that enables systematic measurements of the GC output response to variable input gradients. By measuring at the single molecule level the polarization of GABA A chemoreceptors at the GC membrane, as a function of the external GABA gradient, we find that GCs act as i), signal amplifiers over a narrow range of concentrations, and ii), low-pass temporal filters with a cutoff frequency independent of stimuli conditions. With computational modeling, we determine that these systems-level properties arise at a molecular level from the saturable occupancy response and the lateral dynamics of GABA A receptors. Cop. 2012 Biophysical Society. (10.1016/j.bpj.2012.08.040)
DOI : 10.1016/j.bpj.2012.08.040
Optical activity of metallic helices in the terahertz domain: A theoretical investigation
- Hache François
- Gallot Guilhem
Journal of the Optical Society of America B, Optical Society of America , 2012, 29 (10), pp.2675-2684 . Optical activity in the terahertz spectral domain has recently seen a growing interest, but fine understanding of these phenomena is not yet developed. In this article, we study analytically the response of a metallic helix in the terahertz regime and present a full nonlocal calculation of its chiroptical response. Because we do not use multipolar expansion, this calculation is very general and applies to the case where the helix size is comparable to the wavelength of the light. We calculate the circular birefringence and dichroism in three configurations: propagation along or perpendicular to the helix axis and response of an isotropic distribution of such helices. We obtain analytical expressions and can examine the consequence of the breakdown of the multipolar expansion and the wavelength-dependence of the chiroptical response, as well as give orders of magnitude that compare favorably with experimental data. This calculation is also comforted by a finite element calculation. Cop. 2012 Optical Society of America. (10.1364/JOSAB.29.002675)
DOI : 10.1364/JOSAB.29.002675
Ultrafast Ligand Dynamics in the Heme-Based GAF Sensor Domains of the Histidine Kinases DosS and DosT from Mycobacterium tuberculosis.
- Vos Marten H.
- Bouzhir-Sima Latifa
- Lambry Jean-Christophe
- Luo Hao
- Eaton-Rye Julian J.
- Ioanoviciu Alexandra
- Ortiz de Montellano Paul
- Liebl Ursula
Biochemistry, American Chemical Society , 2012, 51 (1), pp.159-166 . The transcriptional regulator DosR from M. tuberculosis plays a crucial role in the virulence to dormancy transition of the pathogen. DosR can be activated by DosT and DosS, two histidine kinases with heme-containing sensor GAF domains, capable of diatomic ligand binding. To investigate the initial processes occurring upon ligand dissociation, we performed ultrafast time-resolved absorption spectroscopy of the isolated sensor domains ligated with O2, NO, and CO. The results reveal a relatively closed heme pocket for both proteins. For DosT the yield of O2 escape from the heme pocket on the picoseconds time scale upon photodissociation was found to be very low (1.5%), similar to other heme-based oxygen sensor proteins, implying that this sensor acts as an effective O2 trap. Remarkably, this yield is an order of magnitude higher in DosS (18%). For CO, by contrast, the fraction of CO rebinding within the heme pocket is higher in DosS. Experiments with mutant DosT sensor domains and molecular dynamics simulations indicate an important role in ligand discrimination of the distal tyrosine, present in both proteins, which forms a hydrogen bond with heme-bound O2. We conclude that despite their similarity, DosT and DosS display ligand-specific different primary dynamics during the initial phases of intraprotein signaling. The distal tyrosine, present in both proteins, plays an important role in these processes. (10.1021/bi201467c)
DOI : 10.1021/bi201467c
Transcriptional Activation and Cell Cycle Block Are the Keys for 5-Fluorouracil Induced Up-Regulation of Human Thymidylate Synthase Expression
- Ligabue Alessio
- Marverti G.
- Liebl Ursula
- Myllykallio Hannu
PLoS ONE, Public Library of Science , 2012, 7 (10), pp.e47318 . Background: 5-fluorouracil, a commonly used chemotherapeutic agent, up-regulates expression of human thymidylate synthase (hTS). Several different regulatory mechanisms have been proposed to mediate this up-regulation in distinct cell lines, but their specific contributions in a single cell line have not been investigated to date. We have established the relative contributions of these previously proposed regulatory mechanisms in the ovarian cancer cell line 2008 and the corresponding cisplatin-resistant and 5-FU cross-resistant-subline C13*. Methodology/Principal Findings: Using RNA polymerase II inhibitor DRB treated cell cultures, we showed that 70-80% of up-regulation of hTS results from transcriptional activation of TYMS mRNA. Moreover, we report that 5-FU compromises the cell cycle by blocking the 2008 and C13* cell lines in the S phase. As previous work has established that TYMS mRNA is synthesized in the S and G 1 phase and hTS is localized in the nuclei during S and G 2-M phase, the observed cell cycle changes are also expected to affect the intracellular regulation of hTS. Our data also suggest that the inhibition of the catalytic activity of hTS and the up-regulation of the hTS protein level are not causally linked, as the inactivated ternary complex, formed by hTS, deoxyuridine monophosphate and methylenetetrahydrofolate, was detected already 3 hours after 5-FU exposure, whereas substantial increase in global TS levels was detected only after 24 hours. Conclusions/Significance: Altogether, our data indicate that constitutive TYMS mRNA transcription, cell cycle-induced hTS regulation and hTS enzyme stability are the three key mechanisms responsible for 5-fluorouracil induced up-regulation of human thymidylate synthase expression in the two ovarian cancer cell lines studied. As these three independent regulatory phenomena occur in a precise order, our work provides a feasible rationale for earlier observed synergistic combinations of 5-FU with other drugs and may suggest novel therapeutic strategies. Cop. 2012 Ligabue et al. (10.1371/journal.pone.0047318)
DOI : 10.1371/journal.pone.0047318
Multicolor two-photon tissue imaging by wavelength mixing
- Mahou Pierre
- Zimmerley Maxwell
- Loulier Karine
- Matho Katherine S.
- Labroille Guillaume
- Morin Xavier
- Supatto Willy
- Livet Jean
- Débarre Delphine
- Beaurepaire Emmanuel
Nature Methods, Nature Publishing Group , 2012, 9 (8), pp.815-818 . 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. (10.1038/nmeth.2098)
DOI : 10.1038/nmeth.2098
Picosecond time scale modification of forward scattered light induced by absorption inside particles
- Kervella Myriam
- d'Abzac François-Xavier
- Hache François
- Hespel Laurent
- Dartigalongue Thibault
Optics Letters, Optical Society of America - OSA Publishing , 2011, 20 (1), pp.32-41 . The aim of this work is to evaluate the influence of absorption processes on the Time Of Flight (TOF) of the light scattered out of a thick medium in the forward direction. We use a Monte-Carlo simulation with temporal phase function and Debye modes. The main result of our study is that absorption inside the particle induces a decrease of the TOF on a picosecond time scale, measurable with a femtosecond laser apparatus. This decrease, which exhibits a neat sensitivity to the absorption coefficient of particles, could provide an efficient way to measure this absorption. © 2011 OSA (10.1364/OE.20.000032)
DOI : 10.1364/OE.20.000032
Simple characterisation of a deformable mirror inside a high numerical aperture microscope using phase diversity.
- Débarre D.
- Vieille T.
- Beaurepaire Emmanuel
Journal of Microscopy, Wiley , 2011, 244 (2), pp.136-43 . 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. (10.1111/j.1365-2818.2011.03518.x)
DOI : 10.1111/j.1365-2818.2011.03518.x
Saccharomyces cerevisiae chitin biosynthesis activation by N-acetylchitooses depends on size and structure of chito-oligosaccharides.
- Becker Hubert F.
- Piffeteau Annie
- Thellend Annie
BMC Research Notes, BioMed Central , 2011, 4 (1), pp.454 . ABSTRACT: BACKGROUND: To explore chitin synthesis initiation, the effect of addition of exogenous oligosaccharides on in vitro chitin synthesis was studied. Oligosaccharides of various natures and lengths were added to a chitin synthase assay performed on a Saccharomyces cerevisiae membrane fraction. FINDINGS: N-acetylchito-tetra, -penta and -octaoses resulted in 11 to 25 % [14C]-GlcNAc incorporation into [14C]-chitin, corresponding to an increase in the initial velocity. The activation appeared specific to N-acetylchitooses as it was not observed with oligosaccharides in other series, such as beta-(1,4), beta-(1,3) or alpha-(1,6) glucooligosaccharides. CONCLUSIONS: The effect induced by the N-acetylchitooses was a saturable phenomenon and did not interfere with free GlcNAc and trypsin which are two known activators of yeast chitin synthase activity in vitro. The magnitude of the activation was dependent on both oligosaccharide concentration and oligosaccharide size. (10.1186/1756-0500-4-454)
DOI : 10.1186/1756-0500-4-454
Insights into folate/FAD-dependent tRNA methyltransferase mechanism: role of two highly conserved cysteines in catalysis
- Hamdane Djemel
- Argentini Manuela
- Cornu David
- Myllykallio Hannu
- Skouloubris Stéphane
- Hui-Bon-Hoa Gaston
- Golinelli-Pimpaneau Béatrice
Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology , 2011, 286 (42), pp.36268-36280 . The flavoprotein TrmFO methylates specifically the C5 carbon of the highly conserved uridine 54 in tRNAs. Contrary to most methyltransferases, the 1- carbon unit transferred by TrmFO derives from 5,10-methylenetetrahydrofolate and not from S-adenosyl-l-methionine. The enzyme also employs the FAD hydroquinone as a reducing agent of the C5 methylene U54-tRNA intermediate in vitro. By analogy with the catalytic mechanism of thymidylate synthase ThyA, a conserved cysteine located near the FAD isoalloxazine ring was proposed to act as a nucleophile during catalysis. Here, we mutated this residue (Cys-53 in Bacillus subtilis TrmFO) to alanine and investigated its functional role. Biophysical characterization of this variant demonstrated the major structural role of Cys-53 in maintaining both the integrity and plasticity of the flavin binding site. Unexpectedly, gel mobility shift assays showed that, like the wild-type enzyme, the inactive C53A variant was capable of forming a covalent complex with a 5-fluorouridine-containing mini-RNA. This result confirms the existence of a covalent intermediate during catalysis but rules out a nucleophilic role for Cys-53. To identify the actual nucleophile, two other strictly conserved cysteines (Cys-192 and Cys-226) that are relatively far from the active site were replaced with alanine, and a double mutant C53A/C226A was generated. Interestingly, only mutations that target Cys-226 impeded TrmFO from forming a covalent complex and methylating tRNA. Altogether, we propose a revised mechanism for the m5U54 modification catalyzed by TrmFO, where Cys-226 attacks the C6 atom of the uridine, and Cys-53 plays the role of the general base abstracting the C5 proton. (10.1074/jbc.M111.256966)
DOI : 10.1074/jbc.M111.256966
Combined third-harmonic generation and four-wave mixing microscopy of tissues and embryos.
- Mahou Pierre
- Olivier Nicolas
- Labroille Guillaume
- Duloquin Louise
- Sintes Jean-Marc
- Peyriéras Nadine
- Legouis Renaud
- Débarre Delphine
- Beaurepaire Emmanuel
Biomedical optics express, Optical Society of America - OSA Publishing , 2011, 2 (10), pp.2837-49 . 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. (10.1364/BOE.2.002837)
DOI : 10.1364/BOE.2.002837
Monitoring micrometer-scale collagen organization in rat-tail tendon upon mechanical strain using second harmonic microscopy.
- Goulam Houssen Yannick
- Gusachenko Ivan
- Schanne-Klein Marie-Claire
- Allain Jean-Marc
Journal of Biomechanics, Elsevier , 2011, 44 (11), pp.2047-52 . We continuously monitored the microstructure of a rat-tail tendon during stretch/relaxation cycles. To that purpose, we implemented a new biomechanical device that combined SHG imaging and mechanical testing modalities. This multi-scale experimental device enabled simultaneous visualization of the collagen crimp morphology at the micrometer scale and measurement of macroscopic strain-stress response. We gradually increased the ultimate strain of the cycles and showed that preconditioning mostly occurs in the first stretching. This is accompanied by an increase of the crimp period in the SHG image. Our results indicate that preconditioning is due to a sliding of microstructures at the scale of a few fibrils and smaller, that changes the resting length of the fascicle. This sliding can reverse on long time scales. These results provide a proof of concept that continuous SHG imaging performed simultaneously with mechanical assay allows analysis of the relationship between macroscopic response and microscopic structure of tissues. (10.1016/j.jbiomech.2011.05.009)
DOI : 10.1016/j.jbiomech.2011.05.009
Impact of pulse polarization on coherent vibrational ladder climbing signals
- Nuernberger Patrick
- Vieille Thibault
- Ventalon Cathie
- Joffre Manuel
Journal of Physical Chemistry B, American Chemical Society , 2011, 115 (18), pp.5554 . We report a theoretical study that elaborates the influence of the polarization state of both the pump and the probe pulse in ultrafast coherent vibrational ladder climbing experiments in the mid-infrared. Whereas a subensemble in a randomly oriented sample of molecules is excited by the pump pulse in this multiphoton process, further inhomogeneities such as the spatial profile of the laser beams, the longitudinal attenuation in the sample, and the probe beam polarization have to be taken into account. Analytical expressions for a density function describing the number of molecules that are exposed to an effective pump intensity are introduced, and the variation of the population distribution and the actual transient absorption signal in dependence on the polarization-state combinations for pump and probe pulse are discussed in detail. In simulations on the model system carboxy-hemoglobin, it is demonstrated that the polarization states play important roles both for exciting a certain population distribution and for actually observing it. In particular, it will be discussed under which conditions experimental data indicates a population inversion. Cop. 2011 American Chemical Society. (10.1021/jp1113762)
DOI : 10.1021/jp1113762
Biological applications of rare-earth based nanoparticles
- Bouzigues Cédric
- Gacoin T.
- Alexandrou Antigoni
ACS Nano, American Chemical Society , 2011, 5 (11), pp.8488 . Biomedicine and cell and molecular biology require powerful imaging techniques of the single molecule scale to the whole organism, either for fundamental science or diagnosis. These applications are however often limited by the optical properties of the available probes. Moreover, in cell biology, the measurement of the cell response with spatial and temporal resolution is a central instrumental problem. This has been one of the main motivations for the development of new probes and imaging techniques either for biomolecule labeling or detection of an intracellular signaling species. The weak photostability of genetically encoded probes or organic dyes has motivated the interest for different types of nanoparticles for imaging such as quantum dots, nanodiamonds, dye-doped silica particles, or metallic nanoparticles. One of the most active fields of research in the past decade has thus been the development of rare-earth based nanoparticles, whose optical properties and low cytotoxicity are promising for biological applications. Attractive properties of rare-earth based nanoparticles include high photostability, absence of blinking, extremely narrow emission lines, large Stokes shifts, long lifetimes that can be exploited for retarded detection schemes, and facile functionalization strategies. The use of specific ions in their compositions can be moreover exploited for oxidant detection or for implementing potent contrast agents for magnetic resonance imaging. In this review, we present these different applications of rare-earth nanoparticles for biomolecule detection and imaging in vitro, in living cells or in small animals. We highlight how chemical composition tuning and surface functionalization lead to specific properties, which can be used for different imaging modalities. We discuss their performances for imaging in comparison with other probes and to what extent they could constitute a central tool in the future of molecular and cell biology. Cop. 2011 American Chemical Society. (10.1021/nn202378b)
DOI : 10.1021/nn202378b
Toward high-content/high-throughput imaging and analysis of embryonic morphogenesis
- Truong Thai V.
- Supatto Willy
Genesis - The Journal of Genetics and Development, Wiley-Blackwell , 2011, 49 (7), pp.555 . In vivo study of embryonic morphogenesis tremendously benefits from recent advances in live microscopy and computational analyses. Quantitative and automated investigation of morphogenetic processes opens the field to high-content and high-throughput strategies. Following experimental workflow currently developed in cell biology, we identify the key challenges for applying such strategies in developmental biology. We review the recent progress in embryo preparation and manipulation, live imaging, data registration, image segmentation, feature computation, and data mining dedicated to the study of embryonic morphogenesis. We discuss a selection of pioneering studies that tackled the current methodological bottlenecks and illustrated the investigation of morphogenetic processes in vivo using quantitative and automated imaging and analysis of hundreds or thousands of cells simultaneously, paving the way for high-content/high-throughput strategies and systems analysis of embryonic morphogenesis. Cop. 2011 Wiley-Liss, Inc. (10.1002/dvg.20760)
DOI : 10.1002/dvg.20760
Quantitative assessment of collagen i liquid crystal organizations: Role of ionic force and acidic solvent, and evidence of new phases
- de Sa Peixoto Paulo
- Deniset-Besseau Ariane
- Schanne-Klein Marie-Claire
- Mosser Gervaise
Soft Matter, Royal Society of Chemistry , 2011, 7 (23), pp.11203 . Collagen I is the major structural protein in mammals where it exhibits highly organized fibrillar distributions in connective tissues. In vitro, acidic solutions of collagen I display lyotropic liquid crystal organization. These concentrated organized liquid phases can be stabilized by a pH increase to generate in vitro fibrillar matrices with specific organization. The aim of this work is to understand the mechanisms responsible for liquid crystal chirality at acidic pH in order to guide the synthesis of collagen matrices reproducing the great diversity of organizations found in biological tissues. For this purpose, we quantitatively analyze collagen liquid crystal organization by use of multiphoton microscopy, combining fluorescence and second harmonic generation contrasts. The concentration of the isotropic to liquid crystal phase transition and the evolution of the half pitch of the helical phase with collagen concentration are reported in five physico-chemical conditions using hydrochloric and acetic acids at different pHs and ionic strengths. A new phase transition is observed in highly concentrated solutions ranging from 90 mg ml-1 to 300 mg ml-1 depending on the solvent. Our results bring new quantitative information on collagen chemical physics and further substantiate the on-going analysis of the driving parameters generating twists in liquid crystals. These findings could be advantageously exploited to develop new strategies and protocols for tissue engineering. This is crucial for fundamental studies of cell behavior in biomimetic three-dimensional environments and for medical and pharmaceutical applications. Cop. The Royal Society of Chemistry 2011. (10.1039/c1sm06076g)
DOI : 10.1039/c1sm06076g
Sub-picosecond Raman spectrometer for time-resolved studies of structural dynamics in heme proteins
- Kruglik Sergei G.
- Lambry Jean-Christophe
- Martin Jean-Louis
- Vos Marten H.
- Négrerie Michel
Journal of Raman Spectroscopy, Wiley , 2011, 42 (3), pp.265 . We describe a pump-probe Raman spectrometer based on a femtosecond Ti:sapphire laser, an optical parametric generator and two optical parametric amplifiers for time-resolved studies, with emphasis on the structural dynamics in heme proteins. The system provides a 100-fs pump pulse tunable in the range 500-600 nm and a transform-limited sub-picosecond probe pulse tunable in the range 390-450 nm. The spectrometer has spectral (25 cm(-1)) and temporal (similar to 0.7 ps) resolutions which constitute an effective compromise for identifying transient heme protein species and for following their structural evolution by spontaneous Raman scattering in the time range 0.5 ps to 2 ns. This apparatus was applied to time-resolved studies of a broad range of heme proteins, monitoring the primary dynamics of photoinduced heme coordination state and structural changes, its interaction with protein side-chains and diatomic gaseous ligands, as well as heme vibrational cooling. The treatment of transient Raman spectra is described in detail, and the advantages and shortcomings of spontaneous resonance Raman spectroscopy for ultrafast heme proteins studies are discussed. We demonstrate the efficiency of the constructed spectrometer by measuring Raman spectra in the sub-picosecond and picosecond time ranges for the oxygen-storage heme protein myoglobin and for the oxygen-sensor heme protein FixLH in interaction with the diatomic gaseous ligands CO, NO, and O-2. Copyright (C) 2010 John Wiley and Sons, Ltd. (10.1002/jrs.2685)
DOI : 10.1002/jrs.2685
Deep and fast live imaging with two-photon scanned light-sheet microscopy
- Truong T.V.
- Supatto Willy
- Koos D.S.
- Choi J.M.
- Fraser S.E.
Nature Methods, Nature Publishing Group , 2011, 8 (9), pp.757 . We implemented two-photon scanned light-sheet microscopy, combining nonlinear excitation with orthogonal illumination of light-sheet microscopy, and showed its excellent performance for in vivo, cellular-resolution, three-dimensional imaging of large biological samples. Live imaging of fruit fly and zebrafish embryos confirmed that the technique can be used to image up to twice deeper than with one-photon light-sheet microscopy and more than ten times faster than with point-scanning two-photon microscopy without compromising normal biology. Cop. 2011 Nature America, Inc. All rights reserved. (10.1038/nmeth.1652)
DOI : 10.1038/nmeth.1652
Structure and function of a novel endonuclease acting on branched DNA substrates
- Creze C.
- Lestini Roxane
- Kühn Joëlle
- Ligabue Alessio
- Becker Hubert F.
- Czjzek M.
- Flament D.
- Myllykallio Hannu
Biochemical Society Transactions, Portland Press , 2011, 39 (1), pp.145-149 . Branched DNA structures that occur during DNA repair and recombination must be efficiently processed by structure-specific endonucleases in order to avoid cell death. In the present paper, we summarize our screen for new interaction partners for the archaeal replication clamp that led to the functional characterization of a novel endonuclease family, dubbed NucS. Structural analyses of Pyrococcus abyssi NucS revealed an unexpected binding site for ssDNA (single-stranded DNA) that directs, together with the replication clamp, the nuclease activity of this protein towards ssDNA-dsDNA (double-stranded DNA) junctions. Our studies suggest that understanding the detailed architecture and dynamic behaviour of the NucS (nuclease specific for ssDNA)-PCNA (proliferating-cell nuclear antigen) complex with DNA will be crucial for identification of its physiologically relevant activities. ©The Authors Journal compilation ©2011 Biochemical Society. (10.1042/BST0390145)
DOI : 10.1042/BST0390145
Advances in multiphoton microscopy for imaging embryos
- Supatto Willy
- Truong T.V.
- Débarre Delphine
- Beaurepaire Emmanuel
Current Opinion in Genetics and Development, Elsevier , 2011, 21 (5), pp.538 . 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. (10.1016/j.gde.2011.08.003)
DOI : 10.1016/j.gde.2011.08.003
Few femtosecond, few kiloampere electron bunch produced by a laser-plasma accelerator
- Lundh Olle
- Lim J.
- Rechatin Clément
- Ammoura L.
- Ben-Ismaïl Ahmed
- Davoine X.
- Gallot Guilhem
- Goddet Jean-Philippe
- Lefebvre E.
- Malka Victor
- Faure Jérôme
Nature Physics, Nature Publishing Group [2005-....] , 2011, 7 (3), pp.219 . Particle accelerators driven by the interaction of ultraintense and ultrashort laser pulses with a plasma(1) can generate accelerating electric fields of several hundred gigavolts per metre and deliver high-quality electron beams with low energy spread(2-5), low emittance(6) and up to 1 GeV peak energy(7,8). Moreover, it is expected they may soon be able to produce bursts of electrons shorter than those produced by conventional particle accelerators, down to femtosecond durations and less. Here we present wide-band spectral measurements of coherent transition radiation which we use for temporal characterization. Our analysis shows that the electron beam, produced using controlled optical injection(9), contains a temporal feature that can be identified as a 15 pC, 1.4-1.8 fs electron bunch (root mean square) leading to a peak current of 3-4 kA depending on the bunch shape. We anticipate that these results will have a strong impact on emerging applications such as short-pulse and short-wavelength radiation sources(10,11), and will benefit the realization of laboratory-scale free-electron lasers(12-14). (10.1038/NPHYS1872)
DOI : 10.1038/NPHYS1872
Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates
- Botcherby Edward J.
- Smith Christopher W.
- Kohl Michael
- Débarre Delphine
- Booth Martin J.
- Juskaitis Rimas
- Paulsen Ole
- Wilson Tony
Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2011, 109 (8), pp.2919-2924 . 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. (10.1073/pnas.1111662109)
DOI : 10.1073/pnas.1111662109
Rails and anchors: Guiding and trapping droplet microreactors in two dimensions
- Abbyad Paul
- Dangla Rémi
- Alexandrou Antigoni
- Baroud Charles N.
Lab on a Chip, Royal Society of Chemistry , 2011, 11 (5), pp.813-821 . This paper presents a method to control the motion of nanolitre drops in a wide and thin microchannel, by etching fine patterns into the channel's top surface. Such control is possible for drops that are squeezed by the channel roof, by allowing them to reduce their surface energy as they enter into a local depression. The resulting gain in surface energy pulls a drop into the groove such that localized holes can be used as anchors for holding drops, while linear patterns can be used as rails to guide them along complex trajectories. An anchored drop can remain stationary indefinitely, as long as the driving flow rate is below a critical value which depends on the hole and drop sizes. By micro-fabricating holes into a grid pattern, drops can be arrayed and held in the observation field of a microscope against the mean carrier flow. Their contents can then be modulated by gas exchange with the flowing carrier oil. We demonstrate in particular how the pH or the oxygen levels within the drops can be controlled spatially and temporally, either by exposing rows of drops to two streams of oil at different gas concentrations or by periodically switching oil inputs to vary the gas concentration of drops as a function of time. Oxygen control is used to selectively deoxygenate droplets that encapsulate red blood cells from patients suffering from sickle cell disease, in order to study the polymerization of intracellular hemoglobin. Cycles of oxygenation and deoxygenation of anchored droplets induce depolymerization and polymerization of the hemoglobin, thus providing a method to simulate the cycling that takes place in physiological flows. Cop. 2011 The Royal Society of Chemistry. (10.1039/c0lc00104j)
DOI : 10.1039/c0lc00104j