Articles

Sickling of red blood cells through rapid oxygen exchange in microfluidic drops
- Abbyad Paul
- Tharaux Pierre-Louis
- Martin Jean-Louis
- Baroud Charles N.
- Alexandrou Antigoni
Lab on a Chip, Royal Society of Chemistry , 2010, 10 (19), pp.2505 . We have developed a microfluidic approach to study the sickling of red blood cells associated with sickle cell anemia by rapidly varying the oxygen partial pressure within flowing microdroplets. By using the perfluorinated carrier oil as a sink or source of oxygen, the oxygen level within the water droplets quickly equilibrates through exchange with the surrounding oil. This provides control over the oxygen partial pressure within an aqueous drop ranging from 1 kPa to ambient partial pressure, i.e. 21 kPa. The dynamics of the oxygen exchange is characterized through fluorescence lifetime measurements of a ruthenium compound dissolved in the aqueous phase. The gas exchange is shown to occur primarily during and directly after droplet formation, in 0.1 to 0.5 s depending on the droplet diameter and speed. The controlled deoxygenation is used to trigger the polymerization of hemoglobin within sickle red blood cells, encapsulated in drops. This process is observed using polarization microscopy, which yields a robust criterion to detect polymerization based on transmitted light intensity through crossed polarizers. Cop. 2010 The Royal Society of Chemistry. (10.1039/c004390g)
DOI : 10.1039/c004390g
QUANTUM YIELD MEASUREMENTS OF SHORT-LIVED PHOTOACTIVATION INTERMEDIATES IN DNA PHOTOLYASE : TOWARD A DETAILED UNDERSTANDING OF THE TRIPLE TRYPTOPHAN ELECTRON TRANSFER CHAIN
- Byrdin M.
- Lukacs Andras
- Thiagarajan V.
- Apm Eker
- Brettel K.
- Vos Marten H.
Journal of Physical Chemistry A, American Chemical Society , 2010, 114 (9), pp.3207-3214 . The light-dependent DNA repair enzyme photolyase contains a unique evolutionary conserved triple tryptophan electron transfer chain (W382−W359−W306 in photolyase from E. coli) that bridges the ∼15 Å distance between the buried flavin adenine dinucleotide (FAD) cofactor and the surface of the protein. Upon excitation of the semireduced flavin (FADH°), electron transfer through the chain leads to formation of fully reduced flavin (FADH−; required for DNA repair) and oxidation of the most remote tryptophan residue W306, followed by its deprotonation. The thus-formed tryptophanyl radical W306°+ is reduced either by an extrinsic reductant or by reverse electron transfer from FADH−. Altogether the kinetics of these charge transfer reactions span 10 orders of magnitude, from a few picoseconds to tens of milliseconds. We investigated electron transfer processes in the picosecond−nanosecond time window bridging the time domains covered by ultrafast pump−probe and "classical" continuous probe techniques. Using a recent dedicated setup, we directly show that virtually no absorption change between 300 ps and 10 ns occurs in wild-type photolyase, implying that no charge recombination takes place in this time window. In contrast, W306F mutant photolyase showed a partial absorption recovery with a time constant of 0.85 ns. In wild-type photolyase, the quantum yield of FADH− W306°+ was found at 19 ± 4%, in reference to the established quantum yield of the long-lived excited state of [Ru(bpy)3]2+. With this yield, the optical spectrum of the excited state of FADH° can be constructed from ultrafast spectroscopic data; this spectrum is dominated by excited state absorption extending from below 450 to 850 nm. The new experimental results, taken together with previous data, allow us to propose a detailed kinetic and energetic scheme of the electron transfer chain. Copyright © 2009 American Chemical Society (10.1021/jp9093589)
DOI : 10.1021/jp9093589
Quantum yield measurements of short-lived photoactivation intermediates in DNA photolyase: Toward a detailed understanding of the triple tryptophan electron transfer chain
- Byrdin Martin
- Lukacs Andras
- Thiagarajan Viruthachalam
- Eker André P.M.
- Brettel Klaus
- Vos Marten H.
Journal of Physical Chemistry A, American Chemical Society , 2010, 114 (9), pp.3207-3214 . The light-dependent DNA repair enzyme photolyase contains a unique evolutionary conserved triple tryptophan electron transfer chain (W382−W359−W306 in photolyase from E. coli) that bridges the 15 Å distance between the buried flavin adenine dinucleotide (FAD) cofactor and the surface of the protein. Upon excitation of the semireduced flavin (FADH°), electron transfer through the chain leads to formation of fully reduced flavin (FADH−; required for DNA repair) and oxidation of the most remote tryptophan residue W306, followed by its deprotonation. The thus-formed tryptophanyl radical W306°+ is reduced either by an extrinsic reductant or by reverse electron transfer from FADH−. Altogether the kinetics of these charge transfer reactions span 10 orders of magnitude, from a few picoseconds to tens of milliseconds. We investigated electron transfer processes in the picosecond−nanosecond time window bridging the time domains covered by ultrafast pump−probe and "classical" continuous probe techniques. Using a recent dedicated setup, we directly show that virtually no absorption change between 300 ps and 10 ns occurs in wild-type photolyase, implying that no charge recombination takes place in this time window. In contrast, W306F mutant photolyase showed a partial absorption recovery with a time constant of 0.85 ns. In wild-type photolyase, the quantum yield of FADH− W306°+ was found at 19 ± 4%, in reference to the established quantum yield of the long-lived excited state of [Ru(bpy)3]2+. With this yield, the optical spectrum of the excited state of FADH° can be constructed from ultrafast spectroscopic data; this spectrum is dominated by excited state absorption extending from below 450 to 850 nm. The new experimental results, taken together with previous data, allow us to propose a detailed kinetic and energetic scheme of the electron transfer chain. (10.1021/jp9093589)
DOI : 10.1021/jp9093589
Nonlinear beam shaper for femtosecond laser pulses, from Gaussian to flat-top profile
- Mercier Brigitte
- Rousseau Jean-Philippe
- Jullien A.
- Antonucci Laura
Optics Communications, Elsevier , 2010, 283 (14), pp.2900 . We present a straightforward method to transform a spatially Gaussian femtosecond laser beam into a flattop shaped beam. The proposed technique takes advantage of a nonlinear phase induced in positive Kerr medium followed by a simple optical system. The variation of the refractive index with the laser intensity creates a phase plate which induces changes in the beam profile after propagation; flat-top and doughnut profiles are observed. The shaping conditions are computed numerically and confirmed experimentally. The method does not introduce energy losses. The device is very simple, self-regulated, flexible and does not need a manufactured phase plate or precise alignment. This method can be useful for light-matter interaction and laser machining. Cop 2010 Elsevier B.V. All rights reserved. (10.1016/j.optcom.2010.04.004)
DOI : 10.1016/j.optcom.2010.04.004
Multiply excited vibration of carbon monoxide in the primary docking site of hemoglobin following photolysis from the heme
- Nuernberger Patrick
- Lee Kevin F.
- Bonvalet Adeline
- Vos Marten H.
- Joffre Manuel
Journal of Physical Chemistry Letters, American Chemical Society , 2010, 1 (14), pp.2077 . We investigate ultrafast vibrational ligand dynamics in carboxyhemoglobin using chirped pulse upconversion and demonstrate the formation of vibrationally multiply excited carbon monoxide trapped in the primary docking site of hemoglobin after photolysis. The bleach signal due to ligand dissociation and the incipient docking-site absorption signal are about 200 cm-1 apart and differ by more than an order of magnitude in absorbance. In conventional approaches, these signals are monitored individually. Our method allows simultaneous observation of these signals with both high spectral resolution and high sensitivity. The large amount of vibrationally hot CO in the docking site as observed under Soret band excitation of the heme is discussed in the context of excess energy provided by the pump photon and is shown to be in quantitative agreement with predictions based on changes in the CO equilibrium distance upon instantaneous dissociation. Cop 2010 American Chemical Society. (10.1021/jz1006324)
DOI : 10.1021/jz1006324
Density of states and vibrational modes of PDMS studied by terahertz time-domain spectroscopy
- Podzorov Alexander
- Gallot Guilhem
Chemical Physics Letters, Elsevier , 2010, 495 (40969), pp.46 . Using time-domain spectroscopy (TDS), we performed high-precision spectroscopic measurements of polydimethylsiloxane (PDMS) in the terahertz domain. We investigated the influence of crosslinking on the terahertz absorption and refractive index, and modeled the data with vibrational density of states and coupling between photons and vibrational modes of the polymer molecules. We also investigated the influence of the temperature on PDMS, and observed the glass transition temperature, as well as the cold crystallization peak and the melting zone. Cop. 2010 Elsevier B.V. All rights reserved. (10.1016/j.cplett.2010.06.050)
DOI : 10.1016/j.cplett.2010.06.050
Nitric oxide binds to the proximal heme coordination site of the ferrocytochrome c/cardiolipin complex: Formation mechanism and dynamics
- Silkstone G.
- Kapetanaki Sofia M.
- Husu I.
- Vos Marten H.
- Wilson M.T.
Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology , 2010, 285 (26), pp.19785 . Mammalian mitochondrial cytochrome c interacts with cardiolipin to form a complex (cyt. c/CL) important in apoptosis. Here we show that this interaction leads to structural changes in ferrocytochrome c that leads to an open coordinate site on the central iron, resulting from the dissociation of the intrinsic methionine residue, where NO can rapidly bind (k = 1.2 x 10(7) m(-1) s(-1)). Accompanying NO binding, the proximal histidine dissociates leaving the heme pentacoordinate, in contrast to the hexacoordinate nitrosyl adducts of native ferrocytochrome c or of the protein in which the coordinating methionine is removed by chemical modification or mutation. We present the results of stopped-flow and photolysis experiments that show that following initial NO binding to the heme, there ensues an unusually complex set of kinetic steps. The spectral changes associated with these kinetic transitions, together with their dependence on NO concentration, have been determined and lead us to conclude that NO binding to cyt. c/CL takes place via an overall scheme comparable to that described for cytochrome c' and guanylate cyclase, the final product being one in which NO resides on the proximal side of the heme. In addition, novel features not observed before in other heme proteins forming pentacoordinate nitrosyl species, include a high yield of NO escape after dissociation, rapid (<1 ms) dissociation of proximal histidine upon NO binding and its very fast binding (60 ps) after NO dissociation, and the formation of a hexacoordinate intermediate. These features all point at a remarkable mobility of the proximal heme environment induced by cardiolipin. (10.1074/jbc.M109.067736)
DOI : 10.1074/jbc.M109.067736
High Up-Conversion Efficiency of YVO4:Yb,Er Nanoparticles in Water down to the Single-Particle Level
- Mialon Genevieve
- Tuerkcan Silvan
- Dantelle Geraldine
- Collins Daniel P.
- Hadjipanayi Maria
- Taylor Robert A.
- Gacoin Thierry
- Alexandrou Antigoni
- Boilot Jean-Pierre
Journal of Physical Chemistry C, American Chemical Society , 2010, 114 (51), pp.22449 . We report up-conversion emission from an aqueous solution of YVO4:Yb3+,Er3+ nanocrystals synthesized by an original method that produces nanoparticles with excellent crystallinity and no porosity. We show that these YVO4:Yb3+,Er3+ nanocrystals are not very sensitive to nonradiative relaxations, leading to a high green-to-red emission ratio of 6.3. Using a comparison with YVO4:Eu3+ particles, we determined the quantum yield of the up-conversion emission of the aqueous YVO4:Yb3+,Er3+ dispersion to be 0.09 +/- 0.04% for an excitation intensity of only 0.55 kW.cm(-2) at 970 nm. Furthermore, single YVO4:Yb,Er particles with an estimated size down to 10 nm can be detected using a wide-field microscope under a 970 nm, 8 kW.cm(-2) excitation. Because of their unexpectedly high up-conversion emission without intermittency, their water dispersibility, and their photostability, YVO4:Yb3+,Er3+ nanoparticles are highly appropriate both for single-biomolecule and for in vivo imaging. (10.1021/jp107900z)
DOI : 10.1021/jp107900z
Imaging by second harmonic generation: the example of pulmonary and renal fibrosis. [Imagerie par génération de seconde harmonique: l'exemple des fibroses pulmonaires et rénales.]
- Schanne-Klein Marie-Claire
Annales de Pathologie, Elsevier Masson , 2010, 30 (5 Suppl 1), pp.56 .
Polarization-resolved Second Harmonic microscopy in anisotropic thick tissues
- Gusachenko Ivan
- Latour Gaël
- Schanne-Klein Marie-Claire
Optics Express, Optical Society of America - OSA Publishing , 2010, 18 (18), pp.19339-19352 . We thoroughly analyze the linear propagation effects that affect polarization-resolved Second Harmonic Generation imaging of thick anisotropic tissues such as collagenous tissues. We develop a theoretical model that fully accounts for birefringence and diattenuation along the excitation propagation, and polarization scrambling upon scattering of the harmonic signal. We obtain an excellent agreement with polarization-resolved SHG images at increasing depth within a rat-tail tendon for both polarizations of the forward SHG signal. Most notably, we observe interference fringes due to birefringence in the SHG depth profile when excited at π/4 angle from the tendon axis. We also measure artifactual decrease of ρ = χxxx/χxyy with depth due to diattenuation of the excitation. We therefore derive a method that proves reliable to determine both ρ and the tendon birefringence and diattenuation. © 2010 Optical Society of America (10.1364/OE.18.019339)
DOI : 10.1364/OE.18.019339
Heme-heme and heme-ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy
- Rappaport Fabrice
- Zhang Jie
- Vos Marten H.
- Gennis Robert B.
- Borisov Vitaliy B.
Biochimica biophysica acta (BBA) - Bioenergetics, Elsevier , 2010, 1797 (9), pp.1657-1664 . Cytochrome bd is a terminal quinol:O(2) oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b(558), b(595), and d. The role of heme b(595) remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b(595) causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b(595) and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with tau similar to 12 mu s, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with tau similar to 14 ns, 14 mu s, and 280 mu s. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-mu s component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in similar to 4% of the enzyme population. The final, 280-mu s component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b(595), and not that of heme b(558), controls the pathway(s) by which CO migrates between heme d and the medium. Cop 2010 Elsevier B.V. All rights reserved. (10.1016/j.bbabio.2010.05.010)
DOI : 10.1016/j.bbabio.2010.05.010
D1 protein variants in Photosystem II from Thermosynechococcus elongatus studied by low temperature optical spectroscopy
- Hughes Joseph L.
- Cox Nicholas
- Rutherford A.William
- Krausz Elmars
- Lai Thanh-Lan
- Boussac Alain
- Sugiura Miwa
Biochimica et Biophysica Acta (BBA) - Reviews on Bioenergetics, Elsevier , 2010, 1797 (1), pp.11-19 . In Photosystem II (PSII) from Thermosynechococcus elongatus, high-light intensity growth conditions induce the preferential expression of the psbA3 gene over the psbA1 gene. These genes encode for the D1 protein variants labeled D1:3 and D1:1, respectively. We have compared steady state absorption and photo-induced difference spectra at < 10 K of PSII containing either D1:1 or D1:3. The following differences were observed. (i) The pheophytin Qx band was red-shifted in D1:3 (547.3 nm) compared to D1:1 (544.3 nm). (ii) The electrochromism on the PheoD1 Qx band induced by QA− (the C550 shift) was more asymmetric in D1:3. (iii) The two variants differed in their responses to excitation with far red (704 nm) light. When green light was used there was little difference between the two variants. With far red light the stable (t1/2 > 50 ms) QA− yield was ∼ 95% in D1:3, and ∼ 60% in D1:1, relative to green light excitation. (iv) For the D1:1 variant, the quantum efficiency of photo-induced oxidation of side-pathway donors was lower. These effects can be correlated with amino acid changes between the two D1 variants. The effects on the pheophytin Qx band can be attributed to the hydrogen bond from Glu130 in D1:3 to the 131-keto of PheoD1, which is absent for Gln130 in D1:1. The reduced yield with red light in the D1:1 variant could be associated with either the Glu130Gln change, and/or the four changes near the binding site of PD1, in particular Ser153Ala. Photo-induced QA− formation with far red light is assigned to the direct optical excitation of a weakly absorbing charge transfer state of the reaction centre. We suggest that this state is blue-shifted in the D1:1 variant. A reduced efficiency for the oxidation of side-pathway donors in the D1:1 variant could be explained by a variation in the location and/or redox potential of P+. (10.1016/j.bbabio.2009.07.007)
DOI : 10.1016/j.bbabio.2009.07.007
Picosecond primary structural transition of the heme is retarded after nitric oxide binding to heme proteins
- Kruglik Sergei G.
- Yoo Byung-Kuk
- Franzen S.
- Vos Marten H.
- Martin Jean-Louis
- Négrerie Michel
Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2010, 107 (31), pp.13678 . We investigated the ultrafast structural transitions of the heme induced by nitric oxide (NO) binding for several heme proteins by subpicosecond time-resolved resonance Raman and femtosecond transient absorption spectroscopy. We probed the heme iron motion by the evolution of the iron-histidine Raman band intensity after NO photolysis. Unexpectedly, we found that the heme response and iron motion do not follow the kinetics of NO rebinding. Whereas NO dissociation induces quasi-instantaneous iron motion and heme doming (< 0.6 ps), the reverse process results in a much slower picosecond movement of the iron toward the planar heme configuration after NO binding. The time constant for this primary domed-to-planar heme transition varies among proteins (∼30 ps for myoglobin and its H64V mutant, ∼15 ps for hemoglobin, ∼7 ps for dehaloperoxidase, and ∼6 ps for cytochrome c) and depends upon constraints exerted by the protein structure on the heme cofactor. This observed phenomenon constitutes the primary structural transition in heme proteins induced by NO binding. (10.1073/pnas.0912938107)
DOI : 10.1073/pnas.0912938107
Expression and purification of untagged and histidine-tagged folate-dependent tRNA:m5U54 methyltransferase from Bacillus subtilis
- Hamdane Djemel
- Skouloubris Stéphane
- Myllykallio Hannu
- Golinelli-Pimpaneau Béatrice
Protein Expression and Purification, Elsevier , 2010, 73 (1), pp.83-89 . Folate-dependent tRNA m5U methyltransferase TrmFO is a flavoprotein that catalyzes the C5-methylation of uridine at position 54 in the T-Psi-C loop of tRNA in several bacteria. Here we report the cloning and optimization of expression in Escherichia coli BL21 (DE3) of untagged, N-terminus, C-terminus (His)6-tagged TrmFO from Bacillus subtilis. Tagged and untagged TrmFO were purified to homogeneity by metal affinity or ion exchange and heparin affinity, respectively, followed by size-exclusion chromatography. The tag did not significantly alter the expression level, flavin content, activity and secondary structure of the protein. Cop. 2010 Elsevier Inc. All rights reserved. (10.1016/j.pep.2010.04.013)
DOI : 10.1016/j.pep.2010.04.013
Dynamic aberration correction for multiharmonic microscopy.
- Olivier Nicolas
- Débarre Delphine
- Beaurepaire Emmanuel
Optics Letters, Optical Society of America - OSA Publishing , 2009, 34 (20), pp.3145-7 . We demonstrate image-based aberration correction in a third-harmonic generation (THG) microscope. We describe a robust, mostly sample-independent correction scheme relying on prior measurement of the influence of aberration modes produced by a deformable mirror on the quality of THG images. We find that using image sharpness as an image quality metric, correction of N aberration modes is achieved using 2(2N+1) measurements in a variety of samples. We also report aberration correction in combined multiharmonic and two-photon excited fluorescence experiments. Finally, we demonstrate time-dependent adaptive THG imaging in developing embryonic tissue.
The crystal structure of ORF14 from Sulfolobus islandicus filamentous virus
- Goulet Adeline
- Spinelli Silvia
- Blangy Stéphanie
- van Tilbeurgh Herman
- Leulliot Nicolas
- Basta Tamara
- Prangishvili David
- Cambillau Christian
- Campanacci Valérie
Proteins - Structure, Function and Bioinformatics, Wiley , 2009, 76 (4), pp.1020-1022 . (10.1002/prot.22448)
DOI : 10.1002/prot.22448
Multiplexed two-photon microscopy of dynamic biological samples with shaped broadband pulses.
- Pillai Rajesh S.
- Boudoux Caroline
- Labroille Guillaume
- Olivier Nicolas
- Veilleux Israel
- Farge Emmanuel
- Joffre Manuel
- Beaurepaire Emmanuel
Optics Express, Optical Society of America - OSA Publishing , 2009, 17 (15), pp.12741-52 . Coherent control can be used to selectively enhance or cancel concurrent multiphoton processes, and has been suggested as a means to achieve nonlinear microscopy of multiple signals. Here we report multiplexed two-photon imaging in vivo with fast pixel rates and micrometer resolution. We control broadband laser pulses with a shaping scheme combining diffraction on an optically-addressed spatial light modulator and a scanning mirror allowing to switch between programmable shapes at kiloHertz rates. Using coherent control of the two-photon excited fluorescence, it was possible to perform selective microscopy of GFP and endogenous fluorescence in developing Drosophila embryos. This study establishes that broadband pulse shaping is a viable means for achieving multiplexed nonlinear imaging of biological tissues. (10.1364/OE.17.012741)
DOI : 10.1364/OE.17.012741
Structure and function of a novel endonuclease acting on branched DNA substrates.
- Ren Bin
- Kühn Joelle
- Meslet-Cladiere Laurence
- Briffotaux Julien
- Norais Cedric
- Lavigne Regis
- Flament Didier
- Ladenstein Rudolf
- Myllykallio Hannu
EMBO Journal, EMBO Press , 2009, 28 (16), pp.2479-89 . We show that Pyrococcus abyssi PAB2263 (dubbed NucS (nuclease for ss DNA) is a novel archaeal endonuclease that interacts with the replication clamp PCNA. Structural determination of P. abyssi NucS revealed a two-domain dumbbell-like structure that in overall does not resemble any known protein structure. Biochemical and structural studies indicate that NucS orthologues use a non-catalytic ssDNA-binding domain to regulate the cleavage activity at another site, thus resulting into the specific cleavage at double-stranded DNA (dsDNA)/ssDNA junctions on branched DNA substrates. Both 3' and 5' extremities of the ssDNA can be cleaved at the nuclease channel that is too narrow to accommodate duplex DNA. Altogether, our data suggest that NucS proteins constitute a new family of structure-specific DNA endonucleases that are widely distributed in archaea and in bacteria, including Mycobacterium tuberculosis. (10.1038/emboj.2009.192)
DOI : 10.1038/emboj.2009.192
The thermo- and acido-stable ORF-99 from the archaeal virus AFV1
- Goulet Adeline
- Spinelli Silvia
- Blangy Stéphanie
- van Tilbeurgh Herman
- Leulliot Nicolas
- Basta Tamara
- Prangishvili David
- Cambillau Christian
- Campanacci Valérie
Protein Science, Wiley , 2009, 18 (6), pp.1316-1320 . (10.1002/pro.122)
DOI : 10.1002/pro.122
Two-photon microscopy with simultaneous standard and extended depth of field using a tunable acoustic gradient-index lens.
- Olivier Nicolas
- Mermillod-Blondin Alexandre
- Arnold Craig B.
- Beaurepaire Emmanuel
Optics Letters, Optical Society of America - OSA Publishing , 2009, 34 (11), pp.1684-1686 . We describe a simple setup that allows depth of field switching at kilohertz rates in a nonlinear microscope. Beam profile and/or divergence are modulated using a tunable, acoustically driven gradient-index fluid lens. We demonstrate two modulation strategies, one based on fast varifocus scanning during each pixel and the other based on pseudo-Bessel beam excitation. Average beam shape is switched every line during scanning, resulting in the interlaced acquisition of two different images. We apply this approach to the simultaneous standard and 4.5x-extended depth-of-field imaging of developing embryos. © 2009 Optical Society of America (10.1364/OL.34.001684)
DOI : 10.1364/OL.34.001684
New insights into size effects in luminescent oxide nanocrystals
- Mialon G.
- Türkcan Silvan
- Alexandrou Antigoni
- Gacoin Thierry
- Boilot Jean-Pierre
Journal of Physical Chemistry C, American Chemical Society , 2009, 113 (43), pp.18699 . We here investigate the emission properties of rare-earth-doped oxide nanoparticles with the aim to understand the commonly observed altered properties of nanoparticles as compared to the bulk counterparts. This is usually attributed to the detrimental effect of surface states that quench the excited states involved in the emission process. We study the influence of crystalline defects that are present due to the low temperature of the synthesis of 30 nm sized YVO4/Eu nanoparticles. Annealing treatments up to 1000 °C in a porous silica matrix allow the recovery of perfectly crystalline particles as colloidal suspensions and compare their properties to those of the pristine particles obtained by conventional colloid chemistry. Emission properties of pristine and annealed particles are compared with those of the bulk material. A simple model of the emission process allows an accurate fit of the luminescence decay and of the dependence of the quantum yield on europium content. Our results show that pristine particles exhibit altered emission properties mainly due to quenching from defects, among which are surface OH groups, and altered energy transfers within the particle. Annealed particles exhibit properties that are almost the same as those of the bulk material, except that the emission yield for the optimum Eu content is limited to 40 instead of 70% for the bulk material. We show that the difference may be simply explained by the difference of the radiative lifetime that results from the lower effective refractive index in the case of the particles. This effect then seems to be the ultimate limitation for the emission properties of perfectly well-crystallized nanoparticles as compared to those of the bulk material. This work provides an example of a general strategy toward the investigation of the physical properties of nanocrystals which may be altered by crystalline defects. Cop. 2009 American Chemical Society. (10.1021/jp907176x)
DOI : 10.1021/jp907176x
Erratum to : Application of time-resolved circular dichroism to the study of conformational changes in photochemical and photobiological processes
- Hache François
Journal of Photochemistry and Photobiology A: Chemistry, Elsevier , 2009, 205 (1), pp.77 . (10.1016/j.jphotochem.2009.05.017)
DOI : 10.1016/j.jphotochem.2009.05.017
Interaction of carbon monoxide with the apoptosis-inducing cytochrome c-cardiolipin complex
- Kapetanaki Sofia M.
- Silkstone G.
- Husu I.
- Liebl Ursula
- Wilson M.T.
- Vos Marten H.
Biochemistry, American Chemical Society , 2009, 48 (7), pp.1613 . The interaction of mitochondrial cytochrome (cyt) c with cardiolipin (CL) is involved in the initial stages of apoptosis. This interaction can lead to destabilization of the heme−Met80 bond and peroxidase activity [Basova, L. V., et al. (2007) Biochemistry 46, 3423−3434]. We show that under these conditions carbon monoxide (CO) binds to cyt c, with very high affinity (5 × 107 M−1), in contrast to the native cyt c protein involved in respiratory electron shuttling that does not bind CO. Binding of CO to the cyt c−CL complex inhibits its peroxidase activity. Photodissociated CO from the cyt c−CL complex shows <20% picosecond geminate rebinding and predominantly bimolecular rebinding, with a second-order rate constant of 107 M−1 s−1, an order of magnitude higher than in myoglobin. These findings contrast with those of Met80X mutant cyt c, where picosecond geminate recombination dominates due to the rigidity of the protein. Our data imply that CL leads to substantial changes in protein conformation and flexibility, allowing access of ligands to the heme. Together with the findings that (a) 30 CL per cyt c are required for full CO binding and (b) salt-induced dissociation indicates that the two negative headgroup charges interact with 5 positive surface charges of the protein, these results are consistent with a CL anchorage model with an acyl chain impaled in the protein [Kalanxhi, E., and Wallace, C. J. A. (2007) Biochem. J. 407, 179−187]. The affinity of CO for the complex is high enough to envisage an antiapoptotic effect of nanomolar CO concentrations via inhibition of the cyt c peroxidase activity. (10.1021/bi801817v)
DOI : 10.1021/bi801817v
NO formation by neuronal NO-synthase can be controlled by ultrafast electron injection from a nanotrigger
- Beaumont Edward
- Lambry Jean-Christophe
- Blanchard-Desce M.
- Martasek P.
- Panda S.P.
- van Faassen E.E.H.
- Brochon J.-C.
- Deprez E.
- Slama-Schwok Anny
ChemBioChem, Wiley-VCH Verlag , 2009, 10 (4), pp.690 . Nitric oxide synthases (NOSs) are unique flavohemoproteins with various roles in mammalian physiology. Constitutive NOS catalysis is initiated by fast hydride transfer from NADPH, followed by slower structural rearrangements. We used a photoactive nanotrigger (NT) to study the initial electron transfer to FAD in native neuronal NOS (nNOS) catalysis. Molecular modeling and fluorescence spectroscopy showed that selective NT binding to NADPH sites close to FAD is able to override Phe1395 regulation. Ultrafast injection of electrons into the protein electron pathway by NT photoactivation through the use of a femtosecond laser pulse is thus possible. We show that calmodulin, required for NO synthesis by constitutive NOS, strongly promotes intramolecular electron flow (6.2-fold stimulation) by a mechanism involving proton transfer to the reduced FADb site. Site-directed mutagenesis using the S1176A and S1176T mutants of nNOS supports this hypothesis. The NT synchronized the initiation of flavoenzyme catalysis, leading to the formation of NO, as detected by EPR. This NT is thus promising for time-resolved X-ray and other cellular applications. Cop. 2009 Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim. (10.1002/cbic.200800721)
DOI : 10.1002/cbic.200800721
Picosecond Transient Circular Dichroism of the Photoreceptor Protein of the Light-Adapted Form of Blepharisma Japonicum
- Hache François
- Khuc Mai-Thu
- Brazard Johanna
- Plaza Pascal
- Martin Monique M.
- Checcucci Giovanni
- Lenci Francesco
Chemical Physics Letters, Elsevier , 2009, 483, pp.133 . We present a picosecond transient circular dichroism study of OBIP, the putative photoreceptor protein involved in the photophobic response of Blepharisma Japonicum. The probe wavelength was chosen at 230 nm. The results are compared to those of the isolated chromophore, OxyBP, in solution. The CD changes in OBIP and OxyBP do not show the same dynamics: OBIP's signal relaxes in a few ps whereas no such decay is obtained for OxyBP. This observation brings support to the formerly evoked existence of a fast photoinduced reaction in the chromoprotein, and demonstrates the implication of local geometrical changes that accompany this process. (10.1016/j.cplett.2009.10.059)
DOI : 10.1016/j.cplett.2009.10.059