Articles

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
Unobtrusive interferometer tracking by path length oscillation for multidimensional spectroscopy
- Lee Kevin F.
- Bonvalet Adeline
- Nuernberger Patrick
- Joffre Manuel
Optics Express, Optical Society of America - OSA Publishing , 2009, 17 (15), pp.12379-12384 . We track the path difference between interferometer arms with few-nanometer accuracy without adding optics to the beam path. We measure the interference of a helium-neon beam that copropagates through the interferometer with midinfrared pulses used for multidimensional spectroscopy. This can indicate motion, but not direction. By oscillating the path length of one arm with a mirror on a piezoelectric stack and monitoring the oscillations of the recombined helium-neon beam, the direction can be calculated, and the path delay can be continuously tracked. © 2009 Optical Society of America. (10.1364/OE.17.012379)
DOI : 10.1364/OE.17.012379
Apports récents des techniques de quantification de la fibrose pour l'examen anatomopathologique en transplantation rénale
- Servais A.
- Meas-Yedid V.
- Morelon E.
- Strupler Mathias
- Schanne-Klein Marie-Claire
- Legendre C.
- Olivo-Marin J.-C.
- Thervet É.
Médecine/Sciences, EDP Sciences , 2009, 25 (11), pp.945-950 . La néphropathie chronique d'allogreffe constitue la cause principale de perte des greffons rénaux à long terme. Elle peut être détectée précocement par des biopsies de dépistage effectuées de manière systématique. La classification usuelle, semi-quantitative, souffre d'une mauvaise reproductibilité. Diverses méthodes morphométriques ont donc été développées pour quantifier la fibrose interstitielle qui caractérise cette néphropathie. Certaines utilisent la coloration spécifique par le rouge Sirius. L'analyse d'image couleur par segmentation permet une quantification automatique, rapide et robuste de la fibrose interstitielle. Elle utilise une segmentation couleur associée à une analyse de couleur, de localisation spatiale et de forme sur des biopsies colorées au trichrome de Masson. À l'avenir, l'étude des collagènes fibrillaires par la génération de second harmonique pourrait permettre une approche spécifique des composants de la fibrose. (10.1051/medsci/20092511945)
DOI : 10.1051/medsci/20092511945
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
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
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
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
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
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, 204 (2-3), pp.137-143 . Circular dichroism is known to be a very sensitive probe of the molecular conformation and implementation of this technique in a pump-probe experiment is very appealing to access information on the dynamics of conformational changes occurring in photochemical or photobiological processes. In the past years, we have developed such techniques in various ways and applied them to several chemical or biological studies which are presented in this article. Applications concern spectroscopic studies of the excited state in ruthenium tris(bipyridyl) or tris(phenanthroline), dynamics of conformational changes in photoexcited binaphthol and study of the conformational changes occurring in photolyzed carboxy-myoglobin. Extension of these techniques towards biological issues such as protein folding is discussed. Cop. 2009 Elsevier B.V. All rights reserved. (10.1016/j.jphotochem.2009.03.012)
DOI : 10.1016/j.jphotochem.2009.03.012
Femtosecond spectroscopy from the perspective of a global multidimensional response function
- Nuernberger Patrick
- Lee Kevin F.
- Joffre Manuel
Accounts of Chemical Research, American Chemical Society , 2009, 42 (9), pp.1433-1441 . At the microscopic level, multidimensional response functions, such as the nonlinear optical susceptibility or the time-ordered response function, are commonly used tools in nonlinear optical spectroscopy for determining the nonlinear polarization resulting from an arbitrary excitation. In this Account, we point out that the approach successfully developed for the nonlinear polarization can also be used in the case of a directly observable macroscopic quantity. This observable can be, for example, the electric field radiated in a nonlinear mixing experiment, the rate of fluorescence resulting from one- or two-photon absorption, or the rate of a photochemical reaction. For each of these physical processes, perturbation theory can be used to expand the measured quantity in a power series of the exciting field, and an appropriate global response function can be introduced for each order of perturbation. At order n, the multidimensional response function will depend on n variables (either time or frequency) and have the same general properties as the nonlinear susceptibility resulting, for example, from time invariance or causality. The global response function is introduced in this Account in close analogy with the nonlinear susceptibility or the time-ordered microscopic response. We discuss various applications of the global response function formalism. For example, it can be shown that in the weak field limit, a stationary signal induced in a time-invariant system is independent of the spectral phase of the exciting field. Although this result had been demonstrated previously, the global response function enables its derivation in a more general way because no specific microscopic model is needed. Multidimensional spectroscopy is obviously ideally suited to measure the global multidimensional response function. It is shown that the second (or third)-order response can be exactly measured with 2D (or 3D) spectroscopy by taking into account the exact shape of the exciting pulses. In the case of a 2D measurement of the third-order response, a particular projection of the complete 3D response function is actually measured. This projection can be related to a mixed time and frequency representation of the response function when the pulses are assumed to be infinitely short. We thus show that the global response function is a useful tool for deriving general results and that it should help in designing future experimental schemes for femtosecond spectroscopy. Cop. 2009 American Chemical Society. (10.1021/ar900001w)
DOI : 10.1021/ar900001w
Inferring maps of forces inside cell membrane microdomains
- Masson J.-B.
- Casanova Didier
- Türkcan Silvan
- Voisinne G.
- Popoff Michel
- Vergassola M.
- Alexandrou Antigoni
Physical Review Letters, American Physical Society , 2009, 102 (4), pp.48103 . Mapping of the forces on biomolecules in cell membranes has spurred the development of effective labels, e.g., organic fluorophores and nanoparticles, to track trajectories of single biomolecules. Standard methods use particular statistics, namely the mean square displacement, to analyze the underlying dynamics. Here, we introduce general inference methods to fully exploit information in the experimental trajectories, providing sharp estimates of the forces and the diffusion coefficients in membrane microdomains. Rapid and reliable convergence of the inference scheme is demonstrated on trajectories generated numerically. The method is then applied to infer forces and potentials acting on the receptor of the toxin labeled by lanthanide-ion nanoparticles. Our scheme is applicable to any labeled biomolecule and results show its general relevance for membrane compartmentation. Cop. 2009 The American Physical Society. (10.1103/PhysRevLett.102.048103)
DOI : 10.1103/PhysRevLett.102.048103
Multimodal Multiphoton Imaging of Human Eye Tissues
- Aptel Florent
- Olivier Nicolas
- Deniset Ariane
- Plamann Karsten
- Denis P.
- Legeais Jean-Marc
- Schanne-Klein Marie-Claire
- Beaurepaire Emmanuel
Investigative Ophthalmology & Visual Science, Association for Research in Vision and Ophthalmology , 2009, 50, pp.E-Abstract 3693 . Purpose:To evaluate three combined modalities of multiphoton microscopy, second-harmonic generation (SHG), third-harmonic generation (THG), and two-photon-excited fluorescence (2PEF) for imaging cornea and trabecular meshwork in human intact eye tissue. Methods:A tunable femtosecond laser chain ( = 700-1250 nm) comprising a titanium-sapphire laser oscillator and an optical parametric oscillator was used to produce 2PEF (380-620nm), SHG (/2=430 or 600nm) and THG (/3= 400nm). Eight corneoscleral discs from eye bank and seven fresh corneal buttons obtained after penetrating keratoplasty were examined with water-immersion objectives. Forward and backward signals were detected and compared. Results:The three imaging modalities provide complementary information on intact tissue over the entire thickness of the cornea. THG imaging reveals the tissue morphology, including the epithelium structure with sub-cellular resolution. Polarization-resolved THG microscopy reveals stromal birefringent domains. In phenol-stained corneas, THG also reveals the keratocytes network. SHG imaging probes the distribution of stromal collagen lamellas organization. 2PEF imaging reveals the elastic component of the extra-cellular matrix and the distribution of fluorescent organelles (i.e. mitochondria) in stromal and epithelial cells. The trabeculum images show the three-dimensional organisation of the trabecular lamellas. Emission is predominantly forward directed for THG and SHG but in some cases, images can be recorded in the epi-direction. Conclusions:The combined imaging modalities of SHG, THG, and 2PEF microscopy are effective methods to evaluate cornea and trabecular meshwork microstructures in situ. This imaging approach should prove particularly appropriate for assessing corneal and glaucoma physiopathology, and might be amenable to in vivo diagnostics.
Measurement of the Second-Order Hyperpolarizability of the Collagen Triple Helix and Determination of Its Physical Origin
- Deniset-Besseau Ariane
- Duboisset Julien
- Benichou Emmanuel
- Hache François
- Brevet Pierre-Francois
- Schanne-Klein Marie-Claire
Journal of Physical Chemistry B, American Chemical Society , 2009, 113 (40), pp.13437-13445 . We performed Hyper-Rayleigh Scattering (HRS) experiments to measure the second-order nonlinear optical response of the collagen triple helix and determine the physical origin of second harmonic signals observed in collagenous tissues. HRS experiments yielded a second-order hyperpolarizability of 1.25 x 10(-27) esu for rat-tail type I collagen, a Surprisingly large value considering that collagen presents no strong harmonophore in its amino acid sequence. Polarization-resolved experiments showed intramolecular coherent contributions to the HRS signal along with incoherent contributions that are the only contributions for molecules with dimensions much smaller than the excitation wavelength. We therefore modeled the effective second-order hyperpolarizability of the 290 nm long collagen triple helix by summing coherently the nonlinear response of well-aligned moieties along the triple helix axis. This model was confirmed by HRS measurements after denaturation of the collagen triple helix and for a collagen-like short model peptide [(Pro-Pro-Gly)(10)](3). We concluded that the large collagen nonlinear response originates in the tight alignment of a large number of small and weakly efficient harmonophores, presumably the peptide bonds, resulting in a coherent amplification of the nonlinear signal. (10.1021/jp9046837)
DOI : 10.1021/jp9046837
Single europium-doped nanoparticles measure temporal pattern of reactive oxygen species production inside cells
- Casanova Didier
- Bouzigues Cédric
- Nguyên Thanh-Liêm
- Ramodiharilafy Rivo O.
- Bouzhir-Sima Latifa
- Gacoin Thierry
- Boilot Jean-Pierre
- Tharaux Pierre-Louis
- Alexandrou Antigoni
Nature Nanotechnology, Nature Publishing Group , 2009, 4 (9), pp.581 . Low concentrations of reactive oxygen species, notably hydrogen peroxide (H 2 O 2), mediate various signalling processes in the cell. Production of these signals is highly regulated and a suitable probe is needed to measure these events. Here, we show that a probe based on a single nanoparticle can quantitatively measure transient H 2 O 2 generation in living cells. The Y 0.6 Eu 0.4 VO 4 nanoparticles undergo photoreduction under laser irradiation but re-oxidize in the presence of oxidants, leading to a recovery in luminescence. Our probe can be regenerated and reliably detects intracellular H 2 O 2 with a 30-s temporal resolution and a dynamic range of 1-45?M. The differences in the timing of intracellular H 2 O 2 production triggered by different signals were also measured using these nanoparticles. Although the probe is not selective towards H 2 O 2, in many signalling processes H 2 O 2 is, however, the dominant oxidant. In conjunction with appropriate controls, this probe is a powerful tool for unravelling pathways that involve reactive oxygen species. Cop. 2009 Macmillan Publishers Limited. All rights reserved. (10.1038/nnano.2009.200)
DOI : 10.1038/nnano.2009.200
Suppression of perturbed free-induction decay and noise in experimental ultrafast pump-probe data
- Nuernberger Patrick
- Lee Kevin F.
- Bonvalet Adeline
- Polack Thomas
- Vos Marten H.
- Alexandrou Antigoni
- Joffre Manuel
Optics Letters, Optical Society of America - OSA Publishing , 2009, 34 (20), pp.3226-3228 . We apply a Fourier filtering technique for the global removal of coherent contributions, like perturbed freeinduction decay, and noise, to experimental pump-probe spectra. A further filtering scheme gains access to spectra otherwise only recordable by scanning the probe's center frequency with adjustable spectral resolution. These methods cleanse pump-probe data and allow improved visualization and simpler analysis of the contained dynamics. We demonstrate these filters using visible pump/mid-infrared probe spectroscopy of ligand dissociation in carboxyhemoglobin. Cop. 2009 Optical Society of America. (10.1364/OL.34.003226)
DOI : 10.1364/OL.34.003226
Heme ligand binding properties and intradimer interactions in the full-length sensor protein Dos from Escherichia coli and its isolated heme domain
- Lechauve C.
- Bouzhir-Sima Latifa
- Yamashita Taku
- Marden M.C.
- Vos Marten H.
- Liebl Ursula
- Kiger L.
Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology , 2009, 284 (52), pp.36146 . Dos from Escherichia coli is a bacterial gas sensor protein comprising a heme-containing gas sensor domain and a phosphodiesterase catalytic domain. Using a combination of static light scattering and gel filtration experiments, we established that, as are many other sensor proteins, the full-length protein is dimeric. The full-length dimer (association constant <10 nm) is more stable than the dimeric heme domain (association constant ∼1 μm), and the dimer interface presumably includes both sensor and catalytic domains. Ultrafast spectroscopic studies showed little influence of the catalytic domain on kinetic processes in the direct vicinity of the heme. By contrast, the properties of ligand (CO and O2) binding to the heme in the sensor domain, occurring on a microsecond to second time scale, were found to be influenced by (i) the presence of the catalytic domain, (ii) the dimerization state, and in dimers, (iii) the ligation state of the other subunit. These results imply allosteric interactions within dimers. Steady-state titrations demonstrated marked cooperativity in oxygen binding to both the full-length protein and the isolated heme domain, a feature not reported to date for any dimeric sensor protein. Analysis of a variety of time-resolved experiments showed that Met-95 plays a major role in the intradimer interactions. The intrinsic binding and dissociation rates of Met-95 to the heme were modulated ∼10-fold by intradimer and sensor-catalytic domain interactions. Dimerization effects were also observed for cyanide binding to the ferric heme domains, suggesting a similar role for Met-95 in ferric proteins. (10.1074/jbc.M109.066811)
DOI : 10.1074/jbc.M109.066811
Extended fano model of extraordinary electromagnetic transmission through subwavelength hole arrays in the terahertz domain
- Masson Jean-Baptiste
- Podzorov Alexander
- Gallot Guilhem
Optics Express, Optical Society of America - OSA Publishing , 2009, 17 (17), pp.15280-15291 . We developed an extended Fano model describing the Extraordinary Electromagnetic Transmission (EET) through arrays of subwavelength apertures, based on terahertz transmission measurements of arrays of various hole size and shapes. Considering a frequency-dependent coupling between resonant and non-resonant pathways, this model gives access to a simple analytical description of EET, provides good agreement with experimental data, and offers new parameters describing the influence of the hole size and shape on the transmitted signal. Cop. 2009 Optical Society of America. (10.1364/OE.17.015280)
DOI : 10.1364/OE.17.015280
Removing cross-phase modulation from midinfrared chirped-pulse upconversion spectra
- Lee Kevin F.
- Nuernberger Patrick
- Bonvalet Adeline
- Joffre Manuel
Optics Express, Optical Society of America - OSA Publishing , 2009, 17 (21), pp.18738-18744 . We observe that narrow spectral features in mid-infrared spectra obtained by chirped-pulse up-conversion are strongly distorted by crossphase modulation between the mid-infrared field and the chirped pulse. We discuss the consequences of this effect on spectral resolution, and introduce a correction method that recovers masked lines. This simple correction can be applied either when the upconverted field is fully characterized, such as in multidimensional spectroscopy, or when causality can be used, such as in absorption spectroscopy, which we demonstrate experimentally. Cop.2009 Optical Society of America. (10.1364/OE.17.018738)
DOI : 10.1364/OE.17.018738
HIV-1 IN alternative molecular recognition of DNA induced by raltegravir resistance mutations
- Mouscadet J.-F.
- Arora Rakesh
- André J.
- Lambry Jean-Christophe
- Delelis O.
- Malet I.
- Marcelin A.-G.
- Calvez V.
- Tchertanov L.
Journal of Molecular Recognition, Wiley , 2009, 22 (6), pp.480-494 . Virologic failure during treatment with raltegravir, the first effective drug targeting HIV integrase, is associated with two exclusive pathways involving either Q148H/R/K, G140S/A or N155H mutations. We carried out a detailed analysis of the molecular and structural effects of these mutations. We observed no topological change in the integrase core domain, with conservation of a newly identified V-shaped hairpin containing the Q148 residue, in particular. In contrast, the mutations greatly altered the specificity of DNA recognition by integrase. The native residues displayed a clear preference for adenine, whereas the mutant residues strongly favored pyrimidines. Raltegravir may bind to N155 and/or Q148 residues as an adenine bioisoster. This may account for the selected mutations impairing raltegravir binding while allowing alternative DNA recognition by integrase. This study opens up new opportunities for the design of integrase inhibitors active against raltegravir-resistant viruses. Copyright Cop. 2009 John Wiley and Sons, Ltd. Supporting information may be found in the online version of this article. (10.1002/jmr.970)
DOI : 10.1002/jmr.970
A model for thermal exchange in axons during action potential propagation.
- Masson Jean-Baptiste
- Gallot Guilhem
European Biophysics Journal, Springer Verlag (Germany) , 2008, 37 (6), pp.1001-1006 . Several experiments have shown that during propagation of the action potential in axons, thermal energy is locally exchanged. In this paper, we use a simple model based on statistical physics to show that an important part of this exchange comes from the physics of the effusion. We evaluate, during the action potential propagation, the variation of internal energy and of the energy associated with the chemical potential of the effusion of water and ions to extract the thermal energy exchanged. The temperature exchanged is then evaluated on the area where the action potential is active. Results give a good correspondence between experimental work and this model, showing that an important part of the thermal energy exchange comes from the statistical cooling power of the effusion. (10.1007/s00249-008-0329-5)
DOI : 10.1007/s00249-008-0329-5
Ultrafast heme-residue bond formation in six-coordinate heme proteins: implications for functional ligand exchange.
- Vos Marten H.
- Battistoni Andrea
- Lechauve Christophe
- Marden Michael C.
- Kiger Laurent
- Desbois Alain
- Pilet Eric
- de Rosny Eve
- Liebl Ursula
Biochemistry, American Chemical Society , 2008, 47 (21), pp.5718-23 . A survey is presented of picosecond kinetics of heme-residue bond formation after photolysis of histidine, methionine, or cysteine, in a broad range of ferrous six-coordinate heme proteins. These include human neuroglobin, a bacterial heme-binding superoxide dismutase (SOD), plant cytochrome b 559, the insect nuclear receptor E75, horse heart cytochrome c and the heme domain of the bacterial sensor protein Dos. We demonstrate that the fastest and dominant phase of binding of amino acid residues to domed heme invariably takes place with a time constant in the narrow range of 5-7 ps. Remarkably, this is also the case in the heme-binding SOD, where the heme is solvent-exposed. We reason that this fast phase corresponds to barrierless formation of the heme-residue bond from a configuration close to the bound state. Only in proteins where functional ligand exchange occurs, additional slower rebinding takes place on the time scale of tens of picoseconds after residue dissociation. We propose that the presence of these slower phases reflects flexibility in the heme environment that allows external ligands (O2, CO, NO, . . .) to functionally replace the internal residue after thermal dissociation of the heme-residue bond. (10.1021/bi800288z)
DOI : 10.1021/bi800288z
Electron hopping through the 15 Å triple tryptophan molecular wire in DNA photolyase occurs within 30 ps
- Lukacs Andras
- Eker André P.M.
- Byrdin Martin
- Brettel Klaus
- Vos Marten H.
Journal of the American Chemical Society, American Chemical Society , 2008, 130 (44), pp.14394 . DNA photolyase is a photoactive flavoprotein that contains three tryptophan residues between the FAD cofactor and the protein surface, the solvent-exposed Trp being located 14.8 Å from the flavin. Photoreduction of the neutral radical FADH* form to the catalytically active FADH- form occurs via electron transfer through this chain. The first step in this chain takes 30 ps, the second less than 4 ps. Using a combination of site-directed mutagenesis and femtosecond polarization spectroscopy to discriminate the spectroscopically indistinguishable Trp residues, we show that the third step occurs in less than 30 ps. This implies that the first photoreduction step is rate limiting and that the Trp chain effectively acts as molecular "wire" ensuring rapid and directed long-range charge translocation across the protein. This finding is important for the functioning of the large class of cryptochrome blue-light receptors, where the Trp chain is conserved. In DNA photolyase we make use of the natural photoactivation of the process, but more generally chains of aromatic amino acids may allow very fast long-range electron transfer also in nonphotoactive proteins. Copyright Cop. 2008 American Chemical Society. (10.1021/ja805261m)
DOI : 10.1021/ja805261m
Third-harmonic generation microscopy with focus-engineered beams: a numerical study
- Olivier Nicolas
- Beaurepaire Emmanuel
Optics Express, Optical Society of America - OSA Publishing , 2008, 16 (19), pp.14703-14715 . We use a vector field model to analyze third-harmonic generation (THG) from model geometries (interfaces, slabs, periodic structures) illuminated by Hermite-Gaussian (HG) and Laguerre-Gaussian (LG) beams focused by a high NA lens. Calculations show that phase matching conditions are significantly affected by the tailoring of the field distribution near focus. In the case of an interface parallel to the optical axis illuminated by an odd HG mode, the emission patterns and signal level reflect the relative orientation of the interface and the focal field structure. In the case of slabs and periodic structures, the emission patterns reflect the interplay between focal field distribution (amplitude and phase) and sample structure. Forward-to-backward emission ratios using different beam shapes provide sub-wavelength information about sample spatial frequencies. © 2008 Optical Society of America (10.1364/OE.16.014703)
DOI : 10.1364/OE.16.014703
Flavin-dependent thymidylate synthase X limits chromosomal DNA replication
- Escartin Frédéric
- Skouloubris Stéphane
- Liebl Ursula
- Myllykallio Hannu
Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2008, 105 (29), pp.9948-9952 .
Second Harmonic Microscopy to Quantify Renal Interstitial Fibrosis and Arterial Remodeling
- Strupler Mathias
- Hernest Monica
- Fligny Cécile
- Martin Jean-Louis
- Tharaux Pierre-Louis
- Schanne-Klein Marie-Claire
Journal of Biomedical Optics, Society of Photo-optical Instrumentation Engineers , 2008, 13 (5), pp.054041 . Interstitial ﬁbrosis is a powerful pejorative predictor of progression of nephropathies in a variety of chronic renal diseases. It is characterized by the depletion of kidney cells and their replacement by extracellular matrix, in particular, type-I ﬁbrillar collagen, a protein scarce in normal interstitium. However, assessment of ﬁbrosis remains a challenge in research and clinical pathology. We develop a novel methodology based on second harmonic generation SHG microscopy, and we image collagen fibers in human and mouse unstained kidneys. We take into account the variability in renal shape, and we develop automated image processing for quantitative scoring of thick murine tissues. This approach allows quantitative 3-D imaging of interstitial ﬁbrosis and arterial remodeling with high accuracy. Moreover, SHG microscopy helps to raise pathophysiological questions. First, imaging of a large volume within a mouse kidney shows that progression of ﬁbrosis is a heterogeneous process throughout the different renal compartments. Second, SHG from ﬁbrillar collagens does not overlap with the glomerular tuft, despite patent clinical and experimental glomerulosclerosis. Since glomerulosclerosis involves SHG-silent nonﬁbrillar collagens, our work supports pathophysiological differences between interstitial ﬁbrosis and glomerulosclerosis, a clearly nonﬁbrotic process. © 2008 Society of Photo-Optical Instrumentation Engineers (10.1117/1.2981830)
DOI : 10.1117/1.2981830