Polarimetric photoluminescence microscope for strain imaging on semiconductor devices.

Anisotropic strain induces a partial linear polarization of the photo-luminescence (PL) emitted by cubic semiconductor crystals such as GaAs or InP. This paper thus presents a polarimetric PL microscope dedicated to the characterization of semiconductor devices. The anisotropic strain is quantified through the determination of the degree of linear polarization (DOLP) of the PL and the angle of this partial linear polarization. We illustrate the possibilities of this tool by mapping the anisotropic strain generated in GaAs by the presence of a stressor film at its surface, that is, a microstructure defined in a dielectric thin film (SiNx) that has been deposited with a built-in stress and shaped into a narrow stripe by lithography and etching. Our setup shows a DOLP resolution as low as 4.5×10-4 on GaAs.

Non linear elasticity of foam films made of SDS/dodecanol mixtures.

Foam film elasticity plays a significant role in film drainage and film stability and is thus expected to influence foam dynamical properties. It strongly depends on the foaming solution composition and differs from the interface elasticity measured in unconfined geometries. We use a deformable frame to deform an assembly of five films and we measure the tension and extension of each film. This provides a simple and accurate determination of the film elasticity, in the linear and non-linear regimes, for a set of SDS/dodecanol mixtures, at various concentrations. We show that the non-linear elastic behavior is well reproduced by Mysel's model coupled with a Langmuir coadsorption isotherm for a large range of chemical compositions.

Wavy nature of collagen fibrils deduced from the dispersion of their second-order nonlinear optical anisotropy parameters ρ.

From P-SHG experiments, second-order nonlinear optical anisotropy parameters ρ = χZZZ/χZXX of collagen tissues are calculated assuming the same model of supercoiled collagen fibril characterized by a variable angle θ. Dispersion of experimental ρ values is converted into distribution of θ values based on the wavy nature of collagen fibrils deduced from EM studies. For tendon, the results show that the dispersion of experimental ρ values is mainly due to Poisson photonic shot noise assuming a slight fibrillar undulation with θ = 2.2° ± 1.8°. However for skin and vessels, the dispersion of experimental ρ values is mainly due to a stronger fibrillar undulation with θ = 16.2° ± 1.3°. The results highlight that this undulation is reduced during the development of liver fibrosis therefore, contributing to the rigidity of the tissue.

Determination of extracellular matrix collagen fibril architectures and pathological remodeling by polarization dependent second harmonic microscopy.

Polarization dependence second harmonic generation (P-SHG) microscopy is gaining increase popularity for in situ quantification of fibrillar protein architectures. In this report, we combine P-SHG microscopy, new linear least square (LLS) fitting and modeling to determine and convert the complex second-order non-linear optical anisotropy parameter ρ of several collagen rich tissues into a simple geometric organization of collagen fibrils. Modeling integrates a priori knowledge of polyhelical organization of collagen molecule polymers forming fibrils and bundles of fibrils as well as Poisson photonic shot noise of the detection system. The results, which accurately predict the known sub-microscopic hierarchical organization of collagen fibrils in several tissues, suggest that they can be subdivided into three classes according to their microscopic and macroscopic hierarchical organization of collagen fibrils. They also show, for the first time to our knowledge, intrahepatic spatial discrimination between genuine fibrotic and non-fibrotic vessels. CCl4-treated livers are characterized by an increase in the percentage of fibrotic vessels and their remodeling involves peri-portal compaction and alignment of collagen fibrils that should contribute to portal hypertension. This integrated P-SHG image analysis method is a powerful tool that should open new avenue for the determination of pathophysiological and chemo-mechanical cues impacting collagen fibrils organization.

Full characterization of dichroic samples from a single measurement by circular polarization orthogonality breaking.

We report a novel method to unambiguously determine the magnitude and orientation of linear dichroism in a simultaneous way. It is based on the use of a dedicated dual-frequency dual-polarization coherent source providing two orthogonal circularly polarized modes at the output. We show that the interaction of such a beam with dichroic media gives rise to a beatnote signal whose amplitude and phase enable the full determination of the diattenuation coefficient and axis orientation, respectively. The application of this method to polarimetric imaging provides single-shot sample characterization by its diattenuation coefficient and optical axis angle, with potential applications in biomedical imaging.

Myofibrillar misalignment correlated to triad disappearance of mdx mouse gastrocnemius muscle probed by SHG microscopy.

We show that the canonical single frequency sarcomeric SHG intensity pattern (SHG-IP) of control muscles is converted to double frequency sarcomeric SHG-IP in preserved mdx mouse gastrocnemius muscles in the vicinity of necrotic fibers. These double frequency sarcomeric SHG-IPs are often spatially correlated to double frequency sarcomeric two-photon excitation fluorescence (TPEF) emitted from Z-line and I-bands and to one centered spot SHG angular intensity pattern (SHG-AIP) suggesting that these patterns are signature of myofibrillar misalignement. This latter is confirmed with transmission electron microscopy (TEM). Moreover, a good spatial correlation between SHG signature of myofibrillar misalignment and triad reduction is established. Theoretical simulation of sarcomeric SHG-IP is used to demonstrate the correlation between change of SHG-IP and -AIP and myofibrillar misalignment. The extreme sensitivity of SHG microscopy to reveal the submicrometric organization of A-band thick filaments is highlighted. This report is a first step toward future studies aimed at establishing live SHG signature of myofibrillar misalignment involving excitation contraction defects due to muscle damage and disease.

High countrate real-time FCS using F2Cor.

We present a fluorescence correlation spectroscopy setup based on a software correlator. The setup can process autocorrelation curves in real-time at countrate as high as 8 MHz, with time resolution of 1 µs. It uses the F2Cor autocorrelation algorithm, a low cost counting board and a desktop computer. Symmetrical normalization, which improves the signal to noise ratio of the FCS curve for large values of the lag-time, is adapted to the F2Cor algorithm. A new acquisition mode, which we call oscilloscope-mode, is presented. It takes advantage of the flexibility F2Cor, and proves to be very useful for optical setup adjustment. As an application of this setup, we performed FCS measurements on a reference tetramethylrhodamine solution at high concentration, up to 2.5 µM, which extend to the micromolar range the concentration applicable in FCS, using a conventional optical setup. At such high countrates the FCS curves need to be corrected for dead-time of the photo-detector, which was done successfully.

Theoretical and experimental SHG angular intensity patterns from healthy and proteolysed muscles.

SHG angular intensity pattern (SHG-AIP) of healthy and proteolysed muscle tissues are simulated and imaged here for the first time to our knowledge. The role of the spatial distribution of second-order nonlinear emitters on SHG-AIP is highlighted. SHG-AIP with two symmetrical spots is found to be a signature of healthy muscle whereas SHG-AIP with one centered spot in pathological mdx muscle is found to be a signature of myofibrillar disorder. We also show that SHG-AIP provides information on the three-dimensional structural organization of myofibrils in physiological and proteolysed muscle. Our results open an avenue for future studies aimed at unraveling more complex physiological and pathological fibrillar tissues organization.

F2Cor: fast 2-stage correlation algorithm for FCS and DLS.

We present a new multiple-tau correlation algorithm which is the fastest to date. The resulting curve is identical to that obtained with the conventional multiple-tau algorithm, but the calculation time is much shorter. It combines two approaches. For short values of the lag-time a very simple correlation histogram is used, while for higher lag-time values the traditional multiple-tau bin-and-multiply approach is used. The lag-time limit between these two stages depends on the count rate. The computation time scales linearly with the count rate and is as fast as 0.1 µs/photon.

Modeling of supramolecular centrosymmetry effect on sarcomeric SHG intensity pattern of skeletal muscles.

A theoretical far-field second harmonic generation (SHG) imaging radiation pattern is calculated for muscular myosin taking into account both Gouy effect and light diffraction under high focusing excitation. Theoretical analysis, in agreement with experimental results obtained on healthy Xenopus muscles, shows that the increase on intensity at the middle of the sarcomeric SHG intensity pattern is generated by an off-axis constructive interference related to the specific antipolar distribution of myosin molecules within the sarcomere. The best fit of the experimental sarcomeric SHG intensity pattern was obtained with an estimated size of antiparallel, intrathick filaments' packing-width of 115 ± 25 nm localized at the M-band. During proteolysis, experimental sarcomeric SHG intensity pattern exhibits decrease on intensity at the center of the sarcomere. An effective intra- and interthick filaments centrosymmetry of 320 ± 25 nm, in agreement with ultrastructural disorganization observed at the electron microscopy level, was necessary to fit the experimental sarcomeric SHG intensity pattern. Our results show that sarcomeric SHG intensity pattern is very sensitive to misalignment of thick filaments and highlights the potential usefulness of SHG microscopy to diagnose proteolysis-induced muscular disorders.

Skeletal muscle sarcomeric SHG patterns photo-conversion by femtosecond infrared laser.

Femtosecond laser at 780 nm excitation wavelength was used to photo-convert the physiological sarcomeric single band (SB) second harmonic generation (SHG) pattern into double band (DB) in Xenopus laevis premetamorphic tail muscles. This photo-conversion was found to be a third order non-linear optical process and was drastically reduced at 940 nm excitation wavelength. This effect was no longer observed in paraformaldehyde fixed muscles and was enhanced by hydrogen peroxide. The action of hydrogen peroxide suggests that reactive oxygen species (ROS) could contribute to this photo-conversion. These results demonstrate that sarcomeric DB SHG pattern is a marker of sarcomere photodamage in xenopus tadpole muscles and highlight the need of being very careful at using two-photon excitation while observing living tissues. Moreover they open new avenues for in situ intravital investigation of oxidative stress effects in muscle dysfunctions and diseases.

[Carbon monoxide poisoning: an update for 2009]. / L'intoxication au monoxyde de carbone en 2009.

Carbon monoxide (CO) is the leading cause of death by poisoning in developed countries. In 2006, 23 deaths and more than 130 hospitalisations related to CO poisoning have been reported in Switzerland. Impairment in oxygen transport through competitive antagonist action on hemoglobin is well known. Other mechanisms, in particular CO induced mitochondrial dysfunction, participate in CO toxicity. CO intoxications carry a high risk of immediate mortality and long-term neuropsychiatric or cardiac sequelae which justify the need of early diagnosis and treatment. Administration of supplemental oxygen is the cornerstone of treatment of CO poisoning. Hyperbaric oxygen therapy must be considered for the treatment of severe intoxications.

Microbial reactions on 7alpha-hydroxyfrullanolide and evaluation of biotransformed products for antibacterial activity.

7alpha-Hydroxyfrullanolide (1), a known sesquiterpenoid, was isolated from Sphaeranthus indicus using an antibacterial-activity-directed fractionation method. This compound had exhibited a significant antibacterial activity against Gram-positive bacteria. Chemical and microbial reactions were performed to prepare eight different analogues of compound 1 in order to evaluate these newly synthesized compounds for antibacterial activity. These compounds were 1beta,7alpha-dihydroxyfrullanolide (2), 7alpha-hydroxy-1-oxofrullanolide (3), 4,5-dihydro-7alpha-hydroxyfrullanolide (4), 11,13-dihydro-7alpha-hydroxyfrullanolide (5), 13-acetyl-7alpha-hydroxyfrullanolide (6), 2alpha,7alpha-dihydroxysphaerantholide (7), 4alpha,5alpha-epoxy-7alpha-hydroxyfrullanolide (8), and 4beta,5beta-epoxy-7alpha-hydroxyfrullanolide (9). Microbial reactions on 1 using whole-cell cultures of Cunninghamella echinulata and Curvularia lunata yielded compounds 2-4. Incubation of compound 1 with the liquid cultures of Apsergillus niger and Rhizopus circinans yielded metabolites 5-7, while 8 and 9 were prepared by carrying out an epoxidation reaction on 1 using meta-chloroperbenzoic acid (mCPBA). Structures of compounds 2-9 were elucidated with the aid of extensive NMR spectral studies. Compounds 2-4 were found to be new metabolites. Compounds 1-9 were evaluated for antibacterial activity and found to exhibit a wide range of bioactivities. Antibacterial-activity data of 1-9 suggested that the bioactivity of 1 is largely due to the presence of C4=C5, C11=C13, and a gamma-lactone moiety.

Visualization of lipid domains in giant unilamellar vesicles using an environment-sensitive membrane probe based on 3-hydroxyflavone.

We characterized the recently introduced environment-sensitive fluorescent membrane probe based on 3-hydroxyflavone, F2N12S, in model lipid membranes displaying liquid disordered (Ld) phase, liquid ordered (Lo) phase, or their coexistence. Steady-state fluorescence studies in large unilamellar vesicles show that the probe dual emission drastically changes with the lipid bilayer phase, which can be correlated with the difference in their hydration. Using two-photon excitation microscopy on giant unilamellar vesicles, the F2N12S probe was found to bind both Ld and Lo phases, allowing visualization of the individual phases from the fluorescence intensity ratio of its two emission bands. By using a linearly polarized excitation light, a strong photoselection was observed for F2N12S in the Lo phase, indicating that its fluorophore is nearly parallel to the lipid chains of the bilayer. In contrast, the absence of the photoselection with the Ld phase indicated no predominant orientation of the probe in the Ld phase. Comparison of the present results with those reported previously for F2N12S in living cells suggests a high content of the Lo phase in the outer leaflet of the cell plasma membranes. Taking into account the high selectivity of F2N12S for the cell plasma membranes and its suitability for both single- and two-photon excitation, applications of this probe to study membrane lateral heterogeneity in biological membranes are foreseen.

Direct Vpr-Vpr interaction in cells monitored by two photon fluorescence correlation spectroscopy and fluorescence lifetime imaging.

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) encodes several regulatory proteins, notably Vpr which influences the survival of the infected cells by causing a G2/M arrest and apoptosis. Such an important role of Vpr in HIV-1 disease progression has fuelled a large number of studies, from its 3D structure to the characterization of specific cellular partners. However, no direct imaging and quantification of Vpr-Vpr interaction in living cells has yet been reported. To address this issue, eGFP- and mCherry proteins were tagged by Vpr, expressed in HeLa cells and their interaction was studied by two photon fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. RESULTS: Results show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three alpha helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the alpha-helices could perturb the leucine zipper like motifs. The DeltaQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization. CONCLUSION: We report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three alpha helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.

Hydrophobically modified low molecular weight chitosans as efficient and nontoxic gene delivery vectors.

BACKGROUND: Chitosan derivatives are potential candidates for gene delivery because they are biocompatible and low toxic. However, their use has been limited by their moderate transfection efficiency and the rather large sizes of DNA complexes with high molecular weight chitosans. To circumvent these limitations, we used low molecular weight (approximately 5 kDa) chitosans grafted at 3 and 18 mol% with N-/2(3)-(dodec-2-enyl)succinoyl groups (HM-LMW-ch) that exhibit surfactant-like properties. METHODS: The physico-chemical properties of complexes of DNA with the two HM-LMW-ch derivatives and the nonmodified LMW-ch were compared by electrophoresis, dynamic light scattering, fluorescence spectroscopy and fluorescence correlation spectroscopy. Moreover, their transfection efficiencies and cytotoxicity were evaluated and their intracellular trafficking was monitored by confocal microscopy. Finally, their ability to deliver genes in mice kidneys after systemic administration was investigated. RESULTS: Complexes with HM(3%)-LMW-ch, but not with HM(18%)-LMW-ch and LMW-ch, efficiently delivered genes in mice kidneys. HM(3%)-LMW-ch formed small positively charged particles that were resistant to DNases and nucleases and marginally interact with serum components. Moreover, these particles were efficiently internalized in cells and low toxic. By contrast, HM(18%)-LMW-ch formed large and weakly charged aggregates with DNA that were highly susceptible to DNases and nucleases. CONCLUSIONS: HM(3%)-LMW-ch appears to be a promising nonviral vector with low cytotoxicity and efficient transfection properties.

Should the use of modified Jackson Rees T-piece breathing system be abandoned in preschool children?

BACKGROUND: The Jackson Rees breathing system is commonly used for bag and mask ventilation in preschool children, although the lack of a pressure release valve can increase the risk of gastric insufflation. Therefore, we investigated the impact of bag and mask ventilation with a Jackson Rees system on functional residual capacity (FRC) and ventilation homogeneity and evaluated the effect of the level of training of the anesthesiologist in charge. METHODS: Functional residual capacity and ventilation homogeneity were measured in 74 children (1-6 years) undergoing general surgery and the level of training of the anesthesiologist was recorded. FRC was measured (i) after intubation and (ii) after gastric emptying. Sixty-four children were ventilated using a Jackson Rees system, whereas 10 children were ventilated using a circle system to compare these two breathing systems in the second phase of the protocol. RESULTS: Functional residual capacity and ventilation homogeneity increased in all patients following gastric emptying with the highest improvement (25%) being observed when nurse students were in charge of the ventilation with the Jackson Rees system. The lowest changes in FRC and ventilation homogeneity were observed when pediatric consultants were in charge, whereas ventilation by the pediatric nurse anesthetists led to significant gastric gas insufflation. However, the circle system was associated with significantly less gastric insufflation than the Jackson Rees system. CONCLUSIONS: The efficacy of bag and mask ventilation was highly dependent on the training of the anesthesiologist with consultants demonstrating significantly better skills than any of the other groups. As the circle system is associated with a much steeper learning curve than the Jackson Rees system, its use in daily routine practice may prevent ventilatory impairment induced by gastric insufflation.

Conjugation of a new two-photon fluorophore to poly(ethylenimine) for gene delivery imaging.

We report herein the molecular engineering of an efficient two-photon absorbing (TPA) chromophore based on a donor-donor bis-stilbenyl entity to allow conjugation with biologically relevant molecules. The dye has been functionalized using an isothiocyanate moiety to conjugate it with the amine functions of poly(ethylenimine) (PEI), which is a cationic polymer commonly used for nonviral gene delivery. Upon conjugation, the basic architecture and photophysical properties of the active TPA chromophore remain unchanged. At the usual N/P ratio (ratio of the PEI positive charges to the DNA negative charges) of 10 used for transfection, the transfection efficiency and cytotoxicity of the labeled PEI/DNA complexes were found to be comparable to those of the unlabeled PEI/DNA complexes. Moreover, when used in combination with unlabeled PEI (at a ratio of 1 labeled PEI to 3 unlabeled PEI), the labeled PEI does not affect the size of the complexes with DNA. The labeled PEI was successfully used in two-photon fluorescence correlation spectroscopy measurements, showing that at N/P = 10 most PEI molecules are free and the diffusion coefficient of the complexes is consistent with the 360 nm size measured by quasielastic light scattering. Finally, two-photon images of the labeled PEI/DNA complexes confirmed that the complexes enter into the cytoplasm of HeLa cells by endocytosis and hardly escape from the endosomes. As a consequence, the functionalized TPA chromophore appears to be an adequate tool to label the numerous polyamines used in nonviral gene delivery and characterize their complexes with DNA in two-photon applications.

Investigation by fluorescence correlation spectroscopy of the chaperoning interactions of HIV-1 nucleocapsid protein with the viral DNA initiation sequences.

HIV-1 nucleocapsid protein (NC) exhibits nucleic acid chaperone properties that are important during reverse transcription. Herein, we review and extend our recent investigation by fluorescence correlation spectroscopy (FCS) of the NC chaperone activity on the primer binding site sequences (PBS) of the (-) and (+) DNA strands, which are involved in the second strand transfer during reverse transcription. In the absence of NC, the PBS stem-loops exhibited a fraying limited to the terminal G-C base pair. The kinetics of fraying were significantly activated by NC, a feature that may favour (-)PBS/(+)PBS annealing during the second strand transfer. In addition, NC was found to promote the formation of PBS kissing homodimers through interaction between the loops. These kissing complexes may favour secondary contacts between viral sequences and thus, promote recombination and viral diversity.

HIV-1 nucleocapsid protein binds to the viral DNA initiation sequences and chaperones their kissing interactions.

The chaperone properties of the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) are required for the two obligatory strand transfer reactions occurring during viral DNA synthesis. The second strand transfer relies on the destabilization and the subsequent annealing of the primer binding site sequences (PBS) at the 3' end of the (-) and (+) DNA strands. To characterize the binding and chaperone properties of NC on the (-)PBS and (+)PBS sequences, we monitored by steady-state and time-resolved fluorescence spectroscopy as well as by fluorescence correlation spectroscopy the interaction of NC with wild type and mutant oligonucleotides corresponding to the (-)PBS and (+)PBS hairpins. NC was found to bind with high affinity to the loop, the stem and the single-stranded protruding sequence of both PBS sequences. NC induces only a limited destabilization of the secondary structure of both sequences, activating the transient melting of the stem only during its "breathing" period. This probably results from the high stability of the PBS due to the four G-C pairs in the stem. In contrast, NC directs the formation of "kissing" homodimers efficiently for both (-)PBS and (+)PBS sequences. Salt-induced dimerization and mutations in the (-)PBS sequence suggest that these homodimers may be stabilized by two intermolecular G-C Watson-Crick base-pairs between the partly self-complementary loops. The propensity of NC to promote the dimerization of partly complementary sequences may favor secondary contacts between viral sequences and thus, recombination and viral diversity.