Peer-reviewed Publications |
Barroca, T., Balaa, K., Leveque-Fort, S., & Fort, E. (2012). Full-field near-field optical microscope for cell imaging. PHYSICAL REVIEW LETTERS, 108(21), 218101.
Résumé: We report a new full-field fluorescence microscopy method for imaging live cell membranes based on supercritical near-field emission. This technique consists of extracting the self-interference between undercritical and supercritical light by simple image subtraction. In the objective back focal plane, this is equivalent to adding a virtual mask blocking the subcritical emission. We show that this virtual mask is radically different from a real physical mask, enabling a 100 nm axial confinement and enhancing the image sensitivity without damaging the lateral resolution. This technique is easy to implement and simultaneously provides images of the inner parts of the cell and its membrane with standard-illumination light.
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Bulard, E., Fontaine-Aupart, M. - P., Dubost, H., Zheng, W., Bellon-Fontaine, M. - N., Herry, J. - M., & Bourguignon, B. (2012). Competition of Bovine Serum Albumin Adsorption and Bacterial Adhesion onto Surface-Grafted ODT: In Situ Study by Vibrational SFG and Fluorescence Confocal Microscopy. Langmuir, 28(49), 17001–17010.
Résumé: The interaction of hydrophilic and hydrophobic ovococcoid bacteria and bovine serum albumin (BSA) proteins with a well ordered surface of octadecanethiol (ODT) self assembled monolayer (SAM) has been studied in different situations where proteins were either preadsorbed on ODT or adsorbed simultaneously with bacterial adhesion as in life conditions. The two situations lead to very different antimicrobial behavior. Bacterial adhesion on preadsorbed BSA is very limited, while the simultaneous exposure of ODT SAM to proteins and bacteria lead to a markedly weaker antimicrobial effect. The combination of sum frequency generation spectroscopy and fluorescence confocal microscopy experiments allow one to draw conclusions on the factors that govern the ODT SAM or BSA film interaction with bacteria at the molecular level. On the hydrophobic ODT surface, interaction with hydrophobic or hydrophilic biomolecules results in opposite effects on the SAM, namely, a flattening or a raise of the terminal methyl groups of ODT. On an amphiphilic BSA layer, the bacterial adhesion strength is weakened by the negative charges carried by both BSA and bacteria. Surprisingly, preadsorbed BSA that cover part of the bacteria cell walls increase the adhesion strength to the BSA film and reduce hydrophobic interactions with the ODT SAM. Finally, bacterial adhesion on a BSA film is shown to modify the BSA proteins in some way that change their interaction with the ODT SAM. The antimicrobial effect is much stronger in the case of a preadsorbed BSA layer than when BSA and bacteria are in competition to colonize the ODT SAM surface.
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Bulard, E., Fontaine-Aupart, M. - P., Dubost, H., Zheng, W., Herry, J. - M., Bellon-Fontaine, M. - N., Briandet, R., & Bourguignon, B. (2012). The Effect of Bacterial Adhesion on Grafted Chains Revealed by the Non-Invasive Sum Frequency Generation Spectroscopy. Spectrosc.-Int. J., 27(5-6), 571–579.
Résumé: In biomedical and food industry, surface colonization by bacteria is harmful: it leads to biofilm formation, a microbial consortia more resistant to antibiotics than planktonic bacteria. In order to design materials able to limit the biofilm formation, the effect of bacteria on materials has to be well characterized. In this work, a well-defined surface composed of a self-assembled monolayer (SAM) of octadecanethiol (ODT) onto a gold surface is probed in situ. The SAM conformation is obtained using the femtosecond vibrational sum frequency generation (SFG) spectroscopy. This technique provides selectively the molecular vibrational signature of the interface. The behaviour of the ODT SAM is studied in different environments: in air, in water, and upon exposure to hydrophilic or hydrophobic Lactococcus lactis bacteria. Modelling the experimental SFG spectra reveals a measurable change of the SAM conformation which depends on the environment, especially on the hydrophilic-hydrophobic character.
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Daddi Oubekka, S., Briandet, R., Fontaine-Aupart, M. - P., & Steenkeste, K. (2012). Correlative time-resolved fluorescence microscopy to assess antibiotic diffusion-reaction in biofilms. Antimicrobial agents and chemotherapy, 56(6), 3349–58.
Résumé: The failure of antibiotics to inactivate in vivo pathogens organized in biofilms has been shown to trigger chronic infections. In addition to mechanisms involving specific genetic or physiological cell properties, antibiotic sorption and/or reaction with biofilm components may lessen the antibiotic bioavailability and consequently decrease their efficiency. To assess locally and accurately the antibiotic diffusion-reaction, we used for the first time a set of advanced fluorescence microscopic tools (fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and fluorescence lifetime imaging) that offer a spatiotemporal resolution not available with the commonly used time-lapse confocal imaging method. This set of techniques was used to characterize the dynamics of fluorescently labeled vancomycin in biofilms formed by two Staphylococcus aureus human isolates. We demonstrate that, at therapeutic concentrations of vancomycin, the biofilm matrix was not an obstacle to the diffusion-reaction of the antibiotic that can reach all cells through the biostructure.
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Deniset-Besseau, A., Miannay, F. - A., Laplace-Builhe, C., Vielh, P., Lecart, S., Lwaleed, B. A., Eschwege, P., & Fontaine-Aupart, M. - P. (2012). A fluorescence-based assay for monitoring clinical drug resistance. JOURNAL OF CLINICAL PATHOLOGY, 65(11), 1003–1007.
Résumé: Background and aims Multidrug resistance (MDR) limits effectiveness in treating malignancy by modifying internalisation and/or externalisation of drugs through cancer cell membranes. In this study we describe an assay to monitor patients' responses to chemotherapy. Methods The assay is based on the fluorescent properties of doxorubicin alone as well as in combination with methotrexate, vinblastine, doxorubicin and cisplatin (MVAC). The slide-based cell imaging technique was first optimised using a panel of breast and urothelial cancer cell lines and then extended to fine needle breast aspiration biopsy and urine cytology. Results The drug fluorescence behaviour observed on smears of clinical specimens is identical to that obtained using fixed cultured cells. The fluorescence of sensitive cells to chemotherapy is mainly localised in the nucleus, whereas resistant cells show a weak fluorescence signal localised in the cytoplasm. The difference in terms of fluorescence intensity is also highlighted through fluorescence spectra. Conclusions The results suggest that the assay provides clinically valuable information in predicting responses to doxorubicin and/or MVAC therapy. Originally set up on a confocal microscope, the assay was also effective using a standard epifluorescence microscope; as such it is technically simple, reliable and inexpensive.
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Devauges, V., Marquer, C., Lecart, S., Cossec, J. - C., Potier, M. - C., Fort, E., Suhling, K., & Leveque-Fort, S. (2012). Homodimerization of Amyloid Precursor Protein at the Plasma Membrane: A homoFRET Study by Time-Resolved Fluorescence Anisotropy Imaging. PloS one, 7(9), e44434.
Résumé: Classical FRET (Forster Resonance Energy Transfer) using two fluorescent labels (one for the donor and another one for the acceptor) is not efficient for studying the homodimerization of a protein as only half of the homodimers formed can be identified by this technique. We thus resorted to homoFRET detected by time-resolved Fluorescence Anisotropy IMaging (tr-FAIM). To specifically image the plasma membrane of living cells, an original combination of tr-FAIM and Total Internal Reflection Fluorescence Lifetime Imaging Microscope (TIRFLIM) was implemented. The correcting factor accounting for the depolarization due to the high numerical aperture (NA) objective, mandatory for TIRF microscopy, was quantified on fluorescein solutions and on HEK293 cells expressing enhanced Green Fluorescence Protein (eGFP). Homodimerization of Amyloid Precursor Protein (APP), a key mechanism in the etiology of Alzheimer's disease, was measured on this original set-up. We showed, both in epifluorescence and under TIRF excitation, different energy transfer rates associated with the homodimerization of wild type APP-eGFP or of a mutated APP-eGFP, which forms constitutive dimers. This original set-up thus offers promising prospects for future studies of protein homodimerization in living cells in control and pathological conditions.
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Grammenos, A., Fillet, M., Collodoro, M., Lecart, S., Quoilin, C., Fontaine-Aupart, M. - P., & Hoebeke, M. (2012). An ESR, Mass Spectrometry and Fluorescence Microscopy Approach to Study the Stearic Acid Derivatives Anchoring in Cells. APPLIED MAGNETIC RESONANCE, 43(3), 311–320.
Résumé: Lateral phase separations in biological membranes are of great interest, making electron spin resonance (ESR) spectroscopy combined with spin labeling a nondestructive and sensitive technique for the study of lipid rafts. It is currently accepted that the spin probe is localized on the plasma membrane. However, no study confirms this hypothesis. Herein, we report, for the first time, an accurate multispectral method for the quantification of the lipid spin label presence in every subcellular fraction. Cells were incubated with the 5-DOXYL stearic acid derivative and then subfractionated. Results of our multimodal spectroscopy approach ubiquitously demonstrate that the ESR spin label presence only sets in the plasma membranes.
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Le Sech, C., Kobayashi, K., Usami, N., Furusawa, Y., Porcel, E., & Lacombe, S. (2012). Comment on “Enhanced relative biological effectiveness of proton radiotherapy in tumor cells with internalized gold nanoparticles” [Appl. Phys. Lett. 98, 193702 (2011)]. APPLIED PHYSICS LETTERS, 100(2), 026101.
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Le Sech, C., Kobayashi, K., Usami, N., Furusawa, Y., Porcel, E., & Lacombe, S. (2012). Comment on 'Therapeutic application of metallic nanoparticles combined with particle-induced x-ray emission effect'. Nanotechnology, 23(7), 078002–078001; author reply 078002.
Résumé: A recent paper (Kim et al 2010 Nanotechnology 21 425102) presented results on the combination of irradiation by atomic ions of cells loaded by particles made of heavy atoms. They propose that the projectile induced x-rays emission (PIXE) mechanism has an important contribution to the enhancement of the cell death rate. Experiments made in our group to study the effects of such a combination have shown that the Auger effect induced in the high-Z atoms and the following induction of surrounding water radiolysis has an important contribution to the enhancement of the cell death rate. In the light of our studies we propose an alternative interpretation of the results presented in the paper by Kim et al.
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Makhlouf, H., Perronet, K., Dupuis, G., Leveque-Fort, S., & Dubois, A. (2012). Simultaneous optically sectioned fluorescence and optical coherence microscopy with full-field illumination. OPTICS LETTERS, 37(10), 1613–1615.
Résumé: Full-field optical coherence microscopy (FF-OCM) and optically sectioned fluorescence microscopy are two imaging techniques that are implemented here in a novel dual modality instrument. The two imaging modalities use a broad field illumination to acquire the entire field of view without raster scanning. Optical sectioning is achieved in both imaging modalities owing to the coherence gating property of light for FF-OCM, and a structured illumination setup for fluorescence microscopy. Complementary image data are provided by the dual modality instrument in the context of biological tissue screening. FF-OCM imaging modality shows the tissue microarchitecture, while fluorescence microscopy highlights specific tissue features with cellular-level resolution by using targeting contrast agents. Complementary tissue morphology and biochemical features could potentially improve the understanding of cellular functions and disease diagnosis. (C) 2012 Optical Society of America
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Marquer, C., Devauges, V., Cossec, J. - C., Liot, G., Lecart, S., Saudou, F., Duyckaerts, C., Leveque-Fort, S., & Potier, M. - C. (2012). Local cholesterol increase triggers amyloid precursor protein-Bace1 clustering in lipid rafts and rapid endocytosis (vol 25, pg 1295, 2011). FASEB JOURNAL, 26(1), 468.
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Marquer, C., Leveque-Fort, S., & Potier, M. - C. (2012). Determination of lipid raft partitioning of fluorescently-tagged probes in living cells by Fluorescence Correlation Spectroscopy (FCS). Journal of visualized experiments : JoVE, (62), e3513.
Résumé: In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases. Yet their existence is still a matter of controversy. Indeed, lipid raft size has been estimated to be around 20 nm, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells. Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models. FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change.
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Sarsa, A., & Le Sech, C. (2012). Quantum confinement study of the H-2(+) ion with the Monte Carlo approach. Respective role of electron and nuclei confinement. JOURNAL OF PHYSICS B-ATOMIC MOLECULAR AND OPTICAL PHYSICS, 45(20), 205101.
Résumé: A simple approximate trial wavefunction is used to study, within a variational approach and beyond the Born-Oppenheimer approximation, the molecular ion H-2(+) confined by impenetrable spherical surfaces. This wavefunction describes explicitly the motion of the nuclei and the electron of this three-body system in its lowest rovibrational state. Two cut-off functions are introduced to fulfil the Dirichlet boundary conditions, respectively, for the electron and the nuclei. The results show an increase of the energy of the ion when both the confinement radius r(ce) of the electron and R-cN of the nuclei become smaller. The electron constraint is found to be much more efficient to increase the energy than the nuclei confinement. This study shows that a metastable bound state of the ion, above the energy corresponding to the dissociation limit, can be obtained when the electron constraint is present. Expectation values of the internuclear and electron-to-origin distances are also calculated and show a rapid decrease when the confinement is stronger. Finally, the quantum pressure versus the confinement radius is also estimated. A rapid rise of the pressure value when r(ce) is less than 3 au is observed.
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Song, J., Wang, L., Jin, L., Xia, X., Kou, Q., Bouchoule, S., & He, J. - J. (2012). Intensity-Interrogated Sensor Based on Cascaded Fabry-Perot Laser and Microring Resonator. JOURNAL OF LIGHTWAVE TECHNOLOGY, 30(17), 2901–2906.
Résumé: A highly sensitive integrated optical biosensor based on the cascade of a Fabry-Perot cavity laser and a microring resonator is investigated theoretically and experimentally. The free spectral ranges of the microring and the laser cavity are designed to be about the same in order to apply intensity interrogation. The variation of the refractive index of the fluidic sample results in a large shift in the envelope function of the total transmission spectrum of the sensor, and consequently, a large change in the transmitted power. The simple light-source-integrated sensing scheme achieved a high sensitivity of 1000 dB/RIU in the preliminary experiment, demonstrating its principle and potential for low-cost practical applications.
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Actes de Conférences |
Porcel, E., Li, S., Usami, N., Remita, H., Furusawa, Y., Kobayashi, K., Le Sech, C., & Lacombe, S. (2012). Nano-Sensitization under gamma rays and fast ion radiation. In 1ST NANO-IBCT CONFERENCE 2011 – RADIATION DAMAGE OF BIOMOLECULAR SYSTEMS: NANOSCALE INSIGHTS INTO ION BEAM CANCER THERAPY (Vol. 373).
Résumé: The use of heavy compounds to enhance radiation induced damage is a promising approach to improve the therapeutic index of radiotherapy. In order to quantify and control the effects of these radiosensitizers, it is of fundamental interest to describe the elementary processes which take place at the molecular level. Using DNA as a probe, we present a comparison of the damage induced in the presence of platinum compounds exposed to different types of ionizing radiation. We present the results obtained with gamma rays (Linear Energy Transfer (LET) = 0.2 keV.mu m(-1)), fast helium ions He2+ (LET = 2.3 keV.mu m(-1)) and fast carbon ions C6+ (LET = 13 keV.mu m(-1) and LET=110 keV.mu m(-1)). The efficiency of two different sensitizers was measured: platinum based molecules (the chloroterpyridine platinum – PtTC) and platinum nanoparticles (PtNP). These experiments show that the two sensitizers are efficiently amplifying molecular damage under photon or ion irradiation. Experiments with a radical scavenger confirmed that these damages are mediated by free radicals for more than 90%. More interestingly, the induction of complex damage, the most lethal for the cells, is amplified by a factor of 1.5 on average if platinum (PtTC and PtNP) is present. As already known, the induction of complex damages increases also with the radiation LET. So, finally, the most significant enhancement of complex damage is observed when ion radiation is combined with platinum induced sensitization.
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