The book abstracts - Université Paris Saclay
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The book abstracts - Université Paris Saclay
4 - 5 - 6 NOVEMBER 2015 AUDITORIUM INSTITUT CURIE CENTRE UNIVERSITAIRE ORSAY, BÂT. 111 PhysChemCell 2015 PHYSICAL CHEMISTRY OF THE CELL: INNOVATIVE BIOIMAGING Cell signalling and trafficking New cellular reporters Innovative structural and chemical imaging Multimodal imaging In vivo & clinical applications Plenary speakers Crédits photos : CLUPS, IBAIC, ISMO, LCP Philippe Bastiaens (Dortmund) Stefan Jakobs (Göttingen) Kai Johnsson (Lausanne) Abraham Koster (Leiden) Nicolas Beziere (Munich) REGISTRATION AND INFORMATION http://www.cpps.u-psud.fr 2 4 - 5 - 6 NOVEMBER 2015 AUDITORIUM INSTITUT CURIE CENTRE UNIVERSITAIRE ORSAY, BÂT. 111 OBJECTIVES Cell imaging and microscopy play a prominent role in biological and medical research and have considerably progressed in the past fifteen years. Optical imaging techniques track the spatio-temporal transformations, interactions and dynamics of defined biomolecules in their functional context. A variety of chemical mapping methods give access to the biodistribution of cell metabolites as well as of exogenous drugs, nanocarriers or trace elements. Label-free or fast cryogenic methods better preserve living cell and tissue integrity, to analyze their subcellular as well as multicellular organization. Correlative approaches overlay various spectral or chemical information into high-content images. Most of these techniques now operate at or close to the molecular scale, i.e., at nanometric resolution. From the single cell to the living animal, these advances will ultimately give access to a detailed, in situ physical and chemical analysis of biological functions, thereby addressing the challenge of biological complexity and integration. The intimate knowledge of biological processes thus gained at the molecular level brings in turn essential information for understanding and modeling diseases, opening the way to innovative therapeutic strategies. The conference will bring together an interdisciplinary community to discuss the latest advances in the fields of analytical cell imaging. Conference topics will cover cell signaling and trafficking studies, the development of new organic, inorganic or genetically encoded probes, innovative imaging contrasts and instruments, and will explore their potential for in vivo and biomedical applications. 3 ORGANIZING COMMITTEE Larbi Amazit Céline Boutin Cédric Bouzigues Catherine Chapon Ariane Deniset Sandrine Lécart Amélie Leforestier Florence Mahuteau Anne Mantel Rachel Méallet-Renault Fabienne Mérola Valérie Nicolas Oliver Nüsse Robert Pansu Ève Ranvier Matthieu Réfrégiers Béatrice Satiat-Jeunemaître Marie-Noëlle Soler Marie-Paule Teulade-Fichou François Treussart Boris Vauzeilles ACKNOWLEDGMENTS 4 SUMMARY TIME TABLEP. 6 WEDNESDAY 4 NOVEMBER SESSION 1: CELL SIGNALLING AND TRAFFICKING SESSION 2: INNOVATIVE STRUCTURAL AND CHEMICAL IMAGING P. 9 P. 19 THURSDAY 5 NOVEMBER SESSION 1: NEW CELLULAR REPORTERSP. 27 SESSION 2: MULTIMODAL IMAGINGP. 35 FRIDAY 6 NOVEMBER SESSION 1: IN VIVO AND CLINICAL APPLICATIONS P. 43 POSTERS ABSTRACTSP. 51 5 TIME TABLE Wednesday 4 November 8:30 - welcoming session 8:45 - Patricio Leboeuf, Deputy Director for Research, FCS Campus Paris Saclay Session 1 - 9:00-13:00 - Cell signalling and trafficking Chairs : Oliver Nüsse, Béatrice Satiat-Jeunemaître The interdependence of vesicular membrane dynamics and signal processing Philippe Bastiaens, Dortmund Spatio-temporal coordination between DNA replication and mitotic commitment Olivier Gavet, Villejuif Oxidative stress signaling during pathogen phagocytosis Sophie Dupré, Orsay 11:00 - coffee-break Following the autophagosome formation: a live cell imaging challenge Romain le Bars, Gif Oxidant signalling in cells revealed by single rare-earth based nanoparticle imaging Cédric Bouzigues, Palaiseau Industrial session Exploring life science with HORIBA Scientific Chiraz Frydman, Palaiseau Click4Tag markets new tools for labelling membranes of living microorganisms Sam Dukan, Marseille 13:00 - lunch Session 2 - 14:30-18:30 - Innovative structural and chemical imaging Chairs : Marie Erard, François Treussart Live-cell RESOLFT nanoscopy Stefan Jakobs, Göttingen 3D superlocalization microscopy thanks to supercritical angle fluorescence Sandrine Lévêque-Fort, Orsay Label-free deep UV imaging at SOLEIL synchrotron Frédéric Jamme, Saint Aubin 16:30 - coffee-break Biological applications of lipid imaging with cluster-TOF-SIMS and MALDI-TOF mass spectrometry Alain Brunelle, Gif How to investigate bio-molecules in microorganisms by infrared nanoscopy technique Alexandre Dazzi, Orsay Multidisciplinary analysis of the cell membranes electropermeabilization Lluis Mir, Villejuif Thursday 5 November 8:45 - welcoming session Session 1 - 9:00-13:00 - New cellular reporters Chairs : Anton Granzhan, Robert Pansu Synthetic and semisynthetic probes for live-cell imaging Kai Johnsson, Lausanne 6 Metabolic reporters of live bacteria Boris Vauzeilles, Gif Switchable probes for labelling nucleic acids in live cells Marie-Paule Teulade-Fichou, Orsay 11:00 - coffee-break Scanning transmission electron microscopy cathodoluminescence Mathieu Kociak, Orsay Engineering cyan fluorescent proteins with ultimate performances for live cell biosensors Hélène Pasquier, Orsay Laser-polarized xenon for the study of biological cells Céline Boutin, Saclay 13:00 - lunch Session 2 - 14:30-18:30 - Multimodal imaging Chairs : Sandrine Lévêque-Fort, Rachel Méallet-Renault Institutional session Jean-Pierre Mahy, Paris-Saclay Chemistry Department work group Nadine Peyriéras, France-BioImaging Sandrine Lévêque-Fort, GDR 2588 Microscopie et Imagerie du Vivant (MIV) Zooming in on cells and macromolecules with correlative light-electron electron microscopy Abraham Koster, Leiden Chemical imaging tools for intracellular tracking of nanoparticles Sergio Marco, Orsay 16:30 - coffee-break Soft X-ray microscopy with synchrotron radiation Bertrand Cinquin, Saint-Aubin In situ quantitation of collagen fibrils diameter using absolute measurements of SHG signals Marie-Claire Schanne-Klein, Palaiseau Diffusion, localization and bioavailability of clinically-used antibiotics in staphylococcus aureus biofilms Karine Steenkeste, Orsay Friday 6 November 8:45 - welcoming session Session - 9:00-13:00 - In vivo and clinical applications Chairs : Emmanuel Beaurepaire, Matthieu Réfrégiers Optoacoustic Imaging: light and sound for in vivo biomedical imaging Nicolas Bézière, Munich Liver graft quality control by infrared spectroscopy François Le Naour, Villejuif In vivo imaging techniques to explore the immune system in non-human primates Catherine Chapon, Fontenay-aux-Roses 11:00 - coffee-break Optical imaging for biodistribution studies in preclinical research Frédéric Ducongé, Fontenay-aux-Roses In vivo imaging of signaling pathways activation in drosophila’s mushroom bodies neurons Nicolas Gervasi, Paris Thermal effects of high power density light stimulation for optogenetics control of deep brain structures Frédéric Pain, Orsay Closing remarks 7 8 WEDNESDAY 4 NOVEMBER SESSION 1: CELL SIGNALLING AND TRAFFICKING SPEAKERS : Philippe Bastiaens, Dortmund Olivier Gavet, Villejuif Sophie Dupré, Orsay Romain Le Bars, Gif-sur-Yvette Cédric Bouzigues, Palaiseau INDUSTRIAL PRESENTATION HORIBA Scientific Click4Tag CHAIRMANS: Olivier Nüsse and Béatrice Satiat-Jeunemaître 9 N-STORM 4.0 Nikon’s Next Generation N-STORM Super-Resolution Microscope System High Power objectives optimized for N-STORM Imaging Newly developed illumination magnifying lens for high quality Super-Resolution images Newly developed optics for expanded flexibility in the imaging field of view Improved laser excitation design and a sCMOS camera to increase acquisition rates from minutes to seconds www.nikoninstruments.eu 10 The interdependence of vesicular membrane dynamics and signal processing Philippe Bastiaens Systemic Cell Biology, Max Planck Institute for Molecular Physiology,, Dortmund, Germany [email protected] Autocatalytic phosphorylation of receptor tyrosine kinases (RTKs) enables diverse, context-dependent responses to extracellular signals but comes at the price of autonomous, ligand-independent activation. Reactions in and on membranes play an important role in extracellular information processing by cells. The local concentration of signaling proteins is maintained by membrane dynamics to tightly control the qualitative response properties of signaling systems. In order to illuminate the relevance of this spatial dimension in signaling, I will describe how vesicular membrane dynamics control the autocatalytic activity of receptor tyrosine kinases such as EGFR and EphA2. Spontaneous RTK activation is suppressed by vesicular recycling and dephosphorylation by protein tyrosine phosphatases (PTPs) at the pericentriolar recycling endosome. This spatial segregation of catalytically superior PTPs from RTKs is essential to preserve ligand responsiveness of receptors at the plasma membrane. Ligand-induced clustering, on the other hand, promotes phosphorylation of cCbl docking sites and ubiquitination of the receptors, thereby redirecting them to the late endosome/lysosome. This switch from cyclic to unidirectional receptor trafficking thereby converts a continuous suppressive safeguard mechanism into a finite ligand-responsive signaling mode. 11 Spatio-‐temporal coordination between DNA replication and mitotic commitment Olivier GAVET1, 2 1 2 Sorbonne Universités, UPMC Paris VI, UFR927, F-‐75005, Paris, France Present address: Institut Gustave Roussy, UMR 8200 CNRS, PR2, 114 rue Edouard Vaillant, F-‐94805 Villejuif Cedex, France Abstract Eucaryotic cell proliferation requires the orderly progression through two major events of the cell cycle, DNA replication and Mitosis, to ensure the stable inheritance of the genetic material to the cell progeny. How this tight coordination is achieved in space and time remains poorly elucidated. We investigated underlying regulatory network by the development of FRET-‐based kinase biosensors and real time live single cell assays. We previously determined that activation of the master mitotic driver, CyclinB1-‐Cdk1, is rapidly initiated in late G2 cells and immediately triggers prophase onset. Here, we investigated upstream regulatory mechanisms focusing on the involvement of Polo-‐like kinase 1 (Plk1). We found that activation of Plk1 pool reproducibly precedes Cyclin-‐B1-‐Cdk1 one by a few minutes during G2 phase progression and that its activity is critical for mitotic commitment. Plk1 activation relies on upstream Aurora-‐A kinase and appears further related to interphasic CyclinA2-‐Cdk1&2 activity, a main regulator of DNA replication. Indeed, we found that stimulating CyclinA2-‐Cdk activity from late S triggered a premature activation of Plk1 preceding unscheduled entry into mitosis. Consistently, using Knock-‐In CyclinA2-‐Venus human cells, we observed that nuclear CyclinA2 translocates to the cytoplasm during G2 phase progression and progressively interacts with Plk1. Therefore, our results uncover a molecular link between DNA replication and activation of the Mitotic Entry Network. 12 Oxidative stress signaling during pathogen phagocytosis Dupré S., Song Z.M., Bouchab L., Hudik E., Le Bars R., Nusse O. Université Paris Sud, LCP, UMR8000/ INSERM UMR1174, 91405 Orsay [email protected] Neutrophils are white blood cells that engulf pathogens at the site of infection. This process named phagocytosis drives the formation of a closed compartment inside the cell: the phagosome. In the phagosome, reactive oxygen species (ROS) are produced. These ROS, produced by the NADPH oxidase (NOX2), are crucial for pathogen killing. The NADPH oxidase is activated when the cytosolic subunits of NOX2 (p67, p47, p40, Rac) assemble with the membrane subunits (gp91 and p22) at the phagosomal membrane. P67 triggers the electron flow from NADPH to O2.-. We stilO GRQ¶W NQRZ ZKLFK IDFWRUV UHJXODWH D VXVWDLQHG 526 SURGXFWLRQ DW WKH phagosome and maintain the key cytosolic subunit p67 at that phagosomal membrane. Some recent data indicate that phosphoinositol-3phosphate (PI3P) activate ROS production in human neutrophils via its binding to p401. In this study, we wanted to understand the role of PI3P in the regulation of phagosomal ROS production. In a first approach we used a pharmacologic inhibitor of PI3P formation, wortmannin, to decrease phagosomal PI3P. Using spinning disk confocal video microscopy and a fluorescent protein-tagged PI3P probe we observed the disappearance of PI3P within 3-4 minutes after wortmannin addition. The addition of wortmannin also dramatically decreased the time of presence of tagged p40 and p67 at the phagosome, which both left the phagosome at the same time. We then conducted a genetic approach to manipulate the PI3P content of the phagosome. Using a siRNA approach we were able to increase the level of PI3P at the phagosome in space and time. Thanks to the ROS biosensor DCFH2-yeast2, we observed that the increase of PI3P at the phagosome triggered increased ROS production inside the phagosome and an extended accumulation of p67 at the phagosome. We also overexpressed the PI 3-phosphatase MTM1 at the phagosomal membrane with the FRB-FKBP dimerization system. This overexpression induced the disappearance of PI3P from the phagosome and a strong decrease in ROS production. In conclusion, PI3P regulates ROS production in the phagosome. Our data suggest that PI3P could regulate the termination of the oxidative burst and the disassembly of NADPH oxidase complex at the phagosome. 1. Matute JD et al, 2009. ³A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40 phox and selective defects in neutrophil NADPH oxidase activity´. Blood, 114(15):3309-15 2. Tlili A, Dupré-Crochet S, Erard M, Nüsse O.,2011. ³Kinetic analysis of phagosomal production of UHDFWLYHR[\JHQVSHFLHV´ Free Radicals in Biology and Medecine, 50(3):438-47 13 Following the autophagosome formation: a live imaging challenge Romain Le Bars1, Jessica Marion1,2, Claire Boulogne1, Béatrice Satiat-Jeunemaitre1,2, and Michele W. Bianchi2. 1 CNRS, I2BC, Pôle d’Imagerie et de Biologie Cellulaire IMAGERIE-GIF, 91198 Gif sur Yvette, France. 2 CNRS, I2BC, Dynamique de la Compartimentation cellulaire dans les cellules de plantes supérieures, 91198 Gif sur Yvette, France. [email protected] Macroautophagy (here referred to as autophagy) is a bulk degradation mechanism that sequesters cytosolic compounds inside a double membrane bound vesicle termed the autophagosome, which is then delivered to the vacuole for breakdown and turnover. Genetic studies in yeast identified genes involved in the different steps of autophagosome biogenesis. Subsequently, homologs of several of the ATG proteins have been identified in plants. These findings allowed a better understanding of the roles of autophagy such as: nutrient recycling, cell detoxification, organelles turnover, remobilization during leaf senescence However the early steps of autophagosome formation are still poorly understood and the origin of the membrane is still a matter of debate. Autophagy is a very dynamic process within plant cells and also highly sensitive to any kind of environmental stress. A precise and accurate study of this process required the use and development of multiple tools coupled with live imaging technics. The use of such an up to date microscopic setup on Arabidopsis roots, allowed us to show that ATG5, a protein involved in the early steps of autophagosome formation, labels a subdomain of the autophagosome membrane. The ATG5 domain marks the growing autophagosome and gave us the opportunity to closely follow their formation in vivo. The interactions between autophagosomes markers (ATG5, ATG8) and markers for endomembrane compartments were also monitored, revealing a close link with the cortical ER network. This observation was also confirmed by correlative microscopy. Our findings will contribute to the understanding of autophagosome formation in plants. References Le Bars et al. (2014) ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants Nat Commun 5, 4121. Le Bars et al. (2014) Folding into an autophagosome: ATG5 sheds light on how plants do it. Autophagy 10, 52-54. 14 Oxidant Signaling in Cells Revealed by Single Rare-Earth Based Nanoparticle Imaging Cedric Bouzigues, Mouna Abdesselem, Thanh-Liêm Nguyên, Rivo Ramodiharilafy, PierreLouis Tharaux and Antigoni Alexandrou Laboratoire Optique et Biosciences Ecole polytechnique, CNRS, INSERM, Université Paris-Saclay Palaiseau France [email protected] For many cell functions, spatio-temporal organization of signaling pathways is important for the cell response regulation. Reactive oxygen species (ROS) are common second messengers involved in the control of numerous normal and pathological cell responses. ROS homeostasis is thus essential for the cell physiology and their local concentration is thus tightly regulated. However, the dynamics of ROS production and organization is so far mostly unknown, due to the lack of efficient probes. 3+ By imaging single Eu -doped nanoparticles, we quantitatively probed the intracellular ROS response with temporal and spatial resolution. We thus measured the absolute ROS concentration in vascular cells and revealed specific temporal patterns of ROS production under different types of stimulation (PDGF and ET-1). We furthermore quantitatively revealed mechanisms of transactivation of the EGFR pathway, which notably control the dynamics of the cell response. These mechanisms of temporal control are impaired in tumoral cells, which may be of great pathophysiological relevance for the metastatic transition. By using a microfluidic system, we furthermore apply spatially controlled stimulations and displayed the maintenance of asymmetric ROS concentration in the cell under a migration inducing PDGF gradient. This likely relies on a balance between ROS diffusion and degradation, whose molecular basis is still to be investigated. Altogether, our results reveal the regulation mechanisms of the ROS organization in the cell, which illustrates how the spatio-temporal control of transduction pathways is crucial for the buildup of the cell response. We now propose to extend these methods, by using different types of nanoparticles in a ratiomectric approach, to detect ROS at high temporal resolution (!2Hz) in order to elucidate the molecular mechanisms responsible for the observed patterns at the cellular scale. Left: HeLa cells in a microfluidic device creating a permanent PDGF gradient. Right: YVO4:Eu nanoparticles in HeLa cells submitted to a PDGF gradient. 15 Exploring Life Science with HORIBA Scientific Mme Chiraz Frydman Horiba Scientific, Avenue de la Vauve, Passage Jobin Yvon, 91120 Palaiseau, France HORIBA Scientific is a worldwide leader in development and production of analytical measurement equipment for research, analysis for laboratories, and quality control. We offer instrumentation for Fluorescence-‐ and Raman-‐Spectroscopy, Ellipsometry and other methods measuring thin films In addition measurement techniques like Particle analysis, methods for the determination of water quality as well as X-‐Ray fluorescence and SPRi (surface plasmon resonance imaging) belong to our expertise. The better part of these instruments can be used not only for macroscopic analysis, but also for micro-‐measuring techniques -‐ often in a non-‐destructive manner, which is a major advantage over many other analytical methods. 16 Click4Tag markets new tools for labeling membranes of living microorganisms Audrey Dumont, Emilie Fugier and Sam Dukan CLICK4TAG, 163 avenue de Luminy, Zone Luminy Biotech, Case 922, 13288 Marseille cedex 09. [email protected] Click4Tag created in December 2014, develops innovative solutions for detection, enumeration, concentration and/or rapid identification of culturable microorganisms using a real technological breakthrough exclusively licensed at the end of 2014. The technology is based on specific functionalization of culturable microorganisms via natural assimilation and integration of a "hook" (probe) into their membrane. This anchorage point permits to associate conventional labels by "Click" reaction, to (i) a fluorescent label to detect, count and/or identify culturable microorganisms of interest, (ii) magnetic beads in order to concentrate/isolate culturable microorganism of interest. Figure 1: The concept of Click4Tag technology Nowadays, Click4Tag commercialized molecules allowing bacterial membranes functionalization for industrial and academic research. Right now we commercialized L. pneumophila lipopolysaccharides labeling and in few month a new tools that will label membrane from all kind of microorganisms. References - Fugier E., Dumont A., Malleron A., Poquet E., Mas Pons J., Baron A., Vauzeilles B. and Dukan S. (2015) Rapid and specific enrichment of culturable Gram negative bacteria using non-lethal copper-free click chemistry coupled with magnetic beads separation. Plos One 10(6) :e0127700. - Pons J. M., Dumont A., Sautejeau G., Fugier E., Baron A., Dukan S. and Vauzeilles B. (2014) Identification of Living Legionella pneumophila Using Species-Specific Metabolic Lipopolysaccharide Labeling. Angew. Chem. Int. Ed. 53, 1275-1278. - Dumont A., Malleron A., Awwad M., Dukan S. and Vauzeilles, B. (2012) Click labeling of bacterial membranes via metabolic modification of the LPS inner-core. Angew. Chem. Int. Ed., 51, 3143-3146. 17 18 WEDNESDAY 4 NOVEMBER SESSION 2: INNOVATIVE STRUCTURAL AND CHEMICAL IMAGING SPEAKERS: Stefan Jakobs, Göttingen Sandrine Lévêque-Fort, Orsay Frédéric Jamme, Saint Aubin Alain Brunelle, Gif-sur-Yvette Alexandre Dazzi, Orsay Lluis Mir, Villejuif CHAIRMANS: Marie Erard and François Treussart 19 THE FIRST MULTI-PROTEINS PRINTING PLATFORM PRIMO is an optical unit docked to an inverted microscope (Nikon Ti) for printing several proteins on cell culture slides. 3 Based on the combination of a UV illumination system and a patented chemistry (PLPP) which catalyzes the proteins patterning. 2 Fibroblasts arranged in ALVÉOLE pattern Projected pattern on culture slide PRIMO BRINGS YOU NEVER SEEN CAPABILITIES Multiple proteins printing (>3) v v Resolution <1 micrometer over the entire field of view v Quantitative printing (255 grayscale gradient) v Fast printing (20s for a 4mm2 pattern) Connection of PRIMO to the microscope UV 400 µm 3 color fluorescence microscopy image. This complex micro pattern combines three different fluorescent proteins (GFP, mCherry and NeutravidinAto647) which have been printed sequentially at the same location PRIMO SOFTWARE KEY FEATURES v Automatic calibration v Multipatterning v Oversized patterns management v Successive patterns alignment MICROSCOPE NIKON ECLIPSE Ti-E 1 20 ALVÉOLE - 68 Bd de Port-Royal, 75005 PARIS - FRANCE - ALVEOLELAB.COM Image file sent to PRIMO Live-cell RESOLFT nanoscopy Stefan Jakobs Max Planck Institute for Biophysical Chemistry, Dept. of NanoBiophotonics & University Medical Center of Göttingen, Dept. of Neurology Am Fassberg 11 37077 Goettingen, Germany www.RSFP.de E-mail: [email protected] All super-resolution microscopy (nanoscopy) concepts that fundamentally overcome the diffraction barrier in far-field optical microscopy utilise fluorophore transitions between two states, typically a fluorescent on- and a non-fluorescent off-state. In RESOLFT (reversible saturable optical fluorescence transition) and the related STED (stimulated emission depletion) microscopy concepts, a doughnut or a pattern is scanned across the sample, determining at any point in time the nanosized coordinate range where the fluorophores are in the on-state. RESOLFT microscopy utilizes reversibly switchable fluorescent proteins (RSFPs) that can be repeatedly photoswitched between fluorescent and non-fluorescent states by irradiation with distinct light wavelengths to overcome the diffraction barrier in live cell imaging. Previous RSFPs allowed only a limited number of switching cycles before photodestruction. We have generated a number of new and improved RSFPs with different photophysical properties; they exhibit strong resistance against photobleaching and switching fatigue, and thus allow dual-color as well as massively parallelized RESOLFT microscopy. Recent progress in the development of new RSFPs as well as their application in live-cell RESOLFT nanoscopy will be discussed. References Chmyrov, A.,Keller, J.,Grotjohann, T.,Ratz, M.,d'Este, E.,Jakobs, S.,Eggeling, C. and Hell, S.W. (2013). Nanoscopy with more than 100,000 'doughnuts'. Nature Methods 10 (8): 737-740 Grotjohann, T., Testa, I., Reuss, M., Brakemann, T., Eggeling, C, Hell, S.W., and Jakobs, S. (2012). rsEGFP2 enables fast RESOLFT nanoscopy of living cells. eLife, DOI:10.7554/eLife.00248 Grotjohann, T., Testa, I., Leutenegger, M., Bock, H., Urban, N.T., Lavoie-Cardinal, F., Willig, K.I., Eggeling, C., Jakobs*, S., and Hell*, S.W., (2011). Diffraction-unlimited all-optical imaging and writing with a photochromic GFP. Nature, 478(7368):204-8 Brakemann, T., Stiel, A.C., Weber, G., Andresen, M., Testa, I., Grotjohann, T., Leutenegger, M., Plessmann, U., Urlaub, H., Eggeling, C., Wahl, M.C., Hell, S.W., and Jakobs, S. (2011). A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching. Nature Biotech, 29(10):942-7. 21 3D superlocalization microscopy thanks to Supercritical Angle Fluorescence N. Bourg1,2, G. Dupuis2, S. Lécart2, E. Fort3, S. Lévêque-Fort1 1 Institut des Sciences Moléculaires d’Orsay (ISMO), CNRS, Univ. Paris-Sud, Université Paris-Saclay, F91405 Orsay, France 2 Centre Laser de l’Université Paris Sud (CLUPS/LUMAT), Univ. Paris-Sud, CNRS, IOGS, Université Paris-Saclay, F-91405 Orsay, France 3 Institut Langevin, EPSCI ParisTech, CNRS, PSL Research University, F-75005 Paris, France [email protected] Point Spread Function (PSF) engineering methods combined to Single Molecule Localization Microscopy (SMLM) allow ones to retrieve 3D position of fluorescent molecules with nanometer accuracy. Unfortunately, achieving high 3D localization precision is often at the cost of an increased experimental complexity. In addition, all these strategies only provide the relative axial positions of the fluorophores with respect to an arbitrary focal plane. Hence, 3D optical nanoscopy would strongly benefit of a method that combines high nanometer axial precision, simplicity of implementation and absolute axial positioning. Here we propose to take advantage of the forbidden light, also called SupercriticalAngle Fluorescence (SAF)[1,2], at the single molecule level. Indeed, when a fluorophore is located in the vicinity of the coverslip interface, its near-field SAF component become propagative and can be collected by a high numerical aperture objective (Fig. 1). In the objective back focal plan, the SAF component appears as a ring beyond the critical angle !c. For a fluorophore at the interface, the number of photons in the SAF SAF represents 50% of all the photons ring N tot SAF As N decreases collected N . exponentially with the dye depth distance from the coverslip surface, absolute axial Figure 1 Principle of the SAF emission position of each fluorescent dye is retrieved SAF tot versus N . In practice, by comparing N only the detection path of our SMLM setup is modified to insert a compact homemade dual view SAF and Ntot, and retrieve in live the absolute module, which permits to simultaneously measured N axial position of each fluorescent dye. This 3D absolute method, called “Direct Optical Nanoscopy with Axially Localized Detection (DONALD)” gives an isotropic 3D localization precision of 20-nm within an axial range of ~150 nm above the coverslip [3]. The localization performances of DONALD allows us to observe the actin network with an axial resolution ~35 nm (Fig.2), and to super-resolve the 3D hollowness of microtubules. Thanks to the absolute localization, 3D reconstruction of biological structures such as adhesion complex composed of several proteins can be easily retrieved. Figure 2 : COS7 cell with actin network labelled with Alexa 488, diffraction limited image on the left, DONALD super-resolved image on the right. References [1] T. Ruckstuhl et al.,Forbidden light detection from single molecules, Analytical chemistry, 2000 [2] T. Barroca et al., Full-field Near-Field Optical Microscope for Cell Imaging, PRL, 2012 [3] N. Bourg et al., Direct Optical Nanoscopy with Axially Localized Detection, Nat. Photon. 2015. 22 Label-free deep UV Imaging at SOLEIL synchrotron Frederic Jamme1, Alexandre Giuliani1,2, Frank Wien1 and Matthieu Réfrégiers1 1 Synchrotron SOLEIL, L’Orme des Merisiers, 91192 Gif sur Yvette, France Cepia, Institut National de la Recherche Agronomique (INRA), BP 71627, 44316 Nantes, France 2 DISCO Beamline is a bending magnet beamilne at synchrotron SOLEIL covering the unusual 1-21 eV energy range. It is composed of two branches and three endstations, SRCD, Atmospheric pressure photoionisation and DUV imaging. I will focus on the Imaging station. Use of deep ultraviolet (DUV, below 350 nm) fluorescence opens up new possibilities in biology because, it does not need external specific probes or labeling, but instead takes profit of the intrinsic fluorescence that arise from many biomolecules under deep ultraviolet excitation. Indeed, observation of label free biomolecules or active drugs ensures that the label will not modify the biolocalisation or any of its properties. UV monophotonic excitation does present real spectral excitation, leading the way to excitation imaging and a better selectivity of the chromophores. DUV excitation may also be used to track exogenous drugs or toxic compounds that present different spectral behaviour. Moreover, due to diffraction limit the lateral resolution is always increased when looking in the UV range allowing nanometric spatial resolution3. Examples in cell biology, enzymology and tissue diagnosis will be presented and compared to multiphotonic excitation. 1. Giuliani, F. Jamme, V.Rouam, F. Wien, J.L. Giorgetta, B. Lagarde,O. Chubar,S. Bac, I. Yao,S. Rey, C. Herbeaux, J.L Marlats, D. Zerbib, F. Polack and M. Réfrégiers, J. Synchrotron Rad. 2009, 16: 835841. 2. Jamme, F., Villette, S., Giuliani, A., Rouam, V., Wien, F., Lagarde, B., & Refregiers, M. Microscopy and Microanalysis, 2010, 16(5): 507-514. 3. Jamme, Kascakova, S., Villette, S., Allouche, F., Pallu, S., Rouam, V., & Réfrégiers, M. Biology of the Cell,, 2013,105(7), 277–288. 23 Biological applications of lipid imaging with cluster-TOF-SIMS and MALDI-TOF mass spectrometry Alain Brunelle Institut de Chimie des Substances Naturelles, CNRS-ICSN UPR2301, Université Paris-Sud, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France [email protected] Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) using keV energy metal cluster beams as primary ions is now recognized as a powerful method for in situ chemical, biological and medical applications. It opens a new field of surface imaging, particularly for biological tissue sections. Compared to the more established MALDI (Matrix Assisted Laser Desorption Ionisation) imaging approach, TOF-SIMS provides the incomparable advantages of a routine sub-micrometre scale resolution and of an easy sample preparation which does not require matrix coating of the surface. However TOF-SIMS suffers from some limitations, such as the narrow mass range, the lack of structural analysis of the species by tandem mass spectrometry, and the fact that mainly lipids are preferentially released from the biological samples. This lecture intends to show the wealth of powerful information that can be obtained from the chemical analysis of biological surfaces, with several examples chosen among various applications such as the localization of xenobiotics, natural substances, lipid markers from neurodegenerative diseases, and even cultural heritage samples. The strengths and weaknesses of lipid imaging using TOF-SIMS and MALDI-TOF will also be compared, showing the complementarity between the two methods, the compatibility with histology, and very recent developments towards increases of sensitivity and resolution. Images recorded by mass spectrometry: (from left to right) swarming of a bacterium colony, drug in a small area of eye, secondary metabolites in wood, lipids in mouse brain, lipid droplets in fat liver. References Touboul D, Laprévote O, Brunelle A (2011) Micrometric molecular histology of lipids by mass spectrometry imaging Curr Opin Chem Biol 15, 725-732 Bich C, Touboul D, Brunelle A (2014) Cluster TOF-SIMS imaging as a tool for micrometric histology of lipids in tissue Mass Spectrom Rev 33, 442-451 Bich C, Touboul D, Brunelle A (2015) Biomedical studies with TOF-SIMS imaging Biointerphases 10, 2329-2335 24 How to investigate bio-molecules in microorganisms by infrared nanoscopy technique A. Dazzi, R. Rebois, J. Mathurin, D. Onidas, A. Deniset-Besseau Laboratoire de Chimie Physique – Université Paris-Sud – 91405 - ORSAY [email protected] Abstract AFM-IR (1) (coupling an Atomic Force Microscope with an IR laser) is a simple and efficient technique to make nanoscale infrared spectroscopy. Using this technique, we were able to obtain exciting data showing the detection of bio-molecules such as bio-polymer produced by Rhodobacter (2,3) and size estimation of triacylglycerol (TAG) vesicles inside dried microorganisms like Streptomyces bacteria (4) or microalgae. The production of bio-plastic is still challenging because their properties are not always optimum for industrial process as polymer from petrochemistry. (PHB) poly(3-hydroxybutyrate) has been receiving more interest because of its thermo-plastic and mechanical properties similar to those of synthetic polymer (isotactic polypropylene). However, the crystallinity of this polymer is often an issue for specific applications. Using the nanoIR, we have estimated the amorphous and crystal phases of PHB granules directly inside bacteria (Rhodobacter) and study the effect of solvent on these granules (figure below). Figure : (Left) Chemical mapping at 1736 cm-1 revealing vesicles (in violet). (Right) Spectra on a vesicles (in red) and on a bacterium (in green). Black spectrum correspond to the FTIR spectrum of the full culture. With the same idea to look for alternatives to petrochemical product, several oleaginous microorganisms have been intensively explored to produce biodiesel. Numerous works focus on microalgae and little on a peticular bacteral specie called Streptomyces. Thanks to nanoscale IR spectroscopy, we were able to investigate the production of those biodiesel precursors (triacylglycerol or TAG) at the subcellular level. References 1. A.Dazzi et al, Opt. Lett. 30, Issue 18, 2388-2390 (2005). 2. C. Mayet et al Analyst 135, 2540-2545 (2010). 3. C. Mayet et al, Biothechnology advances, vol.31, issue 3, 369-374 (2013). 4. A.Deniset-Besseau, et al, J. Phys. Chem. Lett., 5 (4), pp 654–658 (2014). 25 Multidisciplinary approach to the molecular analysis of the cell membranes impermeability rupture caused by electric pulses (electroporation) L.M. Mir1, M. Breton1, A. Silve1,2, A. Azan1, I.Leray1, M. Scherman3, M. Tarek4, B. Attal-Trétout3 1 Vectorology and Anticancer Therapies, UMR 8203, CNRS, Univ.Paris-Sud, Université Paris-Saclay, Gustave Roussy, Villejuif. 2 present address: Institute for Pulsed Power and Microwave Technology, Karlsruhe Institute of Technology, Karlsruhe, Germany. 3 Département de Mesures Physiques, ONERA, Palaiseau. 4 Structure et Réactivité de Systèmes Moléculaires Complexes, UMR 7565, CNRS, Université de Lorraine, Nancy. [email protected] Cell electroporation, also termed cell electropermeabilization, can be achieved both in vitro and in vivo, and is already used in medical treatments such as the antitumor electrochemotherapy. In vitro, ultrashort electric pulses (for example with a 10 nanoseconds duration) can induce the same biological effects as the classical electric pulses of 100 microseconds. Interestingly, the 10 ns pulses allowed to experimentally validate the results of molecular dynamics simulations of the evolution of membrane models exposed to these same electric pulses. We thus showed that electropores are formed in the 5 nm-thick membrane during the pulses and that they close very rapidly after the end of the pulse (M. Breton et al, JACS, 2012). To explain the permeability of the cell membranes which is observed for minutes after the pulse(s) delivery, we elaborated a new model to explain all the steps of the cell membranes impermeability rupture induced by the electric pulses. The originality of the model is the introduction of electrically-facilitated chemical reactions occurring in the pores of the membrane, during the pulses. We have recently proved this model experimentally using chemical approaches, including mass spectrometry (M. Breton et al., submitted). In order to obtain dynamic information on these processes, we have developed a wide-field Coherent Anti-Stockes Raman Scattering (CARS) microscope with a special illumination geometry that minimizes the non-resonant contribution of the bulk water. We will conclude the presentation by showing preliminary results on the changes of the interfacial water before and after the electric pulses delivery. References M. Breton, L. Delemotte, A. Silve, L.M. Mir, M. Tarek. Transport of siRNA through Lipid Membranes driven by Nanosecond Electric Pulses: an Experimental and Computational Study. J. Am. Chem. Soc., 134, 13938-13941, 2012. 26 THURSDAY 5 NOVEMBER SESSION 1: NEW CELLULAR REPORTERS SPEAKERS: Kai Johnsson, Lausanne Boris Vauzeilles, Gif-sur-Yvette Marie-Paule Teulade-Fichou, Orsay Mathieu Kociak, Orsay Hélène Pasquier, Orsay Céline Boutin, Saclay CHAIRMANS: Anton Granzhan and Robert Pansu 27 Innovation in organic synthesis A partnership with Synth-Innove laboratories will accelerate your move from in-vitro concept to medical drug commercialization François Scherninski Research manager Tél. : +33 (0)1 45 58 36 90 Mobile : 33 (0) 6 86 66 49 97 Fax : +33 (0)1 47 00 89 84 Email : [email protected] 33, Boulevard du Général28Martial Valin 75015 Paris “Synthetic and Semisynthetic Probes for live-cell imaging” Kai Johnsson Institute of Chemical Sciences and Engineering; École Polytechnique Fédérale de Lausanne; CH-1015 Lausanne; Switzerland. [email protected] In my presentation I will demonstrate how a combination of protein engineering and synthetic chemistry can be exploited to generate fluorescent probes for live-cell imaging. In the first part of my presentation I will introduce a highly permeable and biocompatible near-infrared fluorophore that can be specifically coupled to intracellular proteins in live cells and tissues using different labeling techniques. The fluorogenic character of the probe and its high brightness permit live-cell imaging experiments without washing steps. The fluorophore is ideally suited for live-cell superresolution microscopy approaches using stimulated emission depletion (STED) microscopy. The excellent spectroscopic properties of the probe combined with its ease of use in live-cell applications make it a powerful new tool for bioimaging. In the second part of my talk, I will talk about our attempts to introduce a new class of fluorescent sensor proteins that permit to visualize drug and metabolite concentrations in living cells. I will also discuss how these sensor proteins can be utilized for point-of-care therapeutic drug monitoring. References 1) “Fluorogenic probes for live-cell imaging of the cytoskeleton” Lukinavicius, G.; Reymond, L.; D'Este, E.; Masharina, A.; Gottfert, F.; Ta, H.; Guther, A.; Fournier, M.; Rizzo, S.; Waldmann, H.; Blaukopf, C.; Sommer, C.; Gerlich, D. W.; Arndt, H. D.; Hell, S. W.; Johnsson, K. Nature Methods 2014, 11, 731 2) “Bioluminescent sensor proteins for point-of-care therapeutic drug monitoring” Griss, R.; Schena, A.; Reymond, L.; Patiny, L.; Werner, D.; Tinberg, C. E.; Baker, D.; Johnsson, K. Nature Chem Biol 2014, 10, 598 3) “A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins” Lukinavicius, G.; Umezawa, K.; Olivier, N.; Honigmann, A.; Yang, G.; Plass, T.; Mueller, V.; Reymond, L.; Correa, I. R., Jr.; Luo, Z. G.; Schultz, C.; Lemke, E. A.; Heppenstall, P.; Eggeling, C.; Manley, S.; Johnsson, K. Nature Chemistry 2013, 5, 132 29 Metabolic reporters of live bacteria Boris Vauzeilles Department of Chemical Biology, ICSN, CNRS UPR 2301, Université Paris-Saclay, 91198 Gif-sur-Yvette (France), Synthesis of Bioactive Molecules and Macromolecules, ICMMO, CNRS UMR 8182, Univ. Paris-Sud, Université Paris-Saclay, 91405 Orsay (France), and Click4Tag, Zone Luminy Biotech, Case 922, 163 avenue de Luminy, 13009 Marseille (France) [email protected] Sudden outbreaks of new epidemics regularly warn us against the severe sanitary and economical impact resistant bacterial infections could have on our industrialized and globalized societies. The rapid identification of viable bacteria is therefore a fundamental challenge. The outer membrane of Gram-negative bacteria is covered by a dense layer of Lipopolysaccharides (LPS), which are considered to participate into cell integrity, as well as to the level of pathogenicity of a given strain. We have recently shown that, when they are metabolically active, Gram-negative bacteria can specifically incorporate into their LPS a monosaccharide, which has been modified by the introduction of an azido anchor. This bioorthogonal chemical reporter can then be further exploited in the click-chemistry mediated labeling of these bacteria. This overall procedure offers an efficient and rapid strategy to identify living, or metabolically active, bacteria. This communication will present our latest results in this field, including specific labeling of a serious pathogen, Legionella pneumophila. References Dumont A, Malleron A, Awwad M, Dukan S, Vauzeilles B (2012) Click-mediated labeling of bacterial membranes through metabolic modification of the lipopolysaccharide inner core Angew Chem Int Ed Engl 51, 3143-3146 Mas Pons J, Dumont A, Sautejeau G, Fugier E, Baron A, Dukan S, Vauzeilles B (2014) Identification of Living Legionella pneumophila Using Species-Specific Metabolic Lipopolysaccharide Labeling Angew Chem Int Ed Engl 53, 1275-1278 Fugier E, Dumont A, Malleron A, Poquet E, Mas Pons J, Baron A, Vauzeilles B, Dukan S (2015) Rapid and Specific Enrichment of Culturable Gram Negative Bacteria Using Non-Lethal Copper-Free Click Chemistry Coupled with Magnetic Beads Separation PLoS ONE 10(6): e0127700 30 Switchable probes for labelling nucleic acids in live cells: To be or not to be in the nucleus ? Marie-Paule Teulade-Fichou Laboratory Chemistry, Modelling and Imaging for Biology, CMRS UMR9187-Inserm U1196, Institut Curie, Research Center of Orsay, University Paris-Sud, Bât 110, 91405 Orsay , France. [email protected] In view of the rapid developments of optical microscopies new fluorescent probes are constantly needed in particular IR-NIR probes compatible with intravital investigations. In this context we launched a program aimed at the design of new fluorophores excitable in the IR range via a two-photon absorption (2PA) process. Based on the non-linear optical (NLO) properties of push-pull triphenylamines (TP), we engineered this chemical family to make it compatible with biological media and in particular cellular context. Several generations of 2 and 3 branched dyes bearing cationic e-acceptors ( pyridinium, benzimidazolium) linked to the strong e-donor TP core via 1,2 These compounds were shown to combine strong 2PA vinyl bonds has been synthesized. absorption and high affinity for AT-rich DNA grooves. In addition, of utmost importance is the switchable off/on fluorescence of the TP dyes that are virtually non-fluorescent in water and strongly 3 emissive when immobilized in DNA which results in a strong contrast when imaging (Fig. 1). A B C ! Figure 1: A) Model of TP-2Bzim after docking in the minor groove of AT-rich DNA. 2PA microscopy imaging!of nuclear DNA in fixed HT29 cells at 1PM B) and 100nM dye C), Oexc: 800nm.!! Finally we observed that the two-photon absorption cross-section of the dyes is dramatically enhanced 5 once bound to DNA (G=1080 GM vs G=110 GM for the free TP-2Bzim compound ). This is attributed to a tight fit of the TP-2Bzim molecule inside the minor groove of the DNA matrix, which induces geometrical rearrangements in the dye ground state and presumably in its excited state. This effect of the DNA matrix on the nonlinear absorption of a bound dye is revealed for the first time and paves the way for studying NLO processes in DNA. (WO 2008/055969, collaboration with. C Fiorini , F.Charra, CEA-Saclay). 6 More recently it was shown that in live cells the TP dyes are trapped in mitochondria. Upon IR-light 7 excitation dye translocation to the nucleus is induced with concomitant induction of apoptosis. The TP dyes thus represent promising drug prototype able to kill cancer cell upon IR light activation (EP12306133.5.WO 09/13, collaboration with. P.Tauc, E. Deprez LBPA-IDA, Cachan). . !!!!! References : 1- G. Bordeau et al. 2008, J. Am.Chem. Soc., 130, 16836. 2- R.Lartia et al. 2008, J.Org.Chem., 73,1732. 3- B. Dumat et al. 2012 Org.Biomol. Chem.,10, 6054. 5- B. Dumat et al. 2013 J.Amer.Chem.Soc.,135, 12697. 6- R.Chennoufi et al. 2015, Chem Comm, DOI: 10.1039/C5CC05970D; 7- R.Chenouffi et al. submitted. Acknowledgements : We wish to thank ANR, CNRS and Institut Curie for fundings. . 31 Scanning Transmission Electron Microscopy Cathodoluminescence: a powerful tool for combined UltraStructure and luminescence microscopies M. Kociak1, S. Nagarajan1, L. H. G. Tizei1, C-Y Fang2, J-R Bertrand3, C. Durieu4, E. Le Cam4, H-C Chang2, M. de Frutos1, F. Treussart5 1 Laboratoire de Physique des Solides, CNRS UMR8502, Université Paris Sud, Université Paris Saclay, 91405 Orsay, France 2 Institute of Atomic and Molecular Sciences, Academia Sinica,Taipei 106, Taiwan 3 Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, CNRS UMR 8203, Université Paris Sud, Université Paris Saclay, 94805 Villejuif, France 4 Signalisations, Noyaux et Innovations en Cancérologie, CNRS UMR 8121, Université Paris Sud and Institut Gustave Roussy, 94805 Villejuif, France 5 Laboratoire Aimé Cotton, CNRS UMR9188, Université Paris Sud, ENS Cachan, Université Paris Saclay, 91405 Orsay, France [email protected] Techniques allowing to image cells are in constant evolution. Light microscopy (LM) is particularly useful when it comes to study dynamics, while Electron Microscopy (EM) gives access to structural details. The combination of both techniques, namely Correlative Light Electron Microscopy (CLEM) has shown impressive success in the past decade [1]. CLEM, however, is a demanding technique, as it requires several handlings as well as a posteriori alignment of the EM and LM images. Here we present a different approach, where luminescence signal and structural information are generated by the same particle – the electron. Indeed, the luminescence created by an electron impinging a fluorophore – Cathodoluminescence, CL – can be tracked with sub-10 nm resolution [2] when recorded in a Scanning Transmission Electron Microscope (STEM), where in addition morphological images with sub-nanometer resolution can be acquired in parallel. The demonstration of the ability of STEM-CL to correlate luminescence and ultrastructure imaging is given in the specific case of the uptake by human Ewing's sarcoma cells in culture, of fluorescent nanodiamonds (NDs) coated with two different cationic polymers (Polyethyleneimine (PEI) and Polyallylamine Hydrochloride (PAH)). The specific advantage of the technique is illustrated with the use of two types of NDs with a distinct spectral signature for each coating. Co-incubation of the cell with NDs coated with both types of polycations resulted in changes of ND density in the endosomal compartments [3]. Beyond this proof of principle, we believe that the technique may be applied to other biological problems. The bottlenecks towards this goal will be disserted, and we hope this workshop will be the place to discuss the way to overcome them. References 1. Caplan, J., et al., (2011) The power of correlative microscopy: multi-modal, multiscale, multi-dimensional. Current Opinion in Structural Biology, 21(5), 686. 2. Zagonel, L., et al. (2011) Nanometer Scale Spectral Imaging of Quantum Emitters in Nanowires and Its Correlation to Their Atomically Resolved Structure, Nano Letters, 11, 568. 3. Nagarajan, S., et al. (2015) Integrated Cathodoluminescence and Transmission Electron Microscopy allows Simultaneous Ultrastructure and Spectral Imaging of endocytosed Nanodiamonds, in preparation 32 Engineering cyan fluorescent proteins with ultimate performances for live cell biosensors . Hélène Pasquier, Asma Fredj, Yasmina Bousmah, Marie Erard, Fabienne Mérola Laboratoire de Chimie Physique, UMR 8000, CNRS Université Paris Sud, Orsay, France [email protected] Over the past fifteen years, genetically encoded reporters based on fluorescent proteins (FPs) have contributed to major advances in biological imaging, from the dynamic study of biochemical cell signaling to super-resolution optical nanoscopies. New FP variants are constantly discovered from natural sources, and submitted to intensive genetic engineering based on large scale random mutagenesis and directed evolution. However, in many cases, these newly developed FPs fail to achieve all the performances and characteristics required for their bioimaging applications, while the large scale remodeling strategies used have lost track of potentially crucial mutagenesis steps. The design of highly optimized FP-based reporters, simultaneously displaying appropriate color, multimeric state, chromophore maturation speed, brightness, photostability and environmental sensitivity will require a better understanding of the structural and dynamic determinants of FP photophysics. The recent development of new cyan fluorescent proteins (CFPs) like mCerulean3 (Markwardt et al, 2011), mTurquoise2 (Goedhart et al, 2012), and Aquamarine (Erard et al, 2013) brings a different view on these questions, as in this particular case, a step by step evaluation of critical mutations has been performed within a family of spectrally identical and evolutionary close variants. These efforts have led to CFPs with quantum yields close to unity, near single exponential emission decays, high photostability and complete insensitivity to pH, making them ideal choices as robust energy transfer donors in FRET and FLIM imaging applications. With the help of structural and theoretical investigations, these works contribute to establish a rationale for the obtained improvements in the context of FPs derived from hydrozoan species. In particular, we have shown that a proper amino-acid choice at only two positions (148 and 65) is sufficient to completely transform the photophysics of CFPs and achieve the above performances. These two positions critically control also the chromophore photoisomerization kinetics, while other amino-acid locations can be targeted to accelerate chromophore maturation and extend long term photostability. Today, these results provide a useful toolbox for a rapid and reliable upgrade of the different CFP donors carried by FRET biosensors. They also open new perspectives towards the de novo engineering of new generations of FPs with ultimate performances by synthetic biology. References Markwardt ML, Kremers G-J, Kraft CA, Ray K, Cranfill PJC, Wilson KA, Day RN, Wachter RM, Davidson MW, Rizzo MA (2011) An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching. PLoS ONE 6:e17896. Goedhart J, von Stetten D, Noirclerc-Savoye M, Lelimousin M, Joosen L, Hink MA, van Weeren L, Gadella TWJ, Royant A (2012) Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nature Communications 3:751. Erard M, Fredj A, Pasquier H, Betolngar D-B, Bousmah Y, Derrien V, Vincent P, Merola F (2013) Minimum set of mutations needed to optimize cyan fluorescent proteins for live cell imaging. Molecular Biosystems 8:258. 33 Laser-Polarized Xenon for the Study of Biological Cells Céline Boutin, Emilie Mari, Estelle Léonce, Guillaume Carret, Patrick Berthault CEA Saclay, IRAMIS, NIMBE, UMR CEA/CNRS 3685, Laboratoire Structure et Dynamique par Résonance Magnétique, 91191 Gif-sur-Yvette, France [email protected] Hyperpolarized xenon is a powerful sensor for magnetic resonance detection of molecular or biological events. This is due to the huge signal obtained by the prior optical pumping step, to its exogenous and gaseous nature, its solubility in biological media and finally its wide chemical shift range. Using these properties, two approaches where the hyperpolarized noble gas is used alone as a tracer or targeted to biological receptors via functionalized host systems are developed in the lab. 129 We have shown that, for various types of cells, either eukaryotic or prokaryotic, the Xe NMR spectra contain a signal specific to the intracellular compartment, very distinct from the signal of xenon in the external water pool.[1] (see figure 1) This signal depends on the cell type, the viability of the cells, the presence of toxics, etc. We have applied this observation to the study of the multidrug 129 Xe NMR, it has been possible to distinguish resistance phenotype on model cell lines. Using resistant and sensitive cell lines.[2,3] Figure 1. The simplicity of the approach is depicted here. In its original version hyperpolarized xenon prepared in the batch mode by optical pumping is introduced in the NMR tube containing the cell 129 suspension under study. The one-scan Xe NMR spectrum displays two signals corresponding to xenon in the cells and xenon in the medium. 129 Also, in collaboration with chemists (ENS Lyon and CEA/DSV), Xe NMR-based biosensors consisting in xenon hosts bearing a biological ligand have been conceived and studied. They enabled sensitive detection of different analytes or biological receptors. Some of these biosensors will be presented. References 129 [1] C. Boutin et al. (2011) Hyperpolarized Xe NMR signature of living biological cells NMR in Biomedicine 24 1264 [2] C. Boutin, P. Berthault (2012) Procédé de détermination de la résistance cellulaire aux médicaments France Patent 12 52922 [3] P. Berthault, C. Boutin, (2015) Hyperpolarized Xenon-129 Magnetic Resonance: Concepts, Production, Techniques and Applications RSC Book Chapter, New Developments in NMR no. 4, Edited by T. Meersmann and E. Brunner [4] P. Berthault et al. (2009) Biosensing using laser polarized xenon NMR/MRI Progress in NMR Spectroscopy 55 35 34 THURSDAY 5 NOVEMBER INSTITUTIONAL PRESENTATION Jean-Pierre Mahy, Paris-Saclay Chemistry Department work group Nadine Peyriéras, France-BioImaging Sandrine Lévêque-Fort, GDR 2588 Microscopie et Imagerie du Vivant (MIV) SESSION 2: MULTIMODAL IMAGING SPEAKERS: Abraham Koster, Leiden Sergio Marco, Orsay Bertrand Cinquin, Saint-Aubin Marie-Claire Schanne-Klein, Palaiseau Karine Steenkeste, Orsay CHAIRMANS: Sandrine Lévêque-Fort and Rachel Méallet-Renault 35 FEMTONICS Ltd microscopes à 2 photons et imagerie de fluorescence IDIL Fibres Optiques, le partenaire et distributeur en France FEMTONICS Ltd est une spin off de l’IEM HAS Institut de médecine Expérimentale de l’Académie des Sciences de Hongrie, créée en 2005. Le microscope multi-photon est basé sur un statif Olympus motorisé. En plus des 400 éléments propres qui composent le hardware du microscope, FEMMTONICS propose leur propre électronique et leur logiciel unique, le MES. Le point fort du MES développé sous Matlab réside dans la combinaison de la partie imagerie avec l’uncaging, l’analyse et nombre d’autres outils. Le software a été testé par l’IEM HAS et continue à être testé en cours d’amélioration pour un retour en temps réel en continu. Femtonics s’appuie sur plusieurs technologies brevetées pour proposer des microscopes les plus rapides en imagerie 2D et 3D, à ce jour. Une des forces des microscopes FEMTONICS est la modularité qui permet une amélioration du système de base en y ajoutant les fonctions et modules telles que : § Optogenetics Uncaging § (Photo-activation) § Kit d’epifluorescence § Confocal (solution 2 en 1) § FLIM, FRET § Modules sur mesure § Seconde tête de scanner : galvano ou résonant Les points forts additionnels sont la flexibilité, le service client de haute qualité, le développement possible de solutions uniques en partenariat avec les besoins des clients, ainsi que le prix abordable des solutions par rapport à la concurrence. FEMTONICS Ltd a été la première à proposer un vrai microscope mutiphoton 3D. Le Femto3D-RC rend possible la mesure en 3D sur des volumes relativement grands, sur des points aléatoires ou sur des trajectoires, ce qui facilite le balayage rapide pour l’analyse de cellules individuelles, de processus cellulaires ou de populations de cellules. De longues trajectoires spatiales, jusque 700 x 700 x 25 um3 à une fréquence de 150 Hz-el and même <0.1 us/pixel de taux de répétition, peuvent être mesurées. Roller Coaster 3D -> http://femtonics.eu/products/femto3d-rc 3D-AO -> http://femtonics.eu/products/femto3d-acoustooptic Des références d’utilisateurs FEMTONICS en Europe, en Australie et aux Etats Unis sont disponibles. IDIL Fibres Optiques cultive depuis vingt ans son expertise et son savoir-faire en ingénierie optoélectronique fibrée. Notre équipe technique est composée essentiellement de collaborateurs possédant une formation d’un niveau élevé en optique, électronique ou encore informatique (ingénieurs, docteurs). Pour les microscopes biphotons, notre équipe sera capable de vous aider sur l’ensemble des éléments optiques du système. Une de nos antennes se trouve à Orsay, non loin de votre site. Cette antenne est équipée de matériel pour le diagnostique et les mesures de vérifications, comme par exemple des minispectromètres fibrés pour le contrôle des lasers et des optiques. Ce type de minispectromètre est intégré dans les lasers de chez Coherent pour donner la longueur d’onde d’émission du laser. Notre partenariat étroit avec notre fournisseur Femtonics permettra un support et une aide efficace. Vous avez également accès directement aux ingénieurs Femtonics qui ont développé et font la maintenance de la partie informatique. Dans le cas où nos solutions auraient un intérêt plus marqué pour vous, il est également possible de prévoir un rendez-vous dédié par téléphone ou lors d’un rendez-vous de présentation d’un équipement d’un de nos utilisateurs Français. Nous pouvons vous inviter chez FEMTONICS (Hongrie) à nos frais pour vous présenter nos solutions, faire des démonstrations de mesures et discuter de vos applications et besoins spécifiques. Des expériences ciblées peuvent être définies au préalable ensemble pour vous démontrer les performances et l’efficacité d’utilisation de nos microscopes. www.idil.fr, http://femtonics.eu/ et [email protected] 02 96 05 40 20 [email protected] 04 50 28 34 81. 36 IDIL Fibres Optiques 21 rue Louis de Broglie F-22300 Lannion Tel : 02 96 05 40 20 - Fax : 02 96 05 40 25 Email : [email protected] - Site : www.idil.fr Zooming in on cells and macromolecules with correlative light-electron electron microscopy A.J. Koster Department of Molecular Cell Biology Leiden University Medical Centre Leiden, The Netherlands. [email protected] In correlative light and electron microscopy (CLEM) imaging modalities are combined to study cellular processes. Fluorescence light microscopy (FM) enables the imaging of dynamic events in relatively large fields of view exploiting a wide range of available fluorescent markers, while electron microscopy (EM) can reveal structural macromolecular arrangements in their cellular context in 1 relatively narrow fields of view at nm-scale resolution . EM specimens prepared by conventional methods that incorporate chemical fixation and metal 2 staining steps can provide a wealth of information on the cellular architecture and processes . 3D morphology of cell systems and tissue can be unravelled in sections of material several hundred nm 3 thick using electron tomography with transmission EM (TEM). 3D imaging of material 100’s µm in 3 size can be obtained with serial block face scanning electron microscopy . At the molecular level however, the fidelity of interpretation of conventionally prepared specimens is limited because of effects related to the fixation and staining. Imaging of cryo-immobilized frozenhydrated specimens excludes the use of stain and as such the molecular resolution is preserved. Images of frozen hydrated specimens have an inherent low contrast and low signal to noise ratio because of their electron dose sensitivity and the lack of heavy atoms. Fortunately, recent technological improvements of image detectors and contrast-enhancing phase plates for TEM have improved both the contrast and signal-to-noise ratio of cryo EM datasets. 4,5 For applications of EM, FM can be instrumental as a tool for selection of areas of interest . An overview of CLEM developments will be given and workflows of different types of CLEM will be 5 illustrated by results obtained on a variety of biological systems, such as on fluorescently labelled 6 bacteria, virus-induced replication structures and blood-filtering structures in tissue . References 1. Patwardhan et al. (2014) A 3D cellular context for the macromolecular world Nat Struct Mol Biol 21(10):841-5. 2. Barcena and Koster. (2009) Electron tomography in life scienc. Semin Cell Dev Biol 20(8):920-30. 3. Mourik et al. (2015) Towards the imaging of Weibel-Palade body biogenesis by Serial Block Face – SEM J Microscopy 259(2):97-104. 4. Briegel et al. (2010) Correlated light and electron cryo-microscopy Methods Enzymol 481:317-4. 5. Faas et al. (2013) Localization of fluorescently labeled structures in frozen hydrated samples using integrated light electron microscopy. J Struct Biol 181(3):283-90. 6. Mourik et al. (2015) Content delivery to newly forming Weibel-Palade bodies is facilitated by multiple connections with the Golgi apparatus Blood 125(22):3509-16. 37 Chemical imaging by electron microscopy and secondary ion mass spectroscopy for intracellular tracking of nanoparticles Marco Sergio, Guerquin-Kern Jean Luc, Wu Ting-Di, Trepout Sylvain, Messaoudi Cédric INSERM U1196/ Institut Curie/CNRS UMR 9187 Campus Universitaire d'Orsay, Rue Becquerel. Bat 110 91405 Orsay cedex FRANCE [email protected] The characteristics of condensed matter depend on its chemical composition and the understanding of its properties frequently involves the study of the spatial distribution of its chemical elements. When the results of this analysis are qualitative or quantitative chemical maps we talk about chemical imaging. The progresses in biological sample preparation and the improvements in methods whose usage is suitable for chemical imaging not only make this approach possible for the study of biological samples, but also makes available the access to the three-dimensional (3D) distribution of chemical elements. Thus chemical imaging offers nowadays large possibilities for a better understanding of biological systems by allowing the identification of chemical components at tissue, cellular and subcellular levels (Marco et al., 2014). Besides the biological questions which can be approached by chemical imaging, nanoparticles characterization and their intracellular tracking is currently a major topic because of their use in cosmetics and nanomedicine as well as their increasing presence in air and water. Since transmission electron microscopy (TEM) provides access to the 3D chemical maps by the use of electron energy filtered (EFTEM) techniques (Messaoudi et al., 2013), having high spatial resolution on biological samples, it is a method of choice to address the aforementioned questions. Moreover, the use of nano-focused electron beam in scanning mode (STEM) provides access to atomic contrast (Sousa et al., 2012), also call Z-contrast, also suitable for nano-particles characterization and tracking. However, analytical TEM applied to biology is not a quantitative approach and it is limited by the surface of the sample accessible to the analysis in a reasonable scale of time. Secondary ion mass spectroscopy (nano-SIMS) imaging (Guerquin-Kern et al. 2005) offers a way to overcome this limits by providing access to the isotopic quantification and the imaging of isotope labelled molecules (Bower et al., 2009). Nevertheless, nano-SIMS imaging has lower spatial resolution than analytical TEM and cannot be used to generate 3D chemical maps. Since TEM and nano-SIMS are complementary approaches for chemical imaging, to study the same sample in a correlative approach can be the way to overcome the limits of these two methods. During this talk, the basis of analytical TEM, nano-SIMS and chemical correlative imaging will be exposed and they will be illustrated by examples on nanoparticles, drugs and isotope labelled molecules characterization and sub-cellular tracking. References x x x x x Boxer SG, Kraft ML, Weber PK. (2009) Advances in imaging secondary ion mass spectrometry for biological samples. Annu Rev Biophys. 38,53-74. Guerquin-Kern JL, Wu TD, Quintana C, Croisy A. (2005) Progress in analytical imaging of the cell by dynamic secondary ion mass spectrometry (SIMS microscopy). Biochim Biophys Acta. 1724,228-38. Marco S. (2014) Towards Three-dimensional Chemical Imaging of Cells. J. Mol. Biol. & Mol. Imaging. 1,2 Messaoudi C, Aschman N, Cunha M, Oikawa T Sorzano C.O.S, Marco S. (2013) EFTEM-TomoJ: 3D chemical mapping by EFTEM including SNR improvement by PCA and volume improvement by noise suppression during the ART reconstruction process. Microsc. Microanal. 28:1-9. Sousa AA, Leapman RD. (2012) Development and application of STEM for the biological sciences. Ultramicroscopy. 123,38-49. 38 Soft X Ray Microscopy with synchrotron radiation Bertrand Cinquin Synchrotron Soleil, Orme des Merisiers, 92192 Gif sur Yvette [email protected] Utilizing soft x-ray in the water window energy range allow microscopic studies of hydrated biological samples close to their near-native states since Oxygen atoms are more transparent than Carbon atoms. Therefore, a natural good contrast may be obtained from carbonaceous species : different compaction of DNA, rich lipid organelles such as mitochondria, lipid droplet! However, living specimen are highly sensitive to Soft X-Rays and radiation damages are a primary concern. 3D observation with high resolution (with a voxel size of 125 Å3, i.e. 50 nm pixel side) need between one to three hundred projections. Therefore, insuring the conservation of the structures of the sample along the acquisition is mandatory. Presently, Cryo freezing and cryo environment is the best solution. Furthermore, being a transmission technique, linear absorption coefficient can be retrieved allowing the characterization of inner cellular features. Soft X rays microscopies offer an in between resolution compared to electronic microscopy and conventional fluorescent techniques. However, super resolution techniques such as SIM or SPIM can offer the same definition for a similar volume about 10 "m x 10 "m x 10 "m where electronic microscopy is limited by the thickness of the sample (about 1 "m). More, being label-free, other Soft X ray density probes (such as TiO2) could be used in combination with other fluorescent proteins. A correlative work between cryofluorescence microscopy and Soft X ray microscopy will be presented. Besides the soft x-ray microscopy technique2, I will talk about some interesting examples such as the inner nucleus organisation along the cellular differentiation or the role of different proteins involved in the lipid droplet autophagy in S. Cerevisiae as a result of using a soft X-ray microscope at the ALS on NCXT beamline. I will conclude by presenting the principal challenges the technique need to address in order to further explore the utility of such a powerful instrument within the biological community. References 1. B Cinquin, R. Boudreau, M. Legros, C. Larabell (2013) 3D Organization of the Interphase Nucleus using Soft X Ray Tomography. Biophysical Journal, 102, p478a 2. MA Le Gros, G McDermott, B Cinquin, E. Smith, M. Do, W. Chao, P Naulleau, C. Larabell (2014) Biological soft X-ray tomography on beamline 2.1 at the Advanced Light Source. Journal synchrotron radiation, 21, 1370-1377 39 In situ quantitation of collagen fibrils diameter using absolute measurements of SHG signals. M.-C. Schanne-Klein1, S. Bancelin1, I. Gusachenko1, G. Latour1, L. Kowalczuk2, T. Coradin3 and C. Aimé3 1. Laboratoire d’Optique et Biosciences, École Polytechnique, CNRS, INSERM U1182, Université Paris-Saclay, F-91 128 Palaiseau, France 2. Centre de Recherche des Cordeliers, Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS 872, F-75 006 Paris, France 3. Laboratoire de Chimie de la Matière Condensée de Paris, Sorbonne Universités, UPMC Univ Paris 06, CNRS, Collège de France, F-75 231 Paris, France. E-mail: [email protected] Type I collagen is a major structural protein in mammals. This biopolymer is synthesized as a triple helix, which self-assembles into fibrils (diameter: 10-300 nm) and further forms various 3D organizations specific to each tissue. In recent years Second Harmonic Generation (SHG) microscopy has emerged as a powerful technique for the in situ investigation of the fibrillar collagen structures in matrices or tissues [1]. However, as an optical technique with typically 300 nm lateral resolution, SHG microscopy cannot resolve most of the collagen fibrils. Moreover, in contrast to incoherent fluorescence signals that scale linearly with the chromophore concentration, SHG is a coherent multiphoton signal that scales quadratically with the density of collagen triple helices aligned in a parallel way in the focal volume. Consequently, quantitative SHG measurements have been limited so far to averaged phenomenological parameters [1]. In this study, we correlated SHG and transmission electron microscopies to determine the sensitivity of SHG microscopy and calibrate SHG signals as a function of the diameter of the collagen fibril [2]. To that end, we synthesized in vitro isolated fibrils with various diameters and successfully imaged the very same fibrils with both techniques, down to 30 nm diameter (see figure 1). We observed that SHG signals scale as the fourth power of the fibril diameter, as expected from analytical and numerical calculations. It validated our quantitative bottom-up approach used to calculate the non-linear response at the fibrillar scale and demonstrated that the high sensitivity of SHG microscopy originates from the parallel alignment of triple helices within the fibrils and the subsequent constructive interference of SHG radiations. This calibration was then applied to Fig. 1 : Correlative SHG-electron imaging of intact rat corneas, where we successfully isolated collagen fibrils. recovered the diameter of hyperglycemia-induced fibrils in the Descemet’s membrane without having to resolve them [2,3]. Importantly, this calibration only applies to isolated fibrils. Nevertheless, complementary techniques can probe the sub-micrometer structure of dense distributions of collagen fibrils. For instance, we showed that the anisotropy ratio measured by polarization-resolved SHG microscopy increases with the orientation disorder within the focal volume [4]. These results therefore represent a major step towards quantitative SHG imaging of collagen biomaterials or tissues. References [1] M. Strupler et al (2008) Second Harmonic Microscopy to Quantify Renal Interstitial Fibrosis and Arterial Remodeling, J. Biomed. Opt. 13, 054041 [2] S. Bancelin et al (2014), Determination of collagen fibril size via absolute measurements of secondharmonic generation signals, Nat Commun 5 [3] G. Latour et al (2012), Hyperglycemia-Induced Abnormalities in Rat and Human Corneas: The Potential of Second Harmonic Generation Microscopy, PLos ONE 7, e48388 [4] I. Gusachenko et al (2012), Polarization-resolved second-harmonic microscopy in tendon upon mechanical stretching, Biophys. J. 102, 2220-2229. 40 Diffusion, localization and bioavailability of clinically-used antibiotics in Staphylococcus aureus biofilms: toward a better understanding of bacterial biofilms tolerance? Karine Steenkeste, Rym Boudjemaa, Matthieu Revest, Cédric Jacqueline, Jocelyne Caillon, Romain Briandet, Marie-Pierre Fontaine-Aupart Institut des Sciences Moléculaires d’Orsay, Université Paris-Sud, Bât. 210, 91405 Orsay Cedex, France [email protected] Staphylococcus aureus is one of the most frequent pathogens associated with biofilm-related infections. Among clinically-used antibiotics for the treatment of S. aureus infections, very few enable long-term successful treatments against biofilms. There is therefore a major need to gain a better understanding of these biological structures tolerance towards antibiotics. Here, we developed an in vitro model which holds particular relevance for assessing antibiotics activities using time-kill studies but also for directly visualizing by fluorescence microscopy the interaction of fluorescently-labelled antibiotics with S. aureus at different phases of growth. MSSA and MRSA clinical and collection strains were grown in vitro at 37°C in TSB enriched with proteins and calcium. Planktonic bacteria and 24h-biofilms grown in polystyrene microplates were exposed to daptomycin 20µg/mL, vancomycin 40µg/mL or their combination with rifampicin 20µg/mL during 72h. Antibiotics efficiency was screened by time-kill studies. Fluorescence confocal microscopy was employed in addition to visualise biofilms three-dimensional structures and dead and live cells distribution over time but also to probe antibiotics diffusion and availability inside biofilms using timelapse and FRAP modules. Vancomycin and daptomycin alone were ineffective against biofilms, but an important part of viable cells were not culturable. This subpopulation might be responsible for recurrent infections. Previously vancomycin has been shown to diffuse through the whole biofilm structure, to interact with its target but still remains inactive. Similarly, we confirmed that daptomycin penetrated, diffused and was bioavailable inside biofilms. Unexpectedly, we showed that daptomycin could enter cell membranes when bacteria were exponentially-growing. But as soon as bacteria secreted a matrix, daptomycin mainly localized in the biofilm matrix, revealing a higher affinity for matrix components. These in vitro biofilms constitute promising high throughput systems to visualize and quantify antibiotics tolerance within biofilms and decipher the associated molecular mechanisms. They are currently being compared with a mouse model of S. aureus infection on a medical implant to assess their in vivo relevance. References Daddi Oubekka S, Briandet R, Fontaine-Aupart M-P, Steenkeste K. (2012) Correlative time-resolved fluorescence microscopy to assess antibiotic diffusion-reaction in biofilms. Antimicrob Agents Chemother; 56, 3349–58. 41 42 FRIDAY 6 NOVEMBER SESSION 1: IN VIVO AND CLINICAL APPLICATIONS SPEAKERS: Nicolas Bézière, Munich François Le Naour, Villejuif Catherine Chapon, Fontenay-aux-Roses Frédéric Ducongé, Fontenay-aux-Roses Nicolas Gervasi, Paris Frédéric Pain, Orsay CHAIRMANS: Emmanuel Beaurepaire and Matthieu Réfrégiers 43 44 Optoacoustic Imaging: light and sound for in vivo biomedical imaging. Nicolas Bézière, Vasilis Ntziachristos Institute for Biological and Medical Imaging Technische Universität München and Helmholtz Zentrum München Ingolstädter Landstr.1 85764 Neuherberg, Germany [email protected] By relying on the conversion of light energy into ultrasound waves through thermo-elastic expansion, resulting from light absorbance, optoacoustic imaging has effectively provided a new modality to observe, time and quantify molecular events in preclinical and clinical settings. Its latest implementations, built on Multispectral Optoacoustic Tomography (MSOT) approaches, have allowed spectral identification of a variety of intrinsic (blood, melanin…) and extrinsic (optical reporters, metallic nanoparticles, genetic tags) contrast sources(1), yielding unprecedented insight on physiology and pharmacology(2). Being able to follow simultaneously multiple biomarkers, MSOT provided a new perspective on cancer imaging in particular, with accurate oxygenation status monitoring along with drug-delivery system distribution tracking. Labeled liposomes specifically have highlighted the importance of nanoparticle size in relation to tumor type for efficient drug delivery systems, a behavior that could also be seen using gold nanoparticles, both paving the way for real-time theranostic applications. Lastly, the emergence of suitable genetic tags for optoacoustic imaging, either photoabsorbing proteins or through enzymatic activity has recently allowed for longitudinal studies of cell behavior at high resolution and increased imaging depth. Application in different cancer models using either genetically labeled cells or in vivo viral infection highlighted the potential of genetic labels in modern in vivo biomedical imaging. Through these specific applications, current state of the art in preclinical optoacoustic imaging will be discussed, and built upon to provide a glimpse of the future of optoacoustic imaging, from bench to bedside. IndoCyanine Green labelled liposomes (LipoICG, left panel). Transverse optoacoustic image of the distribution of LipoICG (hot scale), overlayed on an anatomic image (grey scale) of a mouse bearing a 4T1 tumor on its back 24h after LipoICG iv injection (right panel). References 1. 2. Ntziachristos V & Razansky D (2010) Molecular imaging by means of multispectral optoacoustic tomography (MSOT). Chemical reviews 110(5):2783-2794. Taruttis A & Ntziachristos V (2015) Advances in real-time multispectral optoacoustic imaging and its applications. Nature photonics 9(4):219-227. 45 Liver graft quality control by infrared spectroscopy François Le Naour INSERM, Unité 1193, Univ. Paris-Sud, hôpital Paul Brousse, F-94807 Villejuif, France [email protected] Liver transplantation constitutes a major treatment of patients with cirrhosis or hepatocellular carcinoma. Due to the shortage of grafts, transplantation teams are using marginal grafts from expanded criteria donors. These marginal grafts constitute an additional source of organs but also an increased risk factor of primary non-function or dysfunction of the graft mainly due to their poor quality. Steatosis is one of the most important factors affecting liver allograft function, mostly because of more severe ischemia-reperfusion injury. Steatosis is characterized by the intra-cellular accumulation of triacylglycerides (TG) resulting in the formation of lipid vesicles in the hepatocytes. The gold standard to assess hepatic steatosis in liver grafts during the transplantation procedure is the histological examination of frozen sections by a pathologist. The quantitative histological evaluation of steatosis is based on the percentage of hepatocytes containing cytoplasm fat inclusions. The major issue is that assessment of hepatic steatosis on histological sections is an imperfect and not reproducible method. Furthermore, the histological estimation of steatosis is poorly correlated with the true lipid content of the liver tissue. Thus, an alternative method to histopathology providing an objective assessment of steatosis is an unmet need. We addressed the potential of infrared (IR) microspectroscopy for grading steatosis on frozen tissue sections. Infrared microspectroscopy is based on the determination of absorption of infrared light due to resonance with vibrational motions of functional molecular groups. As such IR microspectroscopy constitutes a valuable tool for biochemical investigations. The use of the bright infrared source emitted by the synchrotron radiation (SR) allowed investigating the biochemical composition at the cellular level. The variance in the huge number of spectra acquired was addressed by principal component analysis (PCA). The study demonstrated that the progression of steatosis corresponds not only to the accumulation of lipids but also to dramatic changes in the qualitative composition of the tissue. Furthermore, the approach highlighted that dramatic biochemical changes occur also in the non-steatotic part of the tissue despite normal histological aspect suggesting that the whole tissue reflects the grade of steatosis. This latest observation opened the possibility to address the grade of steatosis using a laboratory IR microscope. Thus, acquisition of IR spectra on unstained frozen sections was achieved further using a commercially available laboratory IR microscope that allows scanning a whole tissue section ranking between 1 to 5 mm! (up to 10 000 spectra with 50 x 50 2 "m aperture size). The quantification of the lipid content was addressed from the IR spectra by -1 calculating the ratio of integrated intensity of bands attributed to lipids (2800-3000 cm ) related to -1 proteins (Amide II: 1485-1595 cm ). This ratio allows normalizing the intensity variation of the bands due to variations in thickness of the tissue section. For each patient, the average ratio lipids/proteins measured by IR microspectroscopy was plotted as a function of the concentration of TG leading to exhibit a marked linearity. A standard curve was further established to quantify the lipid content and thus the level of steatosis. Finally, we took advantage of attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy that allows by a close contact with the sample the acquisition of an 2 average IR spectrum corresponding to a huge area of 2 x 2 mm in only 1 minute. In conclusion, infrared microspectroscopy allows accurate quantification of lipid content on tissue section. This rapid method can be easily used at the hospital for reliable assessment of graft quality control in liver transplantation. References Peng C*, Chiappini F*, Ka#$áková S, Danulot M, Sandt C, Samuel D, Dumas P, Guettier C, Le Naour F. Vibrational signatures to discriminate liver steatosis grades. Analyst. 2015, 140:1107-1118. (*) Equal contribution. Le Naour F, Gadea L, Danulot M, Yousef I, Vibert E, Wavelet M, Ka#$áková S, Castaing D, Samuel D, Dumas P*, Guettier C*. Quantitative assessment of liver steatosis on tissue section using infrared spectroscopy. Gastroenterology. 2015, 148:295-297. (*) Equal contribution. 46 In vivo imaging techniques to explore the immune system in non-human primates: application to the study of the immune response to vaccination against HIV 1-2-3 1-2-3 1-2-3 4 3-5 Biliana Todorova , Nina Salabert , Frédéric Martinon , Raphaël Boisgard , Gerard Zurawski , Sandra 3-5 1-2-3 1-2-3 6 3-5 3 , Antonio Cosma , Thierry Kortulewski , Jacques Banchereau , Yves Levy , Zurawski , Nathalie Bosquet 1-2-3 1-2-3 and Catherine Chapon Roger Le Grand [email protected] 1Immunology of viral infections and autoimmune diseases, IDMIT Infrastructure, CEA –iMETI/Division of ImmunoVirology, Université Paris Sud, Inserm U1184, Fontenay-aux-Roses, France; 2- Université Paris-Sud, UMR E1, Orsay, France; 3- Vaccine research institute (VRI), Créteil, France; 4- CEA, Institute of Biomedical Imaging (I2BM), DSV/I2BM/SHFJ/INSERM U1023, CEA, Orsay, France; 5- Baylor Institute for Immunology Research, Dallas, 75204 TX, USA; 6- CEA, Photonic microscopy platform, Institut de Radiobiologie Cellulaire et Moléculaire (IRCM), DSV, Fontenay aux Roses, France Introduction: Non invasive and longitudinal imaging approaches are required to study the behavior of antigen presenting cells (APCs) following immunization in order to better understand the mechanisms leading to the induction of cellular and humoral immune responses after vaccination. Here we describe in vivo fluorescence imaging approaches to track APCs and evaluate the local effects of an intradermal (id) vaccination against HIV with different vaccine vectors (an anti-langerin-HIVgag fusion protein in association with an adjuvant, the resiquimod (R-848); or a DNA plasmid combined with electroporation (EP)). Methods: Monoclonal fluorescent labelled anti-HLA-DR antibody (mAb) was injected id to specifically target and track skin APCs in non-human primates (NHP). Two different vaccine vectors were injected id to study the role of cutaneous APC at the site of vaccination. Anti-langerin-HIVgag labelled with near infrared (NIR) fluorochrome (exc. 682nm; em. 710nm) was injected id, with or without R-848. Moreover, animals were immunized with an id auxo-GTU-Luc-EGFP DNA plasmid injection associated with noninvasive EP. Antigen expression was measured by in vivo bioluminescence, and by in vivo fibered confocal fluorescence microscopy (Cellvizio® DualBand, Mauna Kea Technologies) from 24h to 96h post-vaccination. Epidermal APCs were tracked with noninvasive in vivo fibered confocal fluorescence microscopy for up to 96h post-vaccination. In addition, confocal fast laser scanning microscopy was performed on whole skin biopsies to monitor the behavior of fluorescent-labelled APCs in both dermis and epidermis. Three-dimensional image reconstructions and image analysis (cell quantification and tracking) were performed using Volocity software (Perkin Elmer). Results: APCs were visualized by in vivo fluorescence imaging after id injection of fluorescent labelled anti-HLA-DR mAb for at least 48 hours. The NIR fluorescent-labelled anti-langerin-HIVgag vaccine was visualized in the skin and in the draining lymph nodes by in vivo imaging for up to 48 hours post-injection. Confocal videomicroscopy confirmed that the anti-langerin-HIVgag specifically targeted Langerhans cells (LCs). The number of APCs in the epidermis decreased significantly over time, undoubtedly a consequence of migration out of the skin after vaccination with fusion protein and also with DNA plasmid. Ex vivo monitoring confirmed the cell mobilization with a higher mobility and displacement after DNA vaccination with EP compared to the condition without EP. Furthermore, after EP, LCs interact with antigen producing expressing cells and leave the epidermis following directional movement. Conclusions: In vivo fluorescence imaging approaches allowed us to track the skin immune cells noninvasively in their native environment, and to characterize early cellular local events postvaccination. 47 Optical imaging for biodistribution studies in preclinical research. ! Ioanna Théodorou, Nam Nguyen Quang, Benoit Lelandais & Frédéric Ducongé CEA, DSV, I!BM, MIRCen, CNRS UMR-9199, Université Paris Sud & Université Paris-Saclay, 18, route du Panorama - Bât 61 p113 - BP6 - 92265 Fontenay aux roses, France. [email protected] Several types of compounds (antibodies, peptides, aptamers, nanoparticles") are developed to specifically deliver contrast agents or drugs to pathologic cells. However their in vivo use will be dependent of their ADME characteristics (Absorption, Distribution, Metabolism, and Excretion) which remain difficult to predict. To address limitations of in vitro assays, in vivo imaging of vertebrates has emerged as a powerful tool used in virtually all forms of modern biomedical research. Currently, positron emission tomography (PET) and single-photon-emission computed tomography (SPECT) represent well-validated imaging technologies that allow the quantification of radio-labeled probes independently of their localization inside small animals and humans. As an alternative, new imaging systems based on optics have been developed since the 90s and have encountered huge success in preclinical research. Here, several optical imaging technologies will be presented: planar fluorescence imaging, fluorescence diffuse optical tomography (fDOT) and probe based confocal laser endomicroscopy (pCLE). The advantages and limitations of these technologies as well as their complementarity with nuclear imaging will be discussed from experiments to study the biodistribution of chemical compounds(1), biomolecules(2,3), nanoparticles(4-6) and stem cells(7). 1. 2. 3. 4. 5. 6. 7. Garofalakis, A., Dubois, A., Theze, B., Czarny, B., Tavitian, B. and Duconge, F. (2013) Fusion of [(18)F]FDG PET with fluorescence diffuse optical tomography to improve validation of probes and tumor imaging. Mol Imaging Biol, 15, 316-325. Garofalakis, A., Dubois, A., Kuhnast, B., Dupont, D.M., Janssens, I., Mackiewicz, N., Dolle, F., Tavitian, B. and Duconge, F. (2010) In vivo validation of free-space fluorescence tomography using nuclear imaging. Opt Lett, 35, 3024-3026. Cibiel, A., Nguyen Quang, N., Gombert, K., Thézé, B., Garofalakis, A. and Ducongé, F. (2014) From Ugly Duckling to Swan: Unexpected Identification from Cell-SELEX of an Anti-Annexin A2 Aptamer Targeting Tumors. PLoS ONE, 9, e87002. Mackiewicz, N., Gravel, E., Garofalakis, A., Ogier, J., John, J., Dupont, D.M., Gombert, K., Tavitian, B., Doris, E. and Duconge, F. (2011) Tumor-targeted polydiacetylene micelles for in vivo imaging and drug delivery. Small, 7, 27862792. Gravel, E., Ogier, J., Arnauld, T., Mackiewicz, N., Duconge, F. and Doris, E. (2012) Drug delivery and imaging with polydiacetylene micelles. Chemistry, 18, 400-408. Gravel, E., Tanguy, C., Cassette, E., Pons, T., Knittel, F., Bernards, N., Garofalakis, A., Duconge, F., Dubertret, B. and Doris, E. (2013) Compact tridentate ligands for enhanced aqueous stability of quantum dots and in vivo imaging. Chemical Science, 4, 411-417. Lewandowski, D., Barroca, V., Duconge, F., Bayer, J., Van Nhieu, J.T., Pestourie, C., Fouchet, P., Tavitian, B. and Romeo, P.H. (2010) In vivo cellular imaging pinpoints the role of reactive oxygen species in the early steps of adult hematopoietic reconstitution. Blood, 115, 443-452. 48 In vivo imaging of signaling pathways activation in Drosophila’s Mushroom Bodies neurons. Nicolas Gervasi1 and Paul Tchénio2,3 1-INSERM/UPMC UMR-S 839 Institut du Fer à Moulin, Paris, France. 2-CNRS/Université Paris-Sud/ENS-Cachan Laboratoire Aimé Cotton, Orsay, France. 3-CNRS/ESPCI-ParisTech, PSL Research University Laboratoire Plasticité du Cerveau, Paris, France. Drosophila melanogaster can be classically conditioned both aversively and appetitively, by association of an odorant with electric shock or sugar. The mushroom bodies (MBs) was the first brain region to be shown to play a role in Drosophila olfactory learning. In adult Drosophila, the MBs consist of approximately 2500 intrinsic neurons per brain hemisphere, named the Kenyon cells. Signaling pathway activation from synapses to the nucleus are critical for this olfactory memory processing. However, it remains unclear how the different components of this signaling pathways interact dynamically in vivo and, in particular, in which MB neurons compartment they operate. The brain of the fly is a very dense medium and the MBs spread across more than 200 micron deep. We used and developed confocal, two-photon and Hilo microscopy for suitable and noninvasive fluorescence imaging with a high sensitivity and resolution of living animals. We performed real-time measurements of Protein Kinase cAMP depend (PKA) activity using in vivo multiphoton imaging coupled with the genetically encoded FRET probe. Our data show that MB dendrites and axons integrate cAMP/PKA stimulation with different efficiencies and time courses. Furthermore, we demonstrate subcellular compartimentation of PKA in axons. Using confocal spinning-disk and Hilo microscopy with ArcLight voltage sensor, we accessed to the fast membrane potential modification of the cell bodies of the Kenyons cells in response to aversive stimuli. Finally, I will present how spatial mathematical modelling based on the kinetics data obtain by genetically encoded sensors can show unexpected properties of signaling pathways dynamics in neurons. Using advances in vivo microscopy coupled with specific neuronal expression of genetically encoded sensor provide important information on neuronal integration. Modelling approaches can help to decipher the complex signaling spatiotemporal dynamics process that take place during memory formation. 49 Experimental assessment of thermal effects of high power density light stimulation for optogenetics control of deep brain structures. Pain F1, Sisniak I1, Chiang CC1,2, Martin C1, Palfi S3, Chaillet A4, Senova S3 1 Laboratoire Imagerie et Modélisation en Neurobiologie et Cancérologie, Université Paris sud, Université Paris Sacaly, Bat 440, Orsay 2 3 4 National Tsing Hua University, Medical Imaging Physics Lab, Taïwan Hôpital Henri Mondor, Service de Neurochirurgie, Créteil Laboratoire L2S, Supélec, Université Paris Saclay Contact : [email protected] Optogenetics has become ubiquitous in fundamental neuroscience labs as a very powerful tool to unravel brain networks connectivity and cellular mechanisms. Yet, its clinical translation requires a careful assessment of the inocuity of repeated and sustained high power light stimulations. Objectives: We have studied in vivo in anesthetized rats the potential damages and non-physiological effects produced by high power optical neurostimulation in typical optogenetics experiments. 2D surface maps of light distribution and temperature increase were recorded in wild type anesthetized rats brains during 90s light stimulation at 478nm (blue) and 638nm (red) with continuous or pulsed optical stimulations with corresponding power ranging from 100 up to 1200 mW/mm! at the output of an optical fiber. Post mortem maps were recorded in the same animals to assess the cooling effect of blood flow. Post mortem histological analysis were carried out to assess whether high power light stimulations had phototoxic effects or could trigger non physiological functional activation. Temperature increase remains below physiological changes for stimulations up to 400mW/mm! at 40Hz. Histology did not show significant irreversible modifications or damage to the tissues. The spatial profile of light distribution and heat demonstrate as expected a rapid attenuation. On the basis of Monte Carlo the optimal geometry for an optrode dedicated to optical stimulation of deep cortical tissues is proposed. Keywords : optogenetics , red-shifted opsins, neuromodulation, nonhuman primate, heat map, light distribution. Figure1 : Left : distribution of light in the living brain of a mouse. Right Corresponding thermal map. References Boyden, Edward S., Feng Zhang, Ernst Bamberg, Georg Nagel, et Karl Deisseroth. 2005. « Millisecond-Timescale, Genetically Targeted Optical Control of Neural Activity. » Nature Neuroscience 8 (9): 1263̻68. Dai, Ji, Ilker Ozden, Daniel I. Brooks, Fabien Wagner, Travis May, Naubahar S. Agha, Benjamin Brush, David Borton, Arto V. Nurmikko, et David L. Sheinberg. 2015. « Modified Toolbox for Optogenetics in the Nonhuman Primate. » Neurophotonics 2 (3): 031202. Deng, Wei, Ewa M. Goldys, Melissa M. J. Farnham, et Paul M. Pilowsky. 2014. « Optogenetics, the Intersection between Physics and Neuroscience: Light Stimulation of Neurons in Physiological Conditions. » American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 307 (11): R1292̻1302. Detorakis, Georgios Is, Antoine Chaillet, Stéphane Palfi, et Suhan Senova. 2015. « Closed-Loop Stimulation of a Delayed Neural Fields Model of Parkinsonian STN-GPe Network: A Theoretical and Computational Study. » Frontiers in Neuroscience 9: 237. Vandecasteele, Marie, Yann-Suhan Senova, Stéphane Palfi, et Guillaume P. Dugué. 2015. « [Therapeutic potential of optogenetic neuromodulation]. » Médecine Sciences: M/S 31 (4): 404̻16. 50 POSTERS ABSTRACTS 51 52 Rare-earth based nanoparticles for ROS detection and MRI: from cellular signaling to inflammation imaging Mouna Abdesselem1, Rivo Ramodiharilafy1, Pierre-Louis Tharaux2, Thierry Gacoin3, Cedric Bouzigues1 and Antigoni Alexandrou1 1 LOB, INSERM U1182 - CNRS UMR7645, Ecole polytechnique, Palaiseau, France 2 PARCC, Hôpital Européen Georges Pompidou, Paris, France 3 PMC, UMR 7643, Ecole polytechnique, Palaiseau, France [email protected] Medical imaging, using multiple modalities such as MRI or fluorescence is a powerful tool for the diagnosis and the treatment of complex pathologies. Multifunctional probes may be efficiently used to generate simultaneously different signals and are thus crucial for reducing the toxicity and costs of multimodal exams. We developed rare-earth based nanoparticles (Gd0.6Eu0.4VO4) for optical microscopy and magnetic resonance imaging (MRI). These particles combine paramagnetic properties with remarkable luminescence properties: stable narrow emission, no blinking and reactive oxygen (ROS) detection. We successfully used them for the quantitative and time-resolved (1 Hz) study of ROS-related intracellular signaling pathways and contrast enhancing in small animal MRI. We also demonstrated the feasibility of in vivo imaging with this multifunctional probe for the characterization of tissue inflammation with two complementary approaches: (1) functional imaging based on macrophages tracking by MRI in a renal inflammatory disease, and (2) optical molecular imaging of ROS production during acute skin inflammation. The possibility of collecting, at different scales, both molecular and functional information through a single contrast agent (Gd0.6Eu0.4VO4 nanoparticles) opens perspectives for biomedical applications. These nanoparticles are indeed promising candidates for the quantitative diagnosis and monitoring of ROS related disease including inflammation associated pathologies, neurodegerations, immune dysfunctions and cancer. 53 Developing QD-DNA bioconjugates for biological applications a b b a c Anusuya Banerjee , Chloé Grazon , Brice Nadal , Thomas Pons , Yamuna Krishnan and Benoit a Dubertret * a Laboratoire de Physique et d Etude des Matériaux, ESPCI ParisTech, CNRS UMR 8213, Université Pierre et Marie Curie, 10 rue Vauquelin, 75005 Paris, France b Nexdot, 10 rue Vauquelin, 75005 Paris, France c 929E, 57th St, E305, GCIS, Dept of Chemistry, University of Chicago, Chicago 60637, IL, USA Email: [email protected] Abstract: Quantum Dots (QDs) have emerged as novel fluorescent probes for biomedical applications [1]. The photophysical properties of QDs such as broad absorption, narrow emission spectrum, reduced blinking, and enhanced photostability make them advantageous over organic fluorophores. However, for some biological applications, QDs need to be first targeted to specific intracellular locations. It parallel, DNA has been used as a versatile tool for cellular targeting and biosensing [2] and the combination the photophysical properties of QDs and targettability of DNA has yielded fluorescent and targetable nanosensors [3]. In this project, we present the synthesis of QD-DNA bioconjugates and their applications in bioimaging. We present a novel method to conjugate DNA to QDs solubilized in water using an amphiphilic copolymer. We quantify the DNA coupling efficiency, and show that the DNA coupled on the QD remains available for hybridization. We have investigated and optimized the parameters that influence the conjugation reaction and colloidal stability of QD-DNA conjugates.[4]* The coupling strategy we have developed with the QDs has been successfully extended to gold nanoparticles. We also show that using DNA hybridization, proteins can be bound to QDs with controlled stoichiometry. Our work presents a general strategy for binding various biomolecules to different nanoparticles. We will show few examples of these new QD-conjugates for targeted bioimaging in cells. References: [1] Medintz, I.L., Uyeda, H.T., Goldman, E.R. & Mattoussi, H. (2005). Quantum Dot bioconjugates for imaging, labelling and sensing. Nat. Mater. 4, 435-446. [2] Krishnan, Y., Bathe, M. (2012). Designer Nucleic Acids to probe and program the Cell. Trends in Cell Biol. 22, 624-633. [3] Patolsky, F et al. (2003). Lighting-Up the Dynamics of Telomerization and DNA Replication by CdSe ZnS Quantum Dots J. Am. Chem. Soc., 125, 13918 1391. [4]* Banerjee, A., Grazon, C., Nadal, B., Pons, T., Krishnan, Y., and Dubertret, B. (2015). Fast, efficient and stable conjugation of multiple DNA strands on colloidal quantum dots. Bioconjugate Chem. 26, 1582 1589. *This work has been featured as the ACS Editors Choice Manuscript. 54 A fluorogen-based reporter for fluorescence imaging Arnaud Gautier École Normale Supérieure – PSL Research University, Department of Chemistry, UMR 8640 PASTEUR, 24 rue Lhomond, F-75005 Paris, France. [email protected] Selective labeling with fluorogenic probes represents a general strategy for highlighting proteins in living systems. In this approach, a protein of interest (POI) is fused to a protein tag that binds a fluorogenic ligand and activates its fluorescence. As the fluorogenic ligand is non-fluorescent by its own and becomes strongly fluorescent only upon binding its target, unspecific fluorescence background in cells remains minimal even in the presence of an excess of fluorogen, thus ensuring high imaging contrast. Here, we present Y-FAST (Yellow Fluorescence-Activating and absorptionShifting Tag), a small protein tag enabling to fluorescently label proteins through the specific and reversible binding of a cell-permeant and non-toxic fluorogenic ligand HMBR. High labeling selectivity results from a unique fluorogen activation mechanism combining two spectroscopic changes: increase of fluorescence quantum yield and absorption red-shift. Y-FAST was evolved from the 14-kDa photoactive yellow protein (PYP) by directed evolution using yeast display technologies and high throughput fluorescence activated cell sorting (FACS). Y-FAST is photostable, as bright as common fluorescent proteins, and allows for imaging proteins in various subcellular locations and in a large variety of systems, from mammalian cells (including neurons) to microorganisms and zebrafish. YFAST distinguishes itself from other labeling systems because the binding of the fluorogen is not only instantaneous and specific but also highly dynamic: the exchange dynamics is characterized by a binding/unbinding frequency of about 10 Hz enabling in particular (i) to rapidly switch the fluorescence on and off at will by addition or withdrawal of the fluorogen, opening new perspectives for multiplexing imaging, and (ii) to envision various applications for sub-diffraction-limit imaging using Single-molecule Localization Microscopies (SLM) or Super-resolution Optical Fluctuation Imaging (SOFI). References Plamont, M.-A., Billon-Denis, E., Maurin, S., Gauron, C., Pimenta, F. M., Specht, C. G., Shi, J., Querard, J., Pan, B., Rossignol, J., Morellet, N., Volovitch, M., Lescop, E., Chen, Y., Triller, A., Vriz, S., Le Saux, T., Jullien, L.* & Gautier, A.*. A small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo. PNAS in revision. 55 Rapid and reliable diagnosis of Wilson disease using X-ray fluorescence # # Slávka Ka!"áková1 , Cameron M. Kewish , Stéphan Rouzière, Françoise Schmitt, Rodolphe Sobesky, Joël Poupon, Christophe Sandt, Bruno Francou, Andrea Somogyi, Didier Samuel, Emmanuel Jacquemin, Anne Dubart-Kupperschmitt, Tuan Huy Nguyen, Dominique Bazin, Jean-Charles Duclos-Vallée, Catherine Guettier, François Le Naour # These authors contributed equally to this work Affiliation : Univ Paris-Sud XI/ INSERM Unité 1193, Villejuif, F-94800, France Email : [email protected] Wilson’s disease (WD) is a rare autosomal recessive disease due to mutations of the gene encoding the copper-transporter ATP7B. The diagnosis is hampered by the variability of symptoms induced by copper accumulation, the inconstancy of the pathognomonic signs and the absence of reliable diagnostic test. We investigated the potential of X-ray fluorescence (XRF) that allows quantitative analysis of multiple elements. XRF experiments were first performed using synchrotron radiation leading to measure the distribution of copper, iron and zinc on tissue sections at the cellular level. Investigations were further focused on formalinfixed paraffin embedded needle biopsies using a laboratory X-ray source. The intensity of copper related to iron and zinc significantly discriminated WD from other genetic or chronic liver diseases including cholestatic diseases with 97.6% specificity and 100% sensitivity. This study established a definite diagnosis of Wilson’s disease based on XRF that can be easily implemented at the hospital. 56 ! " # $ % &' ( ! ! !)' ) #'* !+! ('!+,+- ! .( / . ! 0 /10 23 ('4 (.$ !5*6' !* 78.! 9:;<<; 9 0 (555 <=()5$9:;<<; 9 0 3$(5$ >=8< ()5$ 9:;<<; 9 0 ) 3 (' 6' !*- 6' ? . !*-( -5$ 3$()3( >;;< :;<<; @9 A #' * $ ! ? * @#$?A 3$( 5$ :78B C #3()$ 5B<;< C . / . (. $ ! 5* C D 9 9 0 5 6 ( 9 0 2 3 (' # ? * ! ) 3 (' @#?)3(A3$(5$>BE:#3()$5B<78(.$ !5*9:;<<; 9 0 3 ' ' *, '/ /0 *F''' *'/' ! '!! * 0G'' ''' '* F! ! F ' F '* 0 " G#G @G! # G' A F!! /' ! ! '!F! ' * - * ' * ''!F! 0B79 !' ! ' * - '! ' ! ! '!F! * !' !* -* !/ 0 F '' G#G , ! ' B< H, - -* F! ' ! ! '*0!'!! ''* $9 ! G#G '/ '0 00 00 00 0 0" 0)' 0+!(0+,+0-0 0.( / .0 07=II7=I:J00 0 0.( /.0 7E=>7E:>0 57 Outsmarting Photobleaching Processes: The Case Study of Y-FAST, a Fluorogenic Protein with a Chromophore with Fast Exchange Dynamics Frederico M. Pimenta,a,b,c Thomas Le Saux a,b,c Ludovic Jullien a,b,c Arnaud Gautier a,b,c École Normale Supérieure ± PSL Research University, Department of Chemistry, 24 rue Lhomond, F-75005 Paris, France. b Sorbonne Universités, UPMC Univ Paris 06, UMR 8640 PASTEUR, F-75005 Paris, France. c CNRS, UMR 8640 PASTEUR, F-75005 Paris, France. a [email protected] Overcoming photobleaching is arguably one of the remaining Holy Grails in Bioimaging. In this combined photophysical and photochemical process, light absorption by a chromophore leads to non-reversible loss of fluorescence through pathways which may radically differ depending on the chromophore at hand. This creates limitations not only in long-term imaging but, more importantly, in the accurate quantification of cellular processes over time.1 The development of genetically-encoded fluorogenic approaches comes with the promise of greatly improving the photostability of genetically-encoded fluorescent proteins )3¶V. The former are based on the semi-rational design of a protein-chromophore duo that, upon interaction, enhance and/or alter the emission and absorption properties of the fluorophore.2 Unlike typical )3¶V, the (externally or endogenously-supplied) chromophore is usually in surplus amount with respect to its protein counterpart, indicating that chromophore recycling in the protein binding pocket could prevent photobleaching. However, studies on the photostability and photochemical processes that occur in these fluorogenic systems lags behind their current development. To understand if fluorogenic systems can be used to reduce photobleaching in fluorescence imaging we made use of the genetically-encoded system YFAST (YellowFluorescence Activating-Shifting Tag), which presents fast dynamics of binding and unbinding of its exogenous chromophore, HMBR (4-hydroxy-3-methylbenzylidene-rhodanine).3 Through a series of photophysical and photochemical studies, which can be extended to other fluorogenic systems, we demonstrate that YFAST exchange dynamics reduce photobleaching to a certain extent. Loss of fluorescence is still observed upon prolonged irradiation, most likely through an electron-driven photooxidation pathway here explored. This study thus provides an experimental starting point for fluorogenic approaches to be probed and improved upon based on the mechanistic information obtained. References 1 Remington, S. James. (2006) Fluorescent proteins: maturation, photochemistry and photophysics, Current Opinion in Structural Biology, 16 (6), 714-721 2 Bruchez, Marcel P. (2015) Dark dyes-bright complexes: fluorogenic protein labelling, Current Opinion in Structural Biology, 27, 18-23 3 Plamont et al. (2015) A small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo, under revision in Proceedings of the National Academy of Sciences 58 Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy Duwé S1, De Zitter E1, Gielen V1, Moeyaert B1, Vandenberg W1, Grotjohann T2, Clays K, Jakobs S2, Van Meervelt L1, Dedecker P1 1 2 Department of Chemistry, KULeuven, Celestijnenlaan 200G, 3001 Heverlee, Belgium Max Planck Institute for Biophysical Chemistry , Am Fassberg 11, 37077 Goettingen, Germany 3 University of Goettingen Medical School , Robert-Koch-Str. 40, 37075 Goettingen, Germany. [email protected] "Smart fluorophores", such as reversibly switchable fluorescent proteins, are crucial for advanced fluorescence imaging. However, only a limited number of such labels is available, and many display reduced biological performance compared to more classical variants. We present the development of robustly photoswitchable variants of enhanced green fluorescent protein (EGFP), named rsGreens, that display up to 30-fold higher fluorescence in E. coli colonies grown at 37 °C and more than 4-fold higher fluorescence when expressed in HEK293T cells compared to their ancestor protein rsEGFP. This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but rather due to enhanced expression levels that allow many more probe molecules to be functional at any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how these can be used for multimodal diffraction-unlimited fluorescence imaging such as pcSOFI and RESOLFT, achieving a spatial resolution of ∼70 nm. By determining the first ever crystal structures of a negative reversibly switchable FP derived from Aequorea victoria in both the "on"- and "off"conformation we were able to confirm the presence of a cis-trans isomerization and provide further insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically encoded "smart fluorophores" can be readily optimized for biological performance and provides a practical strategy for developing maturation- and stability-enhanced photochromic fluorescent proteins. References S. Duwé et al, ACS Nano 2015: 10.1021/acsnano.5b04129. 59 Imaging Drosophila brain by combining cryo-soft X-ray microscopy of thick vitreous sections and cryo-electron microscopy of ultrathin vitreous sections Amélie Leforestiera, Pierre Levitzb, Thomas Preatc, Peter Guttmannd, Laurent J. Michotb, Paul Tchénioc,e a Laboratoire de Physique des Solides, CNRS, Université Paris-Sud, Universiét Paris Saclay, 91405 Orsay Cedex, France b PHENIX Laboratory, UMR 8234, CNRS-Université Pierre et Marie Curie, 75252 Paris, France c Genes and Dynamics of Memory Systems, Brain Plasticity Unit, CNRS, ESPCI-ParisTech, PSL Research University, Paris, France d Helmholtz-Zentrum Berlin, Institute for Soft Matter and Functional Materials, Albert Einstein Str 15, D-12489 Berlin, Germany e Laboratoire Aimé Cotton, CNRS, Université Paris-Sud, Universiét Paris Saclay, 91405 Orsay, France [email protected] Cryo-soft X-ray microscopy is an emerging imaging tool complementary to cryo-electron microscopy, allowing to image frozen hydrated specimens ten to hundred times thicker, but at lower résolution [1, 2] . We describe how the method, so far restricted to isolated small cells or cell monolayers, can be extended to large cells and tissue. We image the synapses of the Kenyon cells in frozen hydrated Drosophila brains combining cryo-soft X-ray microscopy of thick vitreous sections, and cryo-electron microscopy of ultrathin vitreous sections. We show how to obtain frozen hydrated sections of thicknesses ranging from 40 nm up to 2.5 µm, by tuning the sectioning speed of the cryo-microtome. A fluorescent stereo-microscope mounted on the cryo-microtome allowed us to target the regions of interest after GFP-labeling of synapses. Thick cryo-sections were imaged by cryo-soft X-ray microscopy at a resolution better than 25 nm, while ultrathin cryo-sections of the same regions were explored in parallel at the nanometre level of resolution by cryo-electron microscopy [3]. References [1] C.A. Larabell, K.A. Nugent (2010) Imaging cellular architecture with X-rays. Curr. Opin. Struct. Biol., 20 (5) pp. 623–631 [2] W.G. Müller, J.B. Heymann, K. Nagashima, P. Guttmann, S. Werner, et al. (2012) Towards an atlas of mammalian cell ultrastructure by cryo soft X-ray tomography. J. Struct. Biol., 177, pp. 179–192 . [3] A. Leforestier, P. Levitz , T. Preat , P. Guttmann , LJ. Michot, P. Tchénio (2014) Imaging Drosophila brain by combining cryo-soft X-ray microscopy of thick vitreous sections and cryo-electron microscopy of ultrathin vitreous sections. J Struct Biol. 188(2):177-82 60 "CPBM imaging center: confocal, time-resolved and 3D super-resolution fluorescence microscopy setups” 1: S. Lécart, G. Dupuis 2: N. Bourg, C. Cabriel, I. Coto Hernandez, S. Sivankutty2, SY. Bak, S. Lévêque-Fort, 3: E. Fort 1. CPBM/CLUPS/LUMAT, Orsay; 2. Biophotonics group from ISMO, Orsay; 3. Institut Langevin, ESPCI ParisTech, Paris [email protected] Abstract : The biomedical photonic center is an open imaging facility of the Paris-South University. We work together with different development teams (especially the biophotonics group from ISMO, Orsay and the Institut Langevin from ESPCI, Paris) to develop innovative highly-resolved (in space and time) fluorescence microscopes. At the CPBM, users can access these new microscopes and imaging processes and benefit from the presence of a L2 culture laboratory to prepare their sample. Our recent instrumental and methodological developments have been focused on circumventing the diffraction limit in 3D to image nanometric cell’s structure, for precise colocalisation and understanding protein association for exemple. We developed different strategies to enhance lateral and/or axial resolution. - An original approach based on the selective detection of supercritical angle fluorescence (SAF) will be presented. While keeping a classical epifluorescence excitation, we will present how the SAF detection module allows a simultaneous detection of membrane and inner part of the cells, with a high axial discrimination. - We will focus on our new super-resolved microscope: the direct optical nanoscopy with axially localized detection (DONALD) which combines conventional dSTORM with the detection of a fluorescent probe's evanescent light—specifically, its supercritical-angle fluorescence emission. Using this method, we were able to demonstrate isotropic super-resolution imaging of actin and microtubules in mammalian cells. - Finally, the poster will describe our running STED microscope design to be user friendly with a time gated detection and also a better axial resolution using SAF technic. 61 Quantitative analysis of the assembly of the phagocyte NADPH oxidase in live cells using a FLIM-FRET approach C.S.Ziegler, L.Bouchab, S. Dupré, Y. Bousmah, F. Mérola, O. Nüsse, M. Erard, Laboratoire de Chimie Physique, UMR8000, Université Paris Sud [email protected] The phagocyte NADPH oxidase (NOX2) is generating superoxide anions, which are precursors for other reactive oxygen species. NOX2 is a major enzyme of the innate immune response. Accordingly, dysfunctions of the NOX2 are associated with a plethora of diseases and thus detailed knowledge about its regulation is needed. This oxidase is composed of five subunits, the membrane-bound gp91phox and p22phox and the cytosolic p47phox, p67phox, and p40phox. The cytosolic subunits are assumed to be in a ternary complex which translocates together with the small GTPase Rac to the membranous subunits during activation.[1] Our aim is to reveal interactions of the NOX2 subunits in living cells using Förster Resonance Energy Transfer (FRET) between fluorescent protein-tagged subunits. In our study, we took advantage of the improved fluorescent properties of the new cyan fluorescent donor, Aquamarine, which allow a quantitative detection of FRET by fluorescence lifetime imaging microscopy (FLIM).[2] We observed heterodimeric interactions between all cytosolic subunits. Our data allow for the first time an estimation of the fraction of bound subunits and the apparent affinity of these interactions inside living cells. The molecular FRET efficiency allows as well a rough approximation of the distances between the binding partners which give new ideas of the spatial formation of the complex in live cell. B A Figure 1 A. Design of the FRET experiment to probe the interaction between p67phox and p47phox. B. FRET efficiency (Eapp [%]) and fraction of bound proteins (β) for p67phox and p47phox heterodimer. References [1] Dupré-Crochet S; Erard M, Nüsse O (2013) ROS production in phagocytes, why, when and where? Journal of Leukocyte Biology 94:657-670 [2] Erard M, Fredj A, Pasquier H, Betolngar BD, Bousmah Y, Derrien V, Vincent P, Merola F (2013) Minimum set of mutations needed to optimize cyan fluorescent proteins for live cell imaging Molecular Biosystems 9:258-267 62 Fluorescent LbL Self-assembled Films for Bioimaging Applications Yayang Tian1, 2, Rachel Méallet-Renault2, Gilles Clavier1 1 PPSM, CNRS UMR 8531, ENS-Cachan, Cachan, 94235, France 2 ISMO, UMR 8214, Université Paris-Sud, Orsay, 91405, France [email protected] [email protected] [email protected] Abstract: Two oppositely charged BODIPY-based fluorescence polymer chains were alternatively deposited on the substrate by means of electrostatic attraction. The numbers and top layer of fluorescent Layer-by-Layer (LbL) self-assembled films were easy to control by changing the deposition times. In addition, the photophysical and surface properties of LbL films were characterized by steady-state UV-vis and fluorescence spectroscopy, AFM, confocal intensity and lifetime microscopy imaging. After a certain time of growing E. coli bacteria on FPC LbL film surface, fluorescence imaging shown that most of bacteria became fluorescent, while few of them were not fluorescent. Such surfaces possess a potential application in bioimaging. Indeed it seems that E. coli bacteria “eat” or digest the polymer chains from the LbL architecture. 63 64