Publications

2021

• Three-photon microscopy : instrumentation and extension of the observable parameters
  
  • Ferrer Ortas Júlia
  , 2021. Multiphoton microscopy is the reference technique for fluorescence imaging of intact tissues. Three-photon (3P) microscopy was recently demonstrated as providing superior contrast than two-photon (2P) imaging at large depths in densely labelled scattering media. This advantage is due to the superior confinement of the 3P excitation and the reduced scattering experienced at longer wavelengths. Although this promising approach has made it possible to reach imaging depths beyond one millimetre in the mouse brain, it is not yet fully mature technologically and lacks efficient multicolor imaging modalities. In this work, we explore the extension of the number of signals and parameters that can be used in deep-tissue 3P microscopy. In a first chapter, we introduce the concepts underlying 3P microscopy and we analyse the parameters limiting imaging depth. In a second chapter, we describe the construction and optimization of a 3P microscope based on a new generation of multibeam optical parametric amplifier (OPA) source adapted for 3P excitation in the short-wavelength infrared range. We pay particular attention to source stability, dispersion compensation, and beam combination in the microscope. In the two remaining chapters, we present the results of two projects aiming at extending the signals usable in a 3P system. In the third chapter, we explore the 3P excitation efficiency of several blue fluorescent proteins (FPs) with 1030 nm light, in order to complement previous studies reporting 3P excitation of green and red FPs at 1300 and 1700 nm, respectively. We identify efficiently excited blue FPs producing signal levels comparable to green and red proteins. These encouraging results open perspectives for multicolor 3P microscopy. In the fourth chapter, we explore the possibility of multicolor coherent imaging based on third-order sum-frequency generation. We show that this new contrast modality provides label-free contrast highlighting blood vessels with a possibility of functional imaging, and that it can be directly integrated on a 3P microscope. Overall, the work described in this thesis extends the number of signals that can be probed in deep-tissue microscopy.
• hnRNPA1/UP1 Unfolds KRAS G-Quadruplexes and Feeds a Regulatory Axis Controlling Gene Expression
  
  • Ferino Annalisa
  • Marquevielle Julien
  • Choudhary Himanshi
  • Cinque Giorgio
  • Robert Coralie
  • Bourdoncle Anne
  • Picco Raffaella
  • Mergny Jean-Louis
  • Salgado Gilmar
  • Xodo Luigi
  ACS Omega, ACS Publications, 2021, 6 (49), pp.34092-34106. (10.1021/acsomega.1c05538)
  DOI : 10.1021/acsomega.1c05538
• Exploring Sequence Space to Design Controllable G-Quadruplex Topology Switches
  
  • Chen Jielin
  • Cheng Mingpan
  • Stadlbauer Petr
  • Šponer Jiří
  • Mergny Jean-Louis
  • Ju Huangxian
  • Zhou Jun
  CCS Chemistry, Chinese Chemical Society, 2021, pp.3232 - 3246. As nonclassical nucleic acid structures, G-quadruplexes (G4s) not only play important roles in gene regulation and stability maintenance, but are also widely used in nanotechnology. Structural diversity is one of the main factors explaining the popularity of G4s, but a comprehensive and integrated study of different factors determining G4 structural versatility is currently lacking. Herein, starting from a common G4 sequence, (G 3 T) 3 G 3 , as the parent chain, and then taking advantage of G4 versatility, we present a variety of strategies to control G4 structure, based on the regulation of loop length and flanking sequences, cation (type and concentration), and molecular crowding. These strategies allow us to convert the G4 topology from parallel to hybrid, to antiparallel, and then back to parallel. Such structural diversity reveals the coding regulation ability of G4 structures, with potential applications in nanotechnology. (10.31635/ccschem.021.202101357)
  DOI : 10.31635/ccschem.021.202101357
• Analysis of cell movement coordination mechanisms in the axial mesoderm during gastrulation of the zebafish Danio rerio.
  
  • Boutillon Arthur
  , 2021. Embryonic development, wound healing and some cancer metastases involve migration of groups of cells in which guidance of each cell depends on interactions with its neighbours, a process deemed collective cell migration. In addition, migration of these groups of cells often needs to be coordinated with other cell movements taking place concomitantly. This PhD studied how cell-cell interactions can guide migration and how different cell populations can coordinate their movement during early embryonic development of the Teleost fish Danio rerio, also known as zebrafish. During zebrafish gastrulation takes place the extension of the axial mesoderm, a dorsal structure composed of a dense population of mesodermal cell. This extension is led by the polster that migrates towards the animal pole of the embryo, followed by the posterior axial mesoderm that is undergoing convergence and extension. We used polster and its interaction with posterior axial mesoderm to study collective cell migration and coordination of tissue movement respectively. In order to investigate how polster cells are guided towards the animal pole, we developed precise deep laser ablations, advanced transplantation techniques, and used functional genetic approaches as well as numerical simulations. This work established that the directional information guiding polster cells is provided by the anteriorward migration of the following cells. Polster cells detect, through cell-cell contact, a mechanical signal applied by migrating following cells via the E-Cadherin/α-Catenin/Vinculin mechanotransduction pathway. This signal ensures proper alignment of cell migration with followers and propagates from cell to cell over the whole tissue. Such guidance of migrating cells by followers allows long-range coordination of cell movements without the need for external chemoattraction. Furthermore, this behaviour ensures developmental robustness as it guarantees continuous contact between polster and posterior axial mesoderm during gastrulation. This PhD thus contributes to better understand how gastrulation movements are coordinated in zebrafish and identify a new mechanism by which collective cell migration is achieved.
• The beginning and the end: flanking nucleotides induce a parallel G-quadruplex topology
  
  • Chen Jielin
  • Cheng Mingpan
  • Salgado Gilmar F
  • Stadlbauer Petr
  • Zhang Xiaobo
  • Amrane Samir
  • Guédin Aurore
  • He Fangni
  • Šponer Jiří
  • Ju Huangxian
  • Mergny Jean-Louis
  • Zhou Jun
  Nucleic Acids Research, Oxford University Press, 2021, 49 (16), pp.9548-9559. Abstract Genomic sequences susceptible to form G-quadruplexes (G4s) are always flanked by other nucleotides, but G4 formation in vitro is generally studied with short synthetic DNA or RNA oligonucleotides, for which bases adjacent to the G4 core are often omitted. Herein, we systematically studied the effects of flanking nucleotides on structural polymorphism of 371 different oligodeoxynucleotides that adopt intramolecular G4 structures. We found out that the addition of nucleotides favors the formation of a parallel fold, defined as the ‘flanking effect’ in this work. This ‘flanking effect’ was more pronounced when nucleotides were added at the 5′-end, and depended on loop arrangement. NMR experiments and molecular dynamics simulations revealed that flanking sequences at the 5′-end abolish a strong syn-specific hydrogen bond commonly found in non-parallel conformations, thus favoring a parallel topology. These analyses pave a new way for more accurate prediction of DNA G4 folding in a physiological context. (10.1093/nar/gkab681)
  DOI : 10.1093/nar/gkab681
• Phase-modulated rapid-scanning fluorescence-detected two-dimensional electronic spectroscopy
  
  • Agathangelou Damianos
  • Javed Ariba
  • Sessa Francesco
  • Solinas Xavier
  • Joffre Manuel
  • Ogilvie Jennifer
  The Journal of Chemical Physics, American Institute of Physics, 2021, 155 (9), pp.094201. (10.1063/5.0057649)
  DOI : 10.1063/5.0057649
• Fast in vivo multiphoton microscopy using optimized light-sheet illumination
  
  • Maioli Vincent
  • Boniface Antoine
  • Mahou Pierre
  • Ferrer Ortas Júlia
  • Abdeladim Lamiae
  • Beaurepaire Emmanuel
  • Supatto Willy
  Proceedings of SPIE, the International Society for Optical Engineering, SPIE, The International Society for Optical Engineering, 2021, 11648. Light-sheet fluorescence microscopy is a method of choice for multiscale live imaging. Indeed, its orthogonal geometry results in high acquisition speed, large field-of-view and low photodamage. Its combination with multiphoton excited fluorescence improves its imaging depth in biological tissues. However, it appears femtosecond laser sources commonly used in multiphoton microscopy at an 80 MHz repetition rate may not be optimized to take full advantage of light-sheet illumination during live imaging. Hence, we investigated the nature of induced photodamage in multiphoton light-sheet microscopy and the influence of laser parameters on the signal-to-photodamage ratio. To this end, we used zebrafish embryonic heart beat rate and fluorophore photobleaching as sensitive reporters of photoperturbations. We characterized linear and nonlinear disruptions depending on laser parameters such as laser mean power, pulse frequency or wavelength, and determine their order and relative impact. We found an optimal pulse frequency of ~10 MHz for imaging mCherry labeled beating hearts at 1030 nm excitation wavelength. Thus, we achieved high-speed imaging without inducing additional linear heating or reaching nonlinear photodamage compared to previous implementation. We reach an order-ofmagnitude enhancement in two-photon excited fluorescence signal by optimizing the laser pulse frequency while maintaining low both the laser average power and its peak irradiance. It is possible to reach even larger enhancement of 3photon excited fluorescence using such laser parameters. More generally, using low laser pulse frequency in multiphoton light-sheet microscopy results in a drastic improvement in signal level without compromising live sample, which opens new opportunities for fast in vivo imaging. (10.1117/12.2578703)
  DOI : 10.1117/12.2578703
• Multiscale conformational dynamics probed by time‐resolved circular dichroism from seconds to picoseconds
  
  • Hache François
  • Changenet Pascale
  Chirality, Wiley, 2021, 33 (11), pp.747-757. Time-resolved circular dichroism has been developed for a few decades to investigate rapid conformational changes in (bio)molecules. In our group, we have come up with several experimental set-ups allowing us to study pico-nanosecond local phenomena in molecular systems as well as much slower effects occurring in proteins and DNA in the folding processes. After an overview of the worldwide realizations in this domain, we present emblematic experiments that we have carried out, spanning time domain from picoseconds to seconds. (10.1002/chir.23359)
  DOI : 10.1002/chir.23359
• Novel G-quadruplex prone sequences emerge in the complete assembly of the human X chromosome
  
  • Bohálová Natália
  • Mergny Jean-Louis
  • Brázda Václav
  Biochimie, Elsevier, 2021, 191, pp.87-90. (10.1016/j.biochi.2021.09.004)
  DOI : 10.1016/j.biochi.2021.09.004
• Luminescent lanthanide nanoparticle-based imaging enables ultra-sensitive, quantitative and multiplexed <i>in vitro</i> lateral flow immunoassays
  
  • Mousseau F.
  • Féraudet-Tarisse C.
  • Simon S.
  • Gacoin T.
  • Alexandrou A.
  • Bouzigues C I
  Nanoscale, Royal Society of Chemistry, 2021, 13 (35), pp.14814 - 14824. Lateral Flow Assays (LFAs) have been extensively used on-site to rapidly detect analytes, possibly in complex media. However, standard gold nanoparticle-based LFAs lack sensitivity and cannot provide quantitative measurements with high accuracy. To overcome these limitations, we image lanthanidedoped nanoparticles (YVO 4 :Eu 40%) as new luminescent LFA probes, using a homemade reader coupled to a smartphone and propose an original image analysis allowing strip quantification regardless of the shape of the test band signal. This method is demonstrated for the detection of staphylococcal enterotoxins SEA, SEG, SEH, and SEI. A systematic comparison to state-of-the-art gold nanoparticle-based LFA revealed an analytical sensitivity enhancement of at least one order of magnitude. We furthermore provided measurements of absolute toxin concentration over two orders of magnitude and demonstrated simultaneous quantitative detection of multiple toxins with unaltered sensitivity. In particular, we reached concentrations 100 times lower than the ones reported in the literature for on-site multiplexed LFA targeting enterotoxins. Altogether, these results highlight that our luminescent nanoparticle-based method provides a powerful and versatile on-site framework to detect multiple biomolecules with sensitivity approaching that obtained by ELISA. This paves the way to a change of paradigm in the field of analytical immunoassays by providing fast in situ quantitative high sensitivity detection of biomarkers or pathogens. (10.1039/d1nr03358a)
  DOI : 10.1039/d1nr03358a
• High precision dual-modulation differential terahertz ATR sensor for liquid measurements
  
  • Zheng Xiujun
  • Gevart Thomas
  • Gallot Guilhem
  Optics Letters, Optical Society of America - OSA Publishing, 2021, 46 (16), pp.4045-4048. We describe a highly sensitive and stable quantum-cascade laser-based attenuated total reflection (ATR) terahertz sensor for the detection of very low concentration solutions, using a dual-modulation differential approach and ATR geometry. This sensor offers a very high dynamic range and a long-term stability of 40 dB, which extends the potential of terahertz radiation for the analysis of liquid and biological samples. The performance is illustrated by measurements on standard solutions of ions, sugars, and proteins, for concentrations down to 1 M.
• Nanoaggregate-forming lipid-conjugated AS1411 aptamer as a promising tumor-targeted delivery system of anticancer agents in vitro
  
  • Carvalho Josué
  • Lopes-Nunes Jéssica
  • Vialet Brune
  • Rosado Tiago
  • Gallardo Eugenia
  • Vale João
  • Eloy Catarina
  • Ferreira Sofia
  • Palmeira-De-Oliveira Rita
  • Campello Maria Paula Cabral
  • Paulo António
  • Barthélémy Philippe
  • Mergny Jean-Louis
  • Salgado Gilmar
  • Queiroz João
  • Ellington Andrew
  • Cruz Carla
  Nanomedicine: Nanotechnology, Biology and Medicine, Elsevier, 2021, 36 (13), pp.102429. Abstract The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds. (10.1016/j.nano.2021.102429)
  DOI : 10.1016/j.nano.2021.102429
• Probing living cells permeabilization dynamics by terahertz attenuated total reflection
  
  • Gallot Guilhem
  Proceedings of SPIE, the International Society for Optical Engineering, SPIE, The International Society for Optical Engineering, 2021. Using attenuated total reflection (ATR) in the terahertz domain, we demonstrate non-invasive, non-staining real time measurements of cytoplasm leakage during permeabilization of live epithelial cells by saponin detergent and after electropermeabilization. The origin of the contrast observed between cells and culture medium is addressed by both experimental and theoretical approaches, and gives access to permeabilization dynamics of live cells in real time. We show that terahertz modalities are more sensitive than fluorescence microscopy which is the reference optical technique for electropermeabilization. We propose analytical models for the influx and efflux of non-permeant molecules through permeabilized cell membranes. (10.1117/12.2596166)
  DOI : 10.1117/12.2596166
• The role of G-Quadruplex DNA in Paraspeckle formation in cancer
  
  • Bhatt Uditi
  • Kretzmann Amy
  • Guédin Aurore
  • Ou Arnold
  • Kobelke Simon
  • Bond Charles
  • Evans Cameron
  • Hurley Laurence
  • Mergny Jean-Louis
  • Iyer K. Swaminathan
  • Fox Archa
  • Smith Nicole
  Biochimie, Elsevier, 2021. (10.1016/j.biochi.2021.07.008)
  DOI : 10.1016/j.biochi.2021.07.008
• Gene resolution Hi-C reveals Euryarchaeal genomes are folded into SMC-dependent loops and domains, but lack transcription-mediated compartmentalization
  
  • Cockram Charlotte
  • Thierry Agnès
  • Gorlas A.
  • Lestini Roxane
  • Koszul Romain
  , 2021.
• Towards Objective Corneal Transparency Assessment in the Clinical Setting: Correction of Acquisition Artifacts in Spectral-domain OCT Images
  
  • Vilbert Maëlle
  • Bocheux Romain
  • Lama Hugo
  • Georgeon Cristina
  • Borderie Vincent
  • Pernot Pascal
  • Irsch Kristina
  • Plamann Karsten
  , 2021. (10.1364/ISA.2021.ITu1D.5)
  DOI : 10.1364/ISA.2021.ITu1D.5
• Noninvasive quantitative assessment of collagen degradation in parchments by polarization-resolved SHG microscopy
  
  • Schmeltz Margaux
  • Robinet Laurianne
  • Heu-Thao Sylvie
  • Sintès Jean-Marc
  • Teulon Claire
  • Ducourthial Guillaume
  • Mahou Pierre
  • Schanne-Klein Marie-Claire
  • Latour Gaël
  Science Advances, American Association for the Advancement of Science (AAAS), 2021, 7 (29), pp.eabg1090. Nondestructive and noninvasive investigation techniques are highly sought-after to establish the degradation state of historical parchments, which is up to now assessed by thermal techniques that are invasive and destructive. We show that advanced nonlinear optical (NLO) microscopy enables quantitative in situ mapping of parchment degradation at the micrometer scale. We introduce two parameters that are sensitive to different degradation stages: the ratio of two-photon excited fluorescence to second harmonic generation (SHG) signals probes severe degradation, while the anisotropy parameter extracted from polarization-resolved SHG measurements is sensitive to early degradation. This approach is first validated by comparing NLO quantitative parameters to thermal measurements on artificially altered contemporary parchments. We then analyze invaluable parchments from the Middle Ages and show that we can map their conservation state and assess the impact of a restoration process. NLO quantitative microscopy should therefore help to identify parchments most at risk and optimize restoration methods. (10.1126/sciadv.abg1090)
  DOI : 10.1126/sciadv.abg1090
• Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula
  
  • Boutillon Arthur
  • Escot Sophie
  • David Nicolas B.
  Journal of visualized experiments : JoVE, JoVE, 2021 (173). Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly coordinated, and accumulating evidence suggests this is achieved, at least in part, through mechanical interactions. Testing this in the embryo requires direct physical perturbations. Laser ablations are an increasingly used option that allows relieving mechanical constraints or physically isolating two cell populations from each other. However, many ablations are performed with an ultraviolet (UV) laser, which offers limited axial resolution and tissue penetration. A method is described here to ablate deep, significant, and spatially well-defined volumes using a two-photon microscope. Ablations are demonstrated in a transgenic zebrafish line expressing the green fluorescent protein in the axial mesendoderm and used to sever the axial mesendoderm without affecting the overlying ectoderm or the underlying yolk cell. Cell behavior is monitored by live imaging before and after the ablation. The ablation protocol can be used at different developmental stages, on any cell type or tissue, at scales ranging from a few microns to more than a hundred microns. (10.3791/62815)
  DOI : 10.3791/62815
• Caractérisation objective de la transparence cornéenne par analyse d’images d’OCT clinique
  
  • Vilbert Maëlle
  • Bocheux Romain
  • Lama Hugo
  • Georgeon Cristina
  • Borderie Vincent
  • Pernot Pascal
  • Irsch Kristina
  • Plamann Karsten
  , 2021.
• Folding Dynamics of DNA G-Quadruplexes Probed by Millisecond Temperature Jump Circular Dichroism
  
  • Laouer K
  • Schmid M
  • Wien F
  • Changenet Pascale
  • Hache François
  Journal of Physical Chemistry B, American Chemical Society, 2021. (10.1021/acs.jpcb.1c01993)
  DOI : 10.1021/acs.jpcb.1c01993
• Modeling nonlinear microscopy near index-mismatched interfaces
  
  • Morizet Josephine
  • Sartorello Giovanni
  • Dray Nicolas
  • Stringari Chiara
  • Beaurepaire Emmanuel
  • Olivier Nicolas
  Optica, Optical Society of America - OSA Publishing, 2021, 8 (7), pp.944. Nonlinear microscopy is widely used to characterize thick, optically heterogeneous biological samples. While quantitative image analysis requires accurately describing the contrast mechanisms at play, the majority of established numerical models neglect the influence of field distortion caused by sample heterogeneity near focus. In this work, we show experimentally and numerically that finite-difference time-domain (FDTD) methods are applicable to model focused fields interactions in the presence of heterogeneities, typical of nonlinear microscopy. We analyze the ubiquitous geometry of a vertical interface between index-mismatched media (water, glass, and lipids) and consider the cases of two-photon-excited fluorescence (2PEF), third-harmonic generation (THG) and polarized THG contrasts. We show that FDTD simulations can accurately reproduce experimental images obtained on model samples and in live adult zebrafish, in contrast with previous models neglecting field distortions caused by index mismatch at the micrometer scale. Accounting for these effects appears to be particularly critical when interpreting coherent and polarization-resolved microscopy data. (10.1364/OPTICA.421257)
  DOI : 10.1364/OPTICA.421257
• Electric Field Measurements in Plasmas with E-FISH Using Focused Gaussian Beams.
  
  • Chng Tat Loon
  • Starikovskaia Svetlana M
  • Schanne-Klein Marie-Claire
  , 2021. (10.1109/CLEO/Europe-EQEC52157.2021.9542435)
  DOI : 10.1109/CLEO/Europe-EQEC52157.2021.9542435
• Objective Assessment of Corneal Transparency in the Clinical Setting: Correction of Acquisition Arti-facts in SD-OCT Images and Application to Normal Corneas
  
  • Vilbert Maëlle
  • Bocheux Romain
  • Lama Hugo
  • Georgeon Cristina
  • Borderie Vincent
  • Pernot Pascal
  • Irsch Kristina
  • Plamann Karsten
  , 2021, 11924. (10.1117/12.2616041)
  DOI : 10.1117/12.2616041
• Quantitative structural imaging of keratoconic corneas using polarization-resolved SHG microscopy
  
  • Raoux Clothilde
  • Schmeltz Margaux
  • Bied Marion
  • Alnawaiseh Maged
  • Hansen Uwe
  • Latour Gaël
  • Schanne-Klein Marie-Claire
  Biomedical optics express, Optical Society of America - OSA Publishing, 2021, 12. The human cornea is mainly composed of collagen fibrils aligned together within stacked lamellae. This lamellar structure can be affected in pathologies such as keratoconus, which is characterized by progressive corneal thinning and local steepening. In this study, we use polarization-resolved second harmonic generation (P-SHG) microscopy to characterize 8 control and 6 keratoconic human corneas. Automated processing of P-SHG images of transverse sections provides the collagen orientation in every pixel with sub-micrometer resolution. Series of P-SHG images recorded in the most anterior region of the stroma evidence sutural lamellae inclined at 22°± 5°to the corneal surface, but show no significant difference between control and keratoconic corneas. In contrast, series of P-SHG images acquired along the full thickness of the stroma show a loss of order in the lamellar structure of keratoconic corneas, in agreement with their defective mechanical properties. This structural difference is analyzed quantitatively by computing the entropy and the orientation index of the collagen orientation distribution and significant differences are obtained along the full thickness of the stroma. This study shows that P-SHG is an effective tool for automatic quantitative analysis of structural defects of human corneas and should be applied to other collagen-rich tissues. (10.1364/boe.426145)
  DOI : 10.1364/boe.426145
• Additive CHARMM36 Force Field for Nonstandard Amino Acids
  
  • Croitoru Anastasia
  • Park Sang-Jun
  • Kumar Anmol
  • Lee Jumin
  • Im Wonpil
  • Mackerell Alexander
  • Aleksandrov Alexey
  Journal of Chemical Theory and Computation, American Chemical Society, 2021, 17 (6), pp.3554-3570. (10.1021/acs.jctc.1c00254)
  DOI : 10.1021/acs.jctc.1c00254