Nature Photonics 2015

Direct optical nanoscopy with axially localized detection

N. Bourg, C. Mayet, G. Dupuis, T. Barroca, P. Bon, S. Lécart, E. Fort & S. Lévêque-Fort
Article reference
Evanescent light excitation is widely used in super-resolution fluorescence microscopy to confine light and reduce background noise. Here, we propose a method of exploiting evanescent light in the context of emission. When a fluorophore is located in close proximity to a medium with a higher refractive index, its near-field component is converted into light that propagates beyond the critical angle. This so-called supercritical-angle fluorescence can be captured using a high-numerical-aperture objective and used to determine the axial position of the fluorophore with nanometre precision. We introduce a new technique for three-dimensional nanoscopy that combines direct stochastic optical reconstruction microscopy (dSTORM) with dedicated detection of supercritical-angle fluorescence emission. We demonstrate that our approach of direct optical nanoscopy with axially localized detection (DONALD) typically yields an isotropic three-dimensional localization precision of 20 nm within an axial range of ∼150 nm above the coverslip.
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Optics Letters 2017

Aberration-accounting calibration for 3D single-molecule localization microscopy

Clément Cabriel, Nicolas Bourg, Guillaume Dupuis, and Sandrine Lévêque-Fort
Article reference
We propose a straightforward sample-based technique to calibrate the axial detection in 3D single-molecule localization microscopy. Using microspheres coated with fluorescent molecules, the calibration curves of point spread function-shaping or intensity-based measurements can be obtained over the imaging depth range. This experimental method takes into account the effect of the spherical aberration without requiring computational correction. We demonstrate its efficiency for astigmatic imaging in a 1.2 μm range above the coverslip.
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BioRxiv 2018

Combining 3D single molecule localization strategies for reproducible bioimaging

Clément Cabriel, Nicolas Bourg, Pierre Jouchet, Guillaume Dupuis, Christophe Leterrier, Aurélie Baron, Marie-Ange Badet-Denisot, Boris Vauzeilles, Emmanuel Fort, Sandrine Lévêque-Fort
Article reference
We developed a 3D localization-based super-resolution technique providing an almost isotropic 3D resolution over a 1 μm range with precisions down to 15 nm. The axial localization is performed through a combination of point spread function (PSF) shaping and supercritical angle fluorescence (SAF), which yields absolute axial information. Using a dual-view scheme, the axial detection is decoupled from the lateral detection and optimized independently. This method can be readily implemented on most homemade PSF shaping setups and provides drift-free, tilt-insensitive and achromatic results. Its insensitivity to these unavoidable experimental biases is especially adapted for multicolor 3D super-resolution microscopy, as we demonstrate by imaging cell cytoskeleton, living bacteria membranes and axon periodic submembrane scaffolds. We further illustrate the interest of the technique for biological multicolor imaging over a several μm range by direct merging of multiple acquisitions at different depths.
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ACS Nano 2017

Podosome Force Generation Machinery: A Local Balance between Protrusion at the Core and Traction at the Ring

Anaïs Bouissou, Amsha Proag, Nicolas Bourg, Karine Pingris, Clément Cabriel, Stéphanie Balor, Thomas Mangeat, Christophe Thibault, Christophe Vieu, Guillaume Dupuis, Emmanuel Fort, Sandrine Lévêque-Fort, Isabelle Maridonneau-Parini, and Renaud Poincloux
Article reference 10.1021/acsnano.7b00622
Determining how cells generate and transduce mechanical forces at the nanoscale is a major technical challenge for the understanding of numerous physiological and pathological processes. Podosomes are submicrometer cell structures with a columnar F-actin core surrounded by a ring of adhesion proteins, which possess the singular ability to protrude into and probe the extracellular matrix. Using protrusion force microscopy, we have previously shown that single podosomes produce local nanoscale protrusions on the extracellular environment. However, how cellular forces are distributed to allow this protruding mechanism is still unknown. To investigate the molecular machinery of protrusion force generation, we performed mechanical simulations and developed quantitative image analyses of nanoscale architectural and mechanical measurements. First, in silico modeling showed that the deformations of the substrate made by podosomes require protrusion forces to be balanced by local traction forces at the immediate core periphery where the adhesion ring is located. Second, we showed that three-ring proteins are required for actin polymerization and protrusion force generation. Third, using DONALD, a 3D nanoscopy technique that provides 20 nm isotropic localization precision, we related force generation to the molecular extension of talin within the podosome ring, which requires vinculin and paxillin, indicating that the ring sustains mechanical tension. Our work demonstrates that the ring is a site of tension, balancing protrusion at the core. This local coupling of opposing forces forms the basis of protrusion and reveals the podosome as a nanoscale autonomous force generator.
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Antimicrobial Agents and Chemotherapy 2018

Failure of daptomycin to kill Staphylococcus aureus: impact of bacterial membrane fatty acid composition

Rym Boudjemaa, Clément Cabriel, Florence Dubois-Brissonnet, Nicolas Bourg, Guillaume Dupuis, Alexandra Gruss, Sandrine Lévêque-Fort, Romain Briandet, Marie-Pierre Fontaine-Aupart, Karine Steenkeste
Article reference
Daptomycin is a last-resort membrane-targeting lipopeptide approved for the treatment of drug-resistant staphylococcal infections such as bacteraemia and implant-related infections. Although cases of resistance to this antibiotic are rare, increasing clinical, in vitro and animal studies report treatment failure, notably against Staphylococcus aureus. The aim of this study was to identify the features of daptomycin and its target bacteria that lead to daptomycin treatment failure. We show that daptomycin bactericidal activity against S. aureus to significantly varies with the growth state and strain according to membrane fatty acid composition. Daptomycin efficacy as an antibiotic relies on its ability to oligomerize within membranes and form pores that subsequently lead to cell death. Our findings ascertain that daptomycin interacts with tolerant bacteria and reaches its membrane target, regardless of its functionality. However, the final step of pore formation does not occur in cells that are daptomycin-tolerant, strongly suggesting its incapacity to oligomerize. Importantly, membrane fatty acid contents correlated with poor daptomycin bactericidal activity, which could be manipulated by fatty acid addition. In conclusion, daptomycin failure to treat S. aureus is not due to a lack of antibiotic-target interaction but is driven by its capacity to form pores, which depends on membrane composition. Manipulation of membrane fluidity to restore S. aureus daptomycin bactericidal activity in vivocould open the way to novel strategies of antibiotic treatment.
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Scientific Reports 2017

Nuclear pore complex plasticity during developmental process as revealed by super-resolution microscopy

Julien Sellés, May Penrad-Mobayed, Cyndélia Guillaume, Alica Fuger, Loïc Auvray, Orestis Faklaris & Fabien Montel
Article reference
Nuclear Pore Complex (NPC) is of paramount importance for cellular processes since it is the unique gateway for molecular exchange through the nucleus. Unraveling the modifications of the NPC structure in response to physiological cues, also called nuclear pore plasticity, is key to the understanding of the selectivity of this molecular machinery. As a step towards this goal, we use the optical super-resolution microscopy method called direct Stochastic Optical Reconstruction Microscopy (dSTORM), to analyze oocyte development impact on the internal structure and large-scale organization of the NPC. Staining of the FG-Nups proteins and the gp210 proteins allowed us to pinpoint a decrease of the global diameter by measuring the mean diameter of the central channel and the luminal ring of the NPC via autocorrelation image processing. Moreover, by using an angular and radial density function we show that development of the Xenopus laevis oocyte is correlated with a progressive decrease of the density of NPC and an ordering on a square lattice.
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Nature Methods 2018

Quantitative mapping and minimization of super-resolution optical imaging artifacts

Siân Culley, David Albrecht, Caron Jacobs, Pedro Matos Pereira, Christophe Leterrier, Jason Mercer & Ricardo Henriques
Article reference
Super-resolution microscopy depends on steps that can contribute to the formation of image artifacts, leading to misinterpretation of biological information. We present NanoJ-SQUIRREL, an ImageJ-based analytical approach that provides quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same acquisition volume, this approach generates a quantitative map of super-resolution defects and can guide researchers in optimizing imaging parameters.
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Neuron 2017

Localized Myosin II Activity Regulates Assembly and Plasticity of the Axon Initial Segment

Stephen L. Berger, Alejandra Leo-Macias, Stephanie Yuen, Latika Khatri, Sylvia Pfennig, Yanqing Zhang, Esperanza Agullo-Pascual, Ghislaine Caillol, Min-Sheng Zhu, Eli Rothenberg, Carmen V. Melendez-Vasquez, Mario Delmar, Christophe Leterrier, and James L. Salzer
Article reference
The axon initial segment (AIS) is the site of action potential generation and a locus of activity-dependent homeostatic plasticity. A multimeric complex of sodium channels, linked via a cytoskeletal scaffold of ankyrin G and beta IV spectrin to submembranous actin rings, mediates these functions. The mechanisms that specify the AIS complex to the proximal axon and underlie its plasticity remain poorly understood. Here we show phosphorylated myosin light chain (pMLC), an activator of contractile myosin II, is highly enriched in the assembling and mature AIS, where it associates with actin rings. MLC phosphorylation and myosin II contractile activity are required for AIS assembly, and they regulate the distribution of AIS components along the axon. pMLC is rapidly lost during depolarization, destabilizing actin and thereby providing a mechanism for activity-dependent structural plasticity of the AIS. Together, these results identify pMLC/myosin II activity as a common link between AIS assembly and plasticity.
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Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration 2018

Primary fibroblasts derived from sporadic amyotrophic lateral sclerosis patients do not show ALS cytological lesions

Philippe Codron, Julien Cassereau, Patrick Vourc’h, Charlotte Veyrat-Durebex, Hélène Blasco, Selma Kane, Vincent Procaccio, Franck Letournel, Christophe Verny, Guy Lenaers, Pascal Reynier & Arnaud Chevrollier
Article reference
Objective: Sporadic amyotrophic lateral sclerosis (sALS) is a fatal neurodegenerative disorder affecting upper and lower motor neurons. In view of the heterogeneous presentation of the disease, one of the current challenges is to identify diagnostic and prognostic markers in order to diagnose sALS at early stage and to stratify patients in trials. In this study, we sought to identify cytological hallmarks of sALS in patient-derived fibroblasts with the aim of finding new clinical-related markers of the disease. Methods: Primary fibroblasts were prospectively collected from patients affected with classical, rapid, and slow forms of sALS. TDP-43 localization, cytoskeleton distribution, mitochondrial network architecture, and stress granules formation were analyzed using 3D fluorescence microscopy and new super-resolution imaging. Intracellular reactive oxygen species (ROS) production was assessed using live imaging techniques. Results: Six sALS patients (two classical, two rapid, and two slow) and four age-matched controls were included. No difference in fibroblasts cell growth, morphology, and distribution was noticed. The analysis of TDP-43 did not reveal any mislocalization nor aggregation of the protein. The cytoskeleton was harmoniously distributed among the cells, without any inclusion noticed, and no difference was observed regarding the mitochondrial network architecture. Basal ROS production and response to induced stress were similar among patient and control fibroblasts. Conclusions: ALS cytological lesions are absent in patient-derived fibroblasts and thus cannot contribute as diagnostic nor prognostic markers of the disease.
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ArXiv 2018

Multicolor localization microscopy by deep learning

Eran Hershko, Lucien E. Weiss, Tomer Michaeli, Yoav Shechtman
Article reference arXiv:1807.01637
Deep learning has become an extremely effective tool for image classification and image restoration problems. Here, we apply deep learning to microscopy, and demonstrate how neural networks can exploit the chromatic dependence of the point-spread function to classify the colors of single emitters imaged on a grayscale camera. While existing single-molecule methods for spectral classification require additional optical elements in the emission path, e.g. spectral filters, prisms, or phase masks, our neural net correctly identifies static as well as mobile emitters with high efficiency using a standard, unmodified single-channel configuration. Furthermore, we demonstrate how deep learning can be used to design phase-modulating elements that, when implemented into the imaging path, result in further improved color differentiation between species.
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Science Advances 2018

TADs are 3D structural units of higher-order chromosome organization in Drosophila

Quentin Szabo, Daniel Jost, Jia-Ming Chang, Diego I. Cattoni, Giorgio L. Papadopoulos, Boyan Bonev, Tom Sexton, Julian Gurgo, Caroline Jacquier, Marcelo Nollmann, Frédéric Bantignies and Giacomo Cavalli
Article reference DOI: 10.1126/sciadv.aar8082
Deciphering the rules of genome folding in the cell nucleus is essential to understand its functions. Recent chromosome conformation capture (Hi-C) studies have revealed that the genome is partitioned into topologically associating domains (TADs), which demarcate functional epigenetic domains defined by combinations of specific chromatin marks. However, whether TADs are true physical units in each cell nucleus or whether they reflect statistical frequencies of measured interactions within cell populations is unclear. Using a combination of Hi-C, three-dimensional (3D) fluorescent in situ hybridization, super-resolution microscopy, and polymer modeling, we provide an integrative view of chromatin folding in Drosophila. We observed that repressed TADs form a succession of discrete nanocompartments, interspersed by less condensed active regions. Single-cell analysis revealed a consistent TAD-based physical compartmentalization of the chromatin fiber, with some degree of heterogeneity in intra-TAD conformations and in cis and trans inter-TAD contact events. These results indicate that TADs are fundamental 3D genome units that engage in dynamic higher-order inter-TAD connections. This domain-based architecture is likely to play a major role in regulatory transactions during DNA-dependent processes.
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