News Imagerie cellulaire - Cellular imaging
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Automating multimodal microscopy with NanoJ-Fluidics

Automating multimodal microscopy with NanoJ-Fluidics | News Imagerie cellulaire - Cellular imaging | Scoop.it

 

Pedro Almada, Pedro M. Pereira, Siân Culley, Ghislaine Caillol, Fanny Boroni-Rueda, Christina L. Dix, Guillaume Charras, Buzz Baum, Romain F. Laine, Christophe Leterrier & Ricardo Henriques


Combining and multiplexing microscopy approaches is crucial to understand cellular events, but requires elaborate workflows. Here, we present a robust, open-source approach for treating, labelling and imaging live or fixed cells in automated sequences. NanoJ-Fluidics is based on low-cost Lego hardware controlled by ImageJ-based software, making high-content, multimodal imaging easy to implement on any microscope with high reproducibility. We demonstrate its capacity on event-driven, super-resolved live-to-fixed and multiplexed STORM/DNA-PAINT experiments.

 

 

Nat Commun. 2019 Mar 15;10(1):1223

Open access : https://doi.org/10.1038/s41467-019-0923

 

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SAVE THE DATE   --  14 novembre 2019 - Journée RIC Paris-Saclay

SAVE THE DATE   --  14 novembre 2019 - Journée RIC Paris-Saclay | News Imagerie cellulaire - Cellular imaging | Scoop.it

Jeudi 14 novembre prochain aura lieu

la 5ème journée d’imagerie du RIC Paris-Saclay.

 

Trois thèmes : Imagerie ionique - Imagerie de la transcription - Imagerie de la mitochondrie

 

Lieu : Faculté de médecine Paris-Sud 

 

Programme : communiqué très prochainement.

 

Inscription : gratuite mais obligatoire

  

 

SAVE THE DATE : 14 NOVEMBRE 2019 -  (9h00 / 16h30)

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Early mitochondrial fragmentation is a potential in vitro biomarker of environmental stress.

Early mitochondrial fragmentation is a potential in vitro biomarker of environmental stress. | News Imagerie cellulaire - Cellular imaging | Scoop.it

publication from UPSaclay

  

Perdiz Daniel 1, Oziol Lucie 2, Poüs Christian 3

 

1 Univ. Paris-Sud, INSERM UMR-S 1193, Université Paris Sud-Paris Saclay, Faculté de Pharmacie, Châtenay-Malabry, France

2 Univ. Paris-Sud, CNRS UMR 8079, Université Paris Sud-Paris Saclay, Faculté de Pharmacie, Châtenay-Malabry, France

3 Biochimie-Hormonologie, APHP, Hôpitaux Universitaires Paris-Sud, Site Antoine Béclère, Clamart, France

 

 

Mitochondria are essential dynamic organelles that ordinarily balance between fragmentation and fusion. Under stress conditions, a shift toward fragmentation or hyper-fusion is observed as a pro-survival reaction. Fragmentation of mitochondria occurs within minutes or hours after the beginning of the stress and occurs in response to a large number of stress stimuli, including those triggered by environmental contaminants. In this study, we tested whether the change in the mitochondrial phenotype, from tubular to fragmented, could be used as a potential environmental stress biomarker in cells and compared this response with the standard MTT-based viability assay. Firstly, we show that mitochondrial fragmentation induced by selected stressors not only increases with concentrations, but also correlates positively with the cytotoxicity. Secondly, we found that the mitochondrial fragmentation that occurs in the first hour of stress correlated with the viability measured after a 24-h stress, allowing the establishment of a linear relation between mitochondrial fragmentation at 1 h and the predictable associated cytotoxicity of environmental contaminants alone or in mixture. In conclusion, we have succeeded in developing a model of predictable 24 h-cytotoxicity given mitochondrial fragmentation at 1 h with a set of chemicals. This model has been successful applied to three environmental toxicants and to a set of two chemical mixtures. We thus propose that mitochondrial fragmentation is a response that could be used as an early in vitro biomarker of environmental stress for toxicants alone or in mixture.

 

 

Chemosphere. 2019 Feb 15;223:577-587

https://doi.org/10.1016/j.chemosphere.2019.02.044

PubMed PMID: 30797167.

 

 

 

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Événements | Université Paris Saclay

Événements | Université Paris Saclay | News Imagerie cellulaire - Cellular imaging | Scoop.it

Université Paris-Saclay events

 

Paris-Saclay | Meet my Platform : video

 

 

Événements

 

Une journée de rencontres plateformes entreprises organisée par Genopole et le Département Sciences de la Vie de l'Université Paris-Saclay
 

Le 31 janvier 2019,  Meet My Platform | Université Paris-Saclay, les Sciences de la Vie vous font découvrir leurs plateformes.

Vous faites partie d'une plateforme Sciences de la Vie de l'Université Paris-Saclay et vous souhaitez découvrir les autres plateformes du territoire ? Vous souhaitez vous faire connaître auprès d'elles et des entreprises ? Venez présenter vos savoir-faire et équipements lors de cette journée.

 

Vous êtes une entreprise, une unité de recherche et vous avez besoin d'utiliser des équipements de pointe en Sciences de la Vie ? Vous souhaitez élargir votre réseau de partenaires académiques dans ce domaine ? Venez découvrir le potentiel des plateformes technologiques de l'Université Paris-Saclay.

Programme :

9h30 : accueil

 

10h00 : ouverture de l'événement par Genopole et l'Université Paris-Saclay

 

10h20 – 11h00 : table ronde "la vision de trois industriels sur le potentiel des plateformes Sciences de la Vie de l'Université Paris-Saclay" avec :

  • Laurent CHENE, Head of drug discovery, ENTEROME

  • Georges DA VIOLANTE, Metabolism Dept Manager, in charge of academic and industrial partnerships, SERVIER

  • Artem KHLEBNIKOV, Directeur Stratégique Partnerships, DANONE

Table ronde animée par Nathalie Tourret, journaliste

 

11h00 – 12h00 : découverte de 4 réseaux / typologies sélectionnées de plateformes

 

12h00 : buffet

 

12h30 - 14h00 : stands et networking

 

14h00 – 17h00 : rendez-vous B2B

 

14h00 – 17h00 : ateliers pour les plateformes

 

Cette journée est organisée par Genopole et le Département Sciences de la Vie de l'Université Paris-Saclay, avec le soutien du pôle de compétitivité Medicen Paris Région, de la SATT Paris-Saclay et de CentraleSupélec

 

La matinée aura lieu dans l'auditorium Michelin bâtiment Eiffel et l'après-midi au rez-de-chaussée du bâtiment Bouygues de CentraleSupélec (les deux espaces sont l'un en face l'autre).

 

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Quantification of protein mobility and associated reshuffling of cytoplasm during chemical fixation

Quantification of protein mobility and associated reshuffling of cytoplasm during chemical fixation | News Imagerie cellulaire - Cellular imaging | Scoop.it

Jan Huebinger, Jessica Spindler, Kristin J. Holl and  Björn Koos

 

Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str.11, 44227, Dortmund, Germany.

 

To understand cellular functionalities, it is essential to unravel spatio-temporal patterns of molecular distributions and interactions within living cells. The technological progress in fluorescence microscopy now allows in principle to measure these patterns with sufficient spatial resolution. However, high resolution imaging comes with long acquisition times and high phototoxicity. Therefore, physiological live cell imaging is often unfeasible and chemical fixation is employed. Yet, fixation methods have not been rigorously investigated, in terms of pattern preservation, at the resolution at which cells can now be imaged. A key parameter for this is the time required until fixation is complete. During this time, cells are under unphysiological conditions and patterns decay. We demonstrate here that formaldehyde fixation takes more than one hour for cytosolic proteins in cultured cells. Other small aldehydes, glyoxal and acrolein, did not perform better. Associated with this, we found a distinct displacement of proteins and lipids, including their loss from cells. Fixations using glutaraldehyde were faster than four minutes and retained most cytoplasmic proteins. Surprisingly, autofluorescence produced by glutaraldehyde was almost completely absent with supplementary addition of formaldehyde without compromising fixation speed. These findings indicate, which cellular processes can actually be reliably imaged after a certain chemical fixation.

 

Sci Rep. 2018; 8: 17756

doi : 10.1038/s41598-018-36112-w

Open access article : https://www.nature.com/articles/s41598-018-36112-w.pdf

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Multifocal multiphoton volumetric imaging approach for high-speed time-resolved Förster resonance energy transfer imaging in vivo

Multifocal multiphoton volumetric imaging approach for high-speed time-resolved Förster resonance energy transfer imaging in vivo | News Imagerie cellulaire - Cellular imaging | Scoop.it

Simon P. Poland, Grace K. Chan, James A. Levitt, Nikola Krstajić, Ahmet T. Erdogan, Robert K. Henderson, Maddy Parsons, and Simon M. Ameer-Beg

 

In this Letter, we will discuss the development of a multifocal multiphoton fluorescent lifetime imaging system where four individual fluorescent intensity and lifetime planes are acquired simultaneously, allowing us to obtain volumetric data without the need for sequential scanning at different axial depths. Using a phase-only spatial light modulator (SLM) with an appropriate algorithm to generate a holographic pattern, we project a beamlet array within a sample volume of a size, which can be preprogrammed by the user. We demonstrate the capabilities of the system to image live-cell interactions. While only four planes are shown, this technique can be rescaled to a large number of focal planes, enabling full 3D acquisition and reconstruction.

 

Optics Letters Vol. 43, Issue 24, pp. 6057-6060 (2018)

https://doi.org/10.1364/OL.43.006057

Open Access : https://www.osapublishing.org/ol/viewmedia.cfm?uri=ol-43-24-6057&seq=0 

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Super-resolution enhancement by quantum image scanning microscopy

Super-resolution enhancement by quantum image scanning microscopy | News Imagerie cellulaire - Cellular imaging | Scoop.it

Ron Tenne, Uri Rossman, Batel Rephael, Yonatan Israel, Alexander Krupinski-Ptaszek, Radek Lapkiewicz, Yaron Silberberg & Dan Oron

 

The principles of quantum optics have yielded a plethora of ideas to surpass the classical limitations of sensitivity and resolution in optical microscopy. While some ideas have been applied in proof-of-principle experiments, imaging a biological sample has remained challenging, mainly due to the inherently weak signal measured and the fragility of quantum states of light. In principle, however, these quantum protocols can add new information without sacrificing the classical information and can therefore enhance the capabilities of existing super-resolution techniques. Image scanning microscopy, a recent addition to the family of super-resolution methods, generates a robust resolution enhancement without reducing the signal level. Here, we introduce quantum image scanning microscopy: combining image scanning microscopy with the measurement of quantum photon correlation allows increasing the resolution of image scanning microscopy up to twofold, four times beyond the diffraction limit. We introduce the Q-ISM principle and obtain super-resolved optical images of a biological sample stained with fluorescent quantum dots using photon antibunching, a quantum effect, as a resolution-enhancing contrast mechanism.

 

Nature Methods (2018) Published: 17 December 2018

https://doi.org/10.1038/s41566-018-0324-z

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Imaging cellular ultrastructures using expansion microscopy (U-ExM)

Imaging cellular ultrastructures using expansion microscopy (U-ExM) | News Imagerie cellulaire - Cellular imaging | Scoop.it

Davide Gambarotto, Fabian U. Zwettler, Maeva Le Guennec, Marketa Schmidt-Cernohorska, Denis Fortun, Susanne Borgers, Jörn Heine, Jan-Gero Schloetel, Matthias Reuss, Michael Unser, Edward S. Boyden, Markus Sauer, Virginie Hamel & Paul Guichard

 

Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.

 

Nature Methods (2018) Published: 17 December 2018

https://doi.org/10.1038/s41592-018-0238-1

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Single-Cell Imaging of Metastatic Potential of Cancer Cells

Single-Cell Imaging of Metastatic Potential of Cancer Cells | News Imagerie cellulaire - Cellular imaging | Scoop.it
Krishna Midde, Nina Sun, Cristina Rohena, Linda Joosen, Harsharan Dhillon, Pradipta Ghosh
 

Molecular imaging of metastatic “potential” is an unvanquished challenge. To engineer biosensors that can detect and measure the metastatic “potential” of single living cancer cells, we carried out a comprehensive analysis of the pan-cancer phosphoproteome to search for actin remodelers required for cell migration, which are enriched in cancers but excluded in normal cells. Only one phosphoprotein emerged, tyr-phosphorylated CCDC88A (GIV/Girdin), a bona fide metastasis-related protein across a variety of solid tumors. We designed multi-modular biosensors that are partly derived from GIV, and because GIV integrates prometastatic signaling by multiple oncogenic receptors, we named them “‘integrators of metastatic potential (IMP).” IMPs captured the heterogeneity of metastatic potential within primary lung and breast tumors at steady state, detected those few cells that have acquired the highest metastatic potential, and tracked their enrichment during metastasis. These findings provide proof of concept that IMPs can measure the diversity and plasticity of metastatic potential of tumor cells in a sensitive and unbiased way.

 

iScience Volume 10, 21 December 2018, Pages 53-65
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Dynamic Imaging of Small Molecule Induced Protein–Protein Interactions in Living Cells with a Fluorophore Phase Transition Based Approach

Dynamic Imaging of Small Molecule Induced Protein–Protein Interactions in Living Cells with a Fluorophore Phase Transition Based Approach | News Imagerie cellulaire - Cellular imaging | Scoop.it

Chan-I Chung, Qiang Zhang and Xiaokun Shu

 

Protein–protein interactions (PPIs) mediate signal transduction in cells. Small molecules that regulate PPIs are important tools for biology and biomedicine. Dynamic imaging of small molecule induced PPIs characterizes and verifies these molecules in living cells. It is thus important to develop cellular assays for dynamic visualization of small molecule induced protein–protein association and dissociation in living cells. Here we have applied a fluorophore phase transition based principle and designed a PPI assay named SPPIER (separation of phases-based protein interaction reporter). SPPIER utilizes the green fluorescent protein (GFP) and is thus genetically encoded. Upon small molecule induced PPI, SPPIER rapidly forms highly fluorescent GFP droplets in living cells. SPPIER detects immunomodulatory drug (IMiD) induced PPI between cereblon and the transcription factor Ikaros. It also detects IMiD analogue (e.g., CC-885) induced PPI between cereblon and GSPT1. Furthermore, SPPIER can visualize bifunctional molecules (e.g. PROTAC)-induced PPI between an E3 ubiquitin ligase and a target protein. Lastly, SPPIER can be modified to image small molecule induced protein–protein dissociation, such as nutlin-induced dissociation between HDM2 and p53. The intense brightness and rapid kinetics of SPPIER enable robust and dynamic visualization of PPIs in living cells.

 

Anal. Chem., Article ASAP Publication Date (Web): Nov. 15, 2018
Doi : 10.1021/acs.analchem.8b03476
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Multispectral Atomic Force Microscopy-Infrared Nano-Imaging of Malaria Infected Red Blood Cells

Multispectral Atomic Force Microscopy-Infrared Nano-Imaging of Malaria Infected Red Blood Cells | News Imagerie cellulaire - Cellular imaging | Scoop.it

David Perez-Guaita, Kamila Kochan, Mitchell Batty, Christian Doerig, Jose Garcia-Bustos, Shirly Espinoza, Don McNaughton, Phil Heraud and Bayden R. Wood

 

Atomic force microscopy-infrared (AFM-IR) spectroscopy is a powerful new technique that can be applied to study molecular composition of cells and tissues at the nanoscale. AFM-IR maps are acquired using a single wavenumber value: they show either the absorbance plotted against a single wavenumber value or a ratio of two absorbance values. Here, we implement multivariate image analysis to generate multivariate AFM-IR maps and use this approach to resolve subcellular structural information in red blood cells infected with Plasmodium falciparum at different stages of development. This was achieved by converting the discrete spectral points into a multispectral line spectrum prior to multivariate image reconstruction. The approach was used to generate compositional maps of subcellular structures in the parasites, including the food vacuole, lipid inclusions, and the nucleus, on the basis of the intensity of hemozoin, hemoglobin, lipid, and DNA IR marker bands, respectively. Confocal Raman spectroscopy was used to validate the presence of hemozoin in the regions identified by the AFM-IR technique. The high spatial resolution of AFM-IR combined with hyperspectral modeling enables the direct detection of subcellular components, without the need for cell sectioning or immunological/biochemical staining. Multispectral-AFM-IR thus has the capacity to probe the phenotype of the malaria parasite during its intraerythrocytic development. This enables novel approaches to studying the mode of action of antimalarial drugs and the phenotypes of drug-resistant parasites, thus contributing to the development of diagnostic and control measures

 

Anal. Chem., 2018, 90 (5), pp 3140–3148

Doi : 10.1021/acs.analchem.7b04318

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Combined Use of Unnatural Amino Acids Enables Dual Color Super-Resolution Imaging of Proteins via Click Chemistry

Combined Use of Unnatural Amino Acids Enables Dual Color Super-Resolution Imaging of Proteins via Click Chemistry | News Imagerie cellulaire - Cellular imaging | Scoop.it

Kim -A. Saal, Frank Richter, Peter Rehling, and Silvio O. Rizzoli

 

Recent advances in optical nanoscopy have brought the imaging resolution to the size of the individual macromolecules, thereby setting stringent requirements for the fluorescent labels. Such requirements are optimally fulfilled by the incorporation of unnatural amino acids (UAAs) in the proteins of interest (POI), followed by fluorophore conjugation via click chemistry. However, this approach has been limited to single POIs in mammalian cells. Here we solve this problem by incorporating different UAAs in different POIs, which are expressed in independent cell sets. The cells are then fused, thereby combining the different proteins and organelles, and are easily imaged by dual-color super-resolution microscopy. This procedure, which we termed Fuse2Click, is simple, requires only the well-established Amber codon, and allows the use of all previously optimized UAAs and tRNA/RS pairs. This should render it a tool of choice for multi-color click-based imaging.

 

ACS Nano, Just Accepted Manuscript Dec. 10, 2018
Doi : 10.1021/acsnano.8b06047
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Membrane water for probing neuronal membrane potentials and ionic fluxes at the single cell level

Membrane water for probing neuronal membrane potentials and ionic fluxes at the single cell level | News Imagerie cellulaire - Cellular imaging | Scoop.it

M. E. P. Didier, O. B. Tarun, P. Jourdain, P. Magistretti & S. Roke

 

Neurons communicate through electrochemical signaling within a complex network. These signals are composed of changes in membrane potentials and are traditionally measured with the aid of (toxic) fluorescent labels or invasive electrical probes. Here, we demonstrate an improvement in label-free second harmonic neuroimaging sensitivity by ~3 orders of magnitude using a wide-field medium repetition rate illumination. We perform a side-by-side patch-clamp and second harmonic imaging comparison to demonstrate the theoretically predicted linear correlation between whole neuron membrane potential changes and the square root of the second harmonic intensity. We assign the ion induced changes to the second harmonic intensity to changes in the orientation of membrane interfacial water, which is used to image spatiotemporal changes in the membrane potential and K+ ion flux. We observe a non-uniform spatial distribution and temporal activity of ion channels in mouse brain neurons.

 

Nature Communications volume 9, Article number: 5287 (2018)

https://doi.org/10.1038/s41467-018-07713-w

Open Access : https://www.nature.com/articles/s41467-018-07713-w.pdf

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Human cytomegalovirus hijacks the autophagic machinery and LC3 homologs in order to optimize cytoplasmic envelopment of mature infectious particles

Human cytomegalovirus hijacks the autophagic machinery and LC3 homologs in order to optimize cytoplasmic envelopment of mature infectious particles | News Imagerie cellulaire - Cellular imaging | Scoop.it

publication from UPSaclay

 

Clémence Taisne, Marion Lussignol, Eva Hernandez, Arnaud Moris, Lina Mouna & Audrey Esclatine

 

1. Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette , France.

2. Sorbonne Université, Centre d'Immunologie et des Maladies Infectieuses, INSERM U1135, CNRS ERL 8255, Paris, France.

3. Virologie, APHP, Hôpital Paul Brousse, 94800, Villejuif, France.

4. Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France.



During its life cycle, Human cytomegalovirus (HCMV) tightly modulates autophagy, a vesicular pathway allowing degradation and recycling of cellular components. To study the interplay between autophagy and the viral life cycle, we established various autophagy-deficient human fibroblastic cell lines. By knocking down the expression or activity of five autophagy-related proteins, we confirmed the proviral function that the autophagic machinery exerts on HCMV production. Using 3D reconstruction from confocal microscopy and electron microscopy, we demonstrated that lipidated LC3-positive vesicles accumulated at the viral assembly compartment (vAC). The vAC is a juxtanuclear ring-shaped structure containing several organelles and membranes, where assembly and final envelopment of HCMV particles occur. Two LC3 homologs, GABARAPL1 and GATE16, also accumulated during HCMV infection and were associated with the vAC, in proximity with fragmented Golgi stacks. Additionally, we observed the formation of a pre-assembly compartment (PrAC) in infected cells, which consists of a juxtanuclear structure containing both fragmented Golgi and LC3-positive vesicles. Finally, we showed that highly purified extracellular viral particles were associated with various autophagy proteins. Our results thus suggest that autophagy machinery participates to the final cytoplasmic envelopment of HCMV viral particles into the vAC and that autophagy-related proteins can be spotted in the virions.

 

Sci Rep. 2019, 9(1):4560

Open acces : https://doi.org/10.1038/s41598-019-41029-z

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Correlative infrared nanospectroscopy and transmission electron microscopy to investigate nanometric amyloid fibrils: prospects and challenges.

Correlative infrared nanospectroscopy and transmission electron microscopy to investigate nanometric amyloid fibrils: prospects and challenges. | News Imagerie cellulaire - Cellular imaging | Scoop.it

publication from Paris-Saclay University

 

Partouche D, Mathurin J, Malabirade A, Marco S, Sandt C, Arluison V, Deniset-Besseau A, Trépout S.

 

1 Synchrotron SOLEIL, L'Orme des Merisiers Saint Aubin, Gif-sur-Yvette

2 Laboratoire Léon Brillouin LLB, CEA, CNRS UMR12, Université Paris Saclay

3 Laboratoire de Chimie Physique, CNRS, Univ. Paris-Sud, Université Paris-Saclay

4 INSERM, U1196, Université Paris Sud, Université Paris-Saclay

5 Institut Curie, PSL Research University, CNRS, UMR 9187

6 Université Paris Diderot-Paris 7, Sorbonne Paris Cité

 

Lay Description

Propagation of structural information through conformational changes in amyloid proteins is at the root of many neurodegenerative disorders. Amyloids are nanostructures originating from the aggregation of multiple copies of peptide or protein monomers that eventually form fibrils. Often described as being the cause for the development of various diseases, amyloid fibrils are of major significance in the public health domain. While important breakthroughs have been made in the field, fundamental issues like the 3D‐structures of the fibrils implied in some of those disorders are still to be elucidated. To better characterise these fibrils, a broad range of techniques is currently available for the detection and visualisation of amyloid nanostructures. Nevertheless none of them is able to perform direct chemical characterisation of single protein fibrils. In this work, we propose to investigate the structure of model amyloidogenic fibrils using a correlative approach. The complementary techniques used are transmission electron microscopy and a newly developed infrared nanospectroscopy technique called AFM‐IR that allows chemical characterisation at the nanometric scale. The strategy, protocol, challenges and difficulties encountered in this approach are introduced and discussed herein.

 

J Microsc. 2019 Jan 16

PMID : 30649833

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Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived β cells

Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived β cells | News Imagerie cellulaire - Cellular imaging | Scoop.it

Nair GG, Liu JS, Russ HA, Tran S, Saxton MS, Chen R, Juang C, Li ML, Nguyen VQ, Giacometti S, Puri S, Xing Y, Wang Y, Szot GL, Oberholzer J, Bhushan A and Hebrok M.

 

Diabetes Center, University of California San Francisco, San Francisco, CA, USA.

 

Despite advances in the differentiation of insulin-producing cells from human embryonic stem cells, the generation of mature functional β cells in vitro has remained elusive. To accomplish this goal, we have developed cell culture conditions to closely mimic events occurring during pancreatic islet organogenesis and β cell maturation. In particular, we have focused on recapitulating endocrine cell clustering by isolating and reaggregating immature β-like cells to form islet-sized enriched β-clusters (eBCs). eBCs display physiological properties analogous to primary human β cells, including robust dynamic insulin secretion, increased calcium signalling in response to secretagogues, and improved mitochondrial energization. Notably, endocrine cell clustering induces metabolic maturation by driving mitochondrial oxidative respiration, a process central to stimulus–secretion coupling in mature β cells. eBCs display glucose-stimulated insulin secretion as early as three days after transplantation in mice. In summary, replicating aspects of endocrine cell clustering permits the generation of stem-cell-derived β cells that resemble their endogenous counterparts.

 

Nat Cell Biol. 2019 Feb;21(2):263-274.

doi: 10.1038/s41556-018-0271-4

PubMed PMID: 30710150.

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  A new 3D model of the NADPH oxidase cytosolic complex

   A new 3D model of the  NADPH  oxidase cytosolic complex | News Imagerie cellulaire - Cellular imaging | Scoop.it

From UPSaclay Laboratories

 

Cornelia S Ziegler, Leila Bouchab, Marc Tramier, Dominique Durand, Franck Fieschi, Sophie Dupré-Crochet, Fabienne Mérola, Oliver Nüße, Marie Erard

 

Laboratoire de Chimie Physique, CNRS, Univ. Paris-Sud, Université Paris-Saclay

Univ Rennes, CNRS, IGDR (Institut de génétique et développement de Rennes)

I2BC, CNRS, France

Universite Grenoble Alpes, IB

CNRS

Laboratoire de Chimie Physique, CNRS, Univ. Paris-Sud, Université Paris-Saclay

 

 

Phagocyte NADPH oxidase produces superoxide anions, a precursor of reactive oxygen species (ROS) critical for host responses to microbial infections. However, uncontrolled ROS production contributes to inflammation, making NADPH oxidase a major drug target. It consists of two membranous (Nox2 and p22phox) and three cytosolic subunits (p40phox, p47phox, and p67phox) that undergo structural changes during enzyme activation. Unraveling the interactions between these subunits and the resulting conformation of the complex could shed light on NADPH oxidase regulation and help identify inhibition sites. However, the structures and the interactions of flexible proteins comprising several well-structured domains connected by intrinsically disordered protein segments are difficult to investigate by conventional techniques such as X-ray crystallography, NMR, or cryo-EM. Here, we developed an analytical strategy based on FRET–fluorescence lifetime imaging (FLIM) and fluorescence cross-correlation spectroscopy (FCCS) to structurally and quantitatively characterize NADPH oxidase in live cells. We characterized the inter- and intramolecular interactions of its cytosolic subunits by elucidating their conformation, stoichiometry, interacting fraction, and affinities in live cells. Our results revealed that the three subunits have a 1:1:1 stoichiometry and that nearly 100% of them are present in complexes in living cells. Furthermore, combining FRET data with small-angle X-ray scattering (SAXS) models and published crystal structures of isolated domains and subunits, we built a 3D model of the entire cytosolic complex. The model disclosed an elongated complex containing a flexible hinge separating two domains ideally positioned at one end of the complex and critical for oxidase activation and interactions with membrane components.

 

J Biol Chem. 2019 Jan 10. pii: jbc.RA118.006864

doi: 10.1074/jbc.RA118.006864.

Open Access : www.jbc.org/content/early/2019/01/10/jbc.RA118.006864.full.pdf?with-ds=yes

 

 

 

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EB1 contributes to microtubule bundling and organization, along with root growth, in Arabidopsis thaliana

EB1 contributes to microtubule bundling and organization, along with root growth, in Arabidopsis thaliana | News Imagerie cellulaire - Cellular imaging | Scoop.it
From the Université Paris-Saclay
 
Arthur T. Molines1, Jessica Marion1, Salem Chabout2, Laetitia Besse3, Jim P. Dompierre3, Grégory Mouille2, Frédéric M. Coquelle1
 

1  Department of Cell Biology, Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette Cedex, France

2  Institut Jean-Pierre Bourgin (IJPB), INRA - AgroParisTech, 78026 Versailles Cedex, France

3  Light Microscopy Facility, Imagerie-Gif, Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette Cedex, France

 

 

 

Microtubules are involved in plant development and adaptation to their environment, but the sustaining molecular mechanisms remain elusive. Microtubule-end-binding 1 (EB1) proteins participate in directional root growth in Arabidopsis thaliana . However, a connection to the underlying microtubule array has not been established yet. We show here that EB1 proteins contribute to the organization of cortical microtubules in growing epidermal plant cells, without significant modulation of microtubule dynamics. Using super-resolution stimulated emission depletion (STED) microscopy and an original quantification approach, we also demonstrate a significant reduction of apparent microtubule bundling in cytoplasmic-EB1-deficient plants, suggesting a function for EB1 in the interaction between adjacent microtubules. Furthermore, we observed root growth defects in EB1-deficient plants, which are not related to cell division impairment. Altogether, our results support a role for EB1 proteins in root development, in part by maintaining the organization of cortical microtubules.

 

Biol Open. 2018 Aug 2;7(8)

DOI : https://doi.org/10.1242/bio.030510

PubMed : 29945874

Open access : http://bio.biologists.org/content/biolopen/7/8/bio030510.full.pdf

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Optoacoustic microscopy at multiple discrete frequencies

Optoacoustic microscopy at multiple discrete frequencies | News Imagerie cellulaire - Cellular imaging | Scoop.it

Stephan Kellnberger, Dominik Soliman, George J. Tserevelakis, Markus Seeger, Hong Yang, Angelos Karlas, Ludwig Prade, Murad Omar & Vasilis Ntziachristos

 

Optoacoustic (photoacoustic) sensing employs illumination of transient energy and is typically implemented in the time domain using nanosecond photon pulses. However, the generation of high-energy short photon pulses requires complex laser technology that imposes a low pulse repetition frequency (PRF) and limits the number of wavelengths that are concurrently available for spectral imaging. To avoid the limitations of working in the time domain, we have developed frequency-domain optoacoustic microscopy (FDOM), in which light intensity is modulated at multiple discrete frequencies. We integrated FDOM into a hybrid system with multiphoton microscopy, and we examine the relationship between image formation and modulation frequency, showcase high-fidelity images with increasing numbers of modulation frequencies from phantoms and in vivo, and identify a redundancy in optoacoustic measurements performed at multiple frequencies. We demonstrate that due to high repetition rates, FDOM achieves signal-to-noise ratios similar to those obtained by time-domain methods, using commonly available laser diodes. Moreover, we experimentally confirm various advantages of the frequency-domain implementation at discrete modulation frequencies, including concurrent illumination at two wavelengths that are carried out at different modulation frequencies as well as flow measurements in microfluidic chips and in vivo based on the optoacoustic Doppler effect. Furthermore, we discuss how FDOM redefines possibilities for optoacoustic imaging by capitalizing on the advantages of working in the frequency domain.

 

Light: Science & Applications volume 7, Article number: 109 (2018)

https://doi.org/10.1038/s41377-018-0101-2

Open Access : https://www.nature.com/articles/s41377-018-0101-2.pdf

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Deep learning enables cross-modality super-resolution in fluorescence microscopy

Deep learning enables cross-modality super-resolution in fluorescence microscopy | News Imagerie cellulaire - Cellular imaging | Scoop.it

Hongda Wang, Yair Rivenson, Yiyin Jin, Zhensong Wei, Ronald Gao, Harun Günaydın, Laurent A. Bentolila, Comert Kural & Aydogan Ozcan

 

We present deep-learning-enabled super-resolution across different fluorescence microscopy modalities. This data-driven approach does not require numerical modeling of the imaging process or the estimation of a point-spread-function, and is based on training a generative adversarial network (GAN) to transform diffraction-limited input images into super-resolved ones. Using this framework, we improve the resolution of wide-field images acquired with low-numerical-aperture objectives, matching the resolution that is acquired using high-numerical-aperture objectives. We also demonstrate cross-modality super-resolution, transforming confocal microscopy images to match the resolution acquired with a stimulated emission depletion (STED) microscope. We further demonstrate that total internal reflection fluorescence (TIRF) microscopy images of subcellular structures within cells and tissues can be transformed to match the results obtained with a TIRF-based structured illumination microscope. The deep network rapidly outputs these super-resolved images, without any iterations or parameter search, and could serve to democratize super-resolution imaging.

 

Nature Methods (2018) Published: 17 December 2018

https://doi.org/10.1038/s41592-018-0239-0

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CNRS on Instagram: “#RETRO2017 Replis de la #muqueuse intestinale d’une souris observés en #microscopie confocale multi-couleurs. ”

CNRS on Instagram: “#RETRO2017 Replis de la #muqueuse intestinale d’une souris observés en #microscopie confocale multi-couleurs. ” | News Imagerie cellulaire - Cellular imaging | Scoop.it
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Scanning Fluorescence Correlation Spectroscopy for Quantification of the Dynamics and Interactions in Tube Organelles of Living Cells

Scanning Fluorescence Correlation Spectroscopy for Quantification of the Dynamics and Interactions in Tube Organelles of Living Cells | News Imagerie cellulaire - Cellular imaging | Scoop.it

 

 

Joseph D. Unsay, Fabronia Murad, Eduard Hermann, Jonas Ries,
Ana J. García‐Sáez
 

Single‐molecule spectroscopic quantification of protein‐protein interactions directly in the organelles of living cells is highly desirable but remains challenging. Bulk methods, such as Förster resonance energy transfer (FRET), currently only give a relative quantification of the strength of protein‐protein interactions. Here, we introduce tube scanning fluorescence cross‐correlation spectroscopy (tubeSFCCS) for the absolute quantification of diffusion and complex formation of fluorescently labeled molecules in the mitochondrial compartments. We determined the extent of association between the apoptosis regulators Bcl‐xL and tBid at the mitochondrial outer membrane of living cells and discovered that practically all mitochondria‐bound Bcl‐xL and tBid are associated with each other, in contrast to undetectable association in the cytosol. Furthermore, we show further applicability of our method to other mitochondrial proteins, as well as to proteins in the endoplasmic reticulum (ER) membrane.

 

ChemPhysChem 2018, 19, 3273–327

https://doi.org/10.1002/cphc.201800705

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Imaging of Receptor Dimers in Zebrafish and Living Cells via Aptamer Recognition and Proximity-Induced Hybridization Chain Reaction

Imaging of Receptor Dimers in Zebrafish and Living Cells via Aptamer Recognition and Proximity-Induced Hybridization Chain Reaction | News Imagerie cellulaire - Cellular imaging | Scoop.it

Liping Wang, Wei Li, Jin Sun, Su-Yun Zhang, Sheng Yang, Jingying Li, Juan Li and Huang-Hao Yang

 

On cell-membrane surfaces, receptor-protein dimers play fundamental roles in many signaling pathways that are crucial for normal biological processes and cancer development. Efficient and sensitive analysis of receptor dimers in the native environment is highly desirable. Herein, we present a strategy for amplified imaging of receptor dimers in zebrafish and living cells that relies on aptamer recognition and proximity-induced hybridization chain reaction. Taking advantage of specific aptamer recognition and enzyme-free signal amplification, this strategy is successfully applied to the visualization of c-Met-receptor dimers in an HGF-independent or -dependent manner. Therefore, the developed imaging strategy paves the way for further investigation of the dimerization or oligomerization states of cell-surface receptors and their corresponding activation processes in zebrafish and living cells.

 

Anal. Chem., Article ASAP Publication Date (Web): Nov. 16, 2018
Doi : 10.1021/acs.analchem.8b04015
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Fluorescent Benzothiazinone Analogues Efficiently and Selectively Label Dpre1 in Mycobacteria and Actinobacteria

Fluorescent Benzothiazinone Analogues Efficiently and Selectively Label Dpre1 in Mycobacteria and Actinobacteria | News Imagerie cellulaire - Cellular imaging | Scoop.it

Raphael Sommer,  Joao Neres, Jérémie Piton, Neeraj Dhar, Astrid van der Sar, Raju Mukherjee, Thierry Laroche, Paul J. Dyson, John D. McKinney, Wilbert Bitter, Vadim Makarov and Stewart T. Cole

 

Benzothiazinones (BTZ) are highly potent bactericidal inhibitors of mycobacteria and the lead compound, BTZ043, and the optimized drug candidate, PBTZ169, have potential for the treatment of tuberculosis. Here, we exploited the tractability of the BTZ scaffold by attaching a range of fluorophores to the 2-substituent of the BTZ ring via short linkers. We show by means of fluorescence imaging that the most advanced derivative, JN108, is capable of efficiently labeling its target, the essential flavoenzyme DprE1, both in cell-free extracts and after purification as well as in growing cells of different actinobacterial species. DprE1 displays a polar localization in Mycobacterium tuberculosis, M. marinum, M. smegmatis, and Nocardia farcinica but not in Corynebacterium glutamicum. Finally, mutation of the cysteine residue in DprE1 in these species, to which BTZ covalently binds, abolishes completely the interaction with JN108, thereby highlighting the specificity of this fluorescent probe.

 

J. Am. Chem. Soc., Just Accepted Manuscript Nov. 28, 2018
Doi : 10.1021/jacs.8b10783
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Whole-cell, 3D and multi-color STED imaging with exchangeable fluorophores

Whole-cell, 3D and multi-color STED imaging with exchangeable fluorophores | News Imagerie cellulaire - Cellular imaging | Scoop.it

Christoph Spahn, Jonathan B. Grimm, Luke D. Lavis, Marko Lampe, and Mike Heilemann

 

We demonstrate STED microscopy of whole bacterial and eukaryotic cells using fluorogenic labels that reversibly bind to their target structure. A constant exchange of labels guarantees the removal of photobleached fluorophores and their replacement by intact fluorophores, thereby circumventing bleaching-related limitations of STED super-resolution imaging. We achieve a constant labeling density and demonstrate a fluorescence signal for long and theoretically unlimited acquisition times. Using this concept, we demonstrate whole-cell, 3D, multi-color and live cell STED microscopy.

 

Nano Lett., Just Accepted Manuscript Dec. 10, 2018
DOI: 10.1021/acs.nanolett.8b04385
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