Ce travail est financé par le NICHD / NIH (1R01HD053287) pour Hukriede, NCI / NIH (P01 CA78039) Vogt, et NHLBI / NIH (1R01HL088016) pour Tsang.

1) Mettre en place des accouplements de poisson zèbre et le traitement de BCI. <ol><li> Deux jours avant le traitement, identifier les homozygotes mâles Tg (dusp6: d2eGFP) PT6 poisson zèbre et le lieu individuellement dans une réservoirs d&#39;accouplement. Ajouter séparateur et un type de poisson sauvage AB * femelle et laisser dans des cages d&#39;accouplement pendant la nuit. </li><li> Le lendemain matin enlever le séparateur, et de recueillir des embryons après 1 heure. Transfert des embryons à des plats 100mm avec l&#39;E3 (5 mM de NaCl, 0,17 mM de KCl, 0,33 mM de CaCl2, 0,33 mM MgSO4), solution et incuber à 28 ° C pendant 6 heures. Retirer morts et non fécondés embryons, ajouter la solution E3 frais, et incuber pendant la nuit. </li><li> Trier embryons transgéniques pour le stade similaire (24hpf) et expression de la GFP uniforme. Embryons tableau individuellement dans chaque puits d&#39;une plaque de 96 puits. </li><li> Retirer l&#39;excédent de solution E3 avec une micropipette. Ajouter 200 ul de solution E3 avec une pipette multi-canaux dans chaque puits. </li><li> Ajouter les composés énumérés ci-après: <table border="1"><tbody><tr><td></td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td><td> 8 </td><td> 9 </td><td> 10 </td><td> 11 </td><td> 12 </td></tr><tr ><td> Une </td><td> 2μl DMSO </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> B </td><td> 2μl DMSO </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> C </td><td> 2μl DMSO </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> D </td><td> 2μl DMSO </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> E </td><td> 2μl DMSO </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> F </td><td> 2μl DMSO </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> G </td><td> 2μl DMSO </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mM) </td><td> 2μl DMSO </td></tr><tr ><td> H </td><td> 2μl DMSO </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl DMSO </td></tr></tbody></table> Les concentrations finales sont 1 uM, 5 um, 10 uM, 20μM, épaisseur 25 BCI dans le DMSO à 1%. Recouvrir la plaque dans du papier aluminium et laisser incuber pendant 6 heures à 28 ° C. </li></ol> 2) l&#39;imagerie automatisée des plaques de 96 puits et d&#39;analyse. <ol><li> Ajouter 10 pi de tricaïne (0,61 mm) à chaque puits pour anesthésier embryons 30hpf. </li><li> Charge plaque dans Molecular appareils ultra Imagexpress. </li><li> Ouvrez le logiciel Metaxpress. Choisissez une configuration avec un objectif 4x et un diamètre maximum de sténopé, configurer laser autofocus pour détecter plaque inférieure, et de déterminer optimale plan Z pour détecter des régions d&#39;intérêt. Acquérir les images au excitation / émission longueurs d&#39;onde de 488/525 nm (GFP). Set zone de numérisation comme un site unique de 2000x2000 pixels (4 x 4 mm), sans binning. Configurer la puissance du laser et le gain de telle sorte que les régions plus brillants dans les images de contrôle positif ne saturent pas. Confirmer l&#39;acquisition d&#39;image correcte dans plusieurs puits de contrôle positif et négatif. </li><li> Démarrer l&#39;analyse assiette. L&#39;instrument sera automatiquement acquérir des images de tous les puits et les archiver dans une base SQL server. </li><li> Upload images en utilisant Definiens Developer le module d&#39;application Cellenger et un filtre d&#39;importation qui reconnaît le format d&#39;image et de la structure des fichiers générés par la plateforme Molecular Devices Imagexpress Ultra. </li><li> Traiter les images avec un algorithme personnalisé destiné Cognition Network Technology (également appelé un jeu de règles). L&#39;algorithme, qui nous personnalisée développée basée sur un ensemble de règles récemment publié 2, est conçu pour identifier automatiquement l&#39;embryon, le jaune, et la tête, et de quantifier la luminosité et le domaine de la GFP exprimant structures de la tête. Les puits dans lesquels aucun embryon n&#39;est détecté ou si la tête ne peut être affecté sans ambiguïté (par exemple, avec une toxicité élevée ou composé d&#39;images floues) sont exclus de l&#39;analyse. Configurer l&#39;algorithme utilisant plusieurs images de contrôle négatif (traités par le véhicule) et des images de contrôle positif (BCI traités) de telle sorte que la tête et le jaune sont correctement identifiés dans les deux. Régler une «tête de structures« seuil de détection comme des multiples d&#39;intensité jaune signifie que la détection des structures de la tête lumineuse correspond à l&#39;observation visuelle. Définir les paramètres d&#39;exportation. Dans cet exemple, le paramètre le plus informatif a été la luminosité intégrée totale de la GFP-positives dans la tête des régions ci-après appelé «tête de luminosité totale des structures» (figure 1). <img src="/files/ftp_upload/1900/1900fig2.jpg" alt="Figure 1" /> Figure 1 Graphique depicitng les effets dose-dépendante de la BCI sur l&#39;expression de GFP dans les embryons transgéniques. </li><li> Exécuter l&#39;algorithme sur toutes les images plaque à l&#39;aide du planificateur intégré d&#39;emplois. Le logiciel Definiens utilise l&#39;informatique distribuée sur plusieurs processeurs («moteurs»), permettant l&#39;analyse simultanée de plusieurs images. L&#39;algorithme calcule les mesures numériques basés sur les résultats de l&#39;analyse d&#39;image, qui sont enregistrées dans la base de données avec une copie de l&#39;image traitée, avec l&#39;analyse appliquée. </li></ol> Les résultats représentatifs: Après toutes les images sont analysées, les données peuvent être visualisées au sein Cellenger ou exportées vers un logiciel tiers (Excel, GraphPad Prism) pour une analyse approfondie et de représentation graphique. La figure 2 montre les images archivées numériser à partir d&#39;un véhicule et un traité BCI-bien traités, avec et sans CNT analyse appliquée. Figure 1 documents un effet dose-dépendant de la BCI sur l&#39;expression du gène rapporteur GFP dans les Tg (dusp6: d2EGFP) PT6 embryons. La concentration nécessaire pour obtenir une réponse maximale moitié (50) était d&#39;environ 12 um. <img src="/files/ftp_upload/1900/1900fig1.jpg" alt="Figure 2" /> Figure 2 Capture d&#39;image automatisée et l&#39;analyse des embryons de poissons zèbres transgéniques. (A) du véhicule DMSO traitée Tg (dusp6: d2eGFP) PT6 embryon au 30hpf. (B) des embryons transgéniques traitées avec 20μM BCI montrent des augmentations dans l&#39;expression de la GFP. (C) image de l&#39;embryon à partir de (A) après l&#39;exécution de Definiens ruleset de trouver expression de la GFP dans la tête. Taches orange indiquent où le logiciel mesure l&#39;intensité de la GFP. (D) image de l&#39;embryon à partir de (B) après l&#39;exécution de Definiens ruleset de trouver expression de la GFP dans la tête. Taches orange indiquent où le logiciel mesure l&#39;intensité de la GFP. Notez que le jeu de règles a été en mesure de trouver un espace élargi de l&#39;expression de GFP dans les embryons traités.

<ol>
	<li>Molina, G. A., Watkins, S. C., Tsang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+FGF+reporter+transgenic+zebrafish+and+their+utility+in+chemical+screens.">Generation of FGF reporter transgenic zebrafish and their utility in chemical screens.</a> BMC Dev Biol. 7, 62-62 (2007).</li><li>Vogt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+image-based+phenotypic+analysis+in+zebrafish+embryos.">Automated image-based phenotypic analysis in zebrafish embryos.</a> Dev Dyn. 238, 656-663 (2009).</li><li>Molina, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+chemical+screening+reveals+an+inhibitor+of+Dusp6+that+expands+cardiac+cell+lineages.">Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages.</a> Nat Chem Biol. 5, 680-687 (2009).</li></ol>

Un des principaux facteurs limitant le débit d&#39;écrans chimiques poisson zèbre est l&#39;absence de méthodologie pour traiter systématiquement les plaques 96 puits pour l&#39;imagerie et l&#39;analyse. Parce que les images des organismes multicellulaires complexes, faible contraste, et de nature hétérogène, les algorithmes existants d&#39;analyse d&#39;image ne parviennent pas à détecter et quantifier des structures spécifiques au sein de l&#39;organisme. La plupart des écrans chimiques publiée le poisson-zèbre donc impliquer un examen visuel de chaque puits par un personnel dévoué pendant toute la procédure. Cela empêche généralement la génération de lectures numériques et un archivage complet des images d&#39;écran et les données. Par ailleurs, l&#39;évaluation manuelle élimine plusieurs avantages clés de l&#39;image basée sur le dépistage, à savoir la capacité d&#39;effectuer des analyses rétrospectives de performances de l&#39;écran, pour examiner les changements phénotypiques qui ne sont pas l&#39;objectif principal de la campagne de dépistage (par exemple, la toxicité ou des anomalies du développement) et de traverser référence les données de dépistage avec des écrans passées ou à venir en utilisant la bibliothèque même composé. Dans ce rapport, nous mettons en évidence la méthodologie pour l&#39;imagerie automatisée et d&#39;analyse des embryons de poissons zèbres en plaques multipuits, sans intervention de l&#39;utilisateur. Nous capture d&#39;image automatisée sur un laser ultra ImageXpress confocale à balayage lecteur (Molecular Devices, Sunnyvale, CA) à l&#39;image de Tg (dusp6: d2EGFP) PT6 embryons en plaques de 96 puits et développé un algorithme d&#39;analyse d&#39;images basée sur la technologie Definiens «Cognition réseau que la GFP quantifiés expression dans les têtes des embryons transgéniques. La méthode livré des réponses classées et quantifié l&#39;activité in vivo d&#39;une petite molécule d&#39;activateur de FGF de signalisation. Des résultats similaires ont été obtenus avec la II épifluorescence basé ArrayScan (Cellomics Inc, Pittsburgh, PA) documentant qu&#39;il est possible de mettre en oeuvre la capture d&#39;image automatisée des embryons de poissons zèbres sur une variété de lecteurs de plaques disponibles dans le commerce 2. Le développement de la méthodologie pour analyser automatiquement les images organisme multicellulaire éliminé le besoin de notation visuelle par un observateur humain et a permis à la génération et l&#39;archivage des données numériques et des images pour l&#39;analyse rétrospective et des comparaisons avec des écrans de demain.

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development of automated imaging and analysis for zebrafish chemical screens.

Nous démontrons l&#39;application de la base d&#39;images à haute teneur de dépistage (HCS) une méthodologie pour identifier de petites molécules qui peuvent moduler la voie FGF / RAS / MAPK dans des embryons de poissons zèbres. L&#39;embryon de poisson zèbre est un système idéal pour les écrans chimiques in vivo à haute teneur. L&#39;embryon de 1 jour est d&#39;environ vieille 1mm de diamètre et peuvent être facilement rangés dans des boîtes de 96 puits, un format standard pour le criblage haut débit. Durant la première journée de développement, les embryons sont transparents avec la plupart des principaux organes présents, permettant ainsi la visualisation de la formation des tissus durant l&#39;embryogenèse. L&#39;automatisation complète d&#39;écrans chimiques poisson zèbre est toujours un défi, cependant, en particulier dans le développement de l&#39;acquisition automatique d&#39;images et d&#39;analyse. Nous avons déjà généré une ligne journaliste transgénique qui exprime la protéine fluorescente verte (GFP) sous le contrôle de l&#39;activité du FGF et démontré leur utilité dans les écrans chimiques<sup&gt; 1</sup&gt;. Afin d&#39;établir une méthodologie pour les écrans à haut débit organisme tout entier, nous avons développé un système automatisé pour l&#39;imagerie et l&#39;analyse des embryons de poissons zèbres moins 24-48 heures après fécondation (HPF) en plaques de 96 puits<sup&gt; 2</sup&gt;. Dans cette vidéo, nous mettons en évidence les procédures d&#39;rangeant embryons transgéniques en plaques multipuits à 24hpf et l&#39;ajout d&#39;une petite molécule (BCI) qui hyperactivates FGF de signalisation<sup&gt; 3</sup&gt;. Les plaques sont incubées pendant 6 heures, suivie par l&#39;ajout de tricaïne pour anesthésier les larves avant d&#39;imagerie automatisée sur un Molecular Devices ImageXpress Ultra confocale à balayage laser lecteur de HCS. Les images sont traitées par le logiciel Definiens Developer utilisant un algorithme de technologie Cognition réseau que nous avons développé pour détecter et quantifier l&#39;expression de la GFP dans les têtes des embryons transgéniques. Dans cet exemple nous mettons en évidence la capacité de l&#39;algorithme pour mesurer la dose-dépendante des effets de la BCI sur l&#39;expression du gène rapporteur GFP dans les embryons traités.

Watch this Scientific Journal Video about Development of automated imaging and analysis for zebrafish chemical screens. at JoVE.com

Development of automated imaging and analysis for zebrafish chemical screens.

Nous rapportons le développement d&#39;un système automatisé pour l&#39;imagerie et l&#39;analyse des embryons de poisson zèbre transgénique dans des plaques multipuits. Ceci démontre la capacité de mesurer les effets dose-dépendante d&#39;une petite molécule, la BCI, le Fibroblast Growth Factor expression du gène rapporteur et de fournir la technologie pour établir des écrans à haut débit chimiques du poisson zèbre.

Développement de l&#39;imagerie automatisée et d&#39;analyse pour les écrans chimiques du poisson zèbre.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

development-automated-imaging-analysis-for-zebrafish-chemical

Research

JoVE Journal

Biology

11.3K vues.  University of Pittsburgh Drug Discovery Institute. Nous rapportons le développement d'un système automatisé pour l'imagerie et l'analyse des embryons de poisson zèbre transgénique dans des plaques multipuits. Ceci démontre la capacité de mesurer les effets dose-dépendante d'une petite molécule, la BCI, le Fibroblast Growth Factor expression du gène rapporteur et de fournir la technologie pour établir des écrans à haut débit chimiques du poisson zèbre.

Vidéo: Development of automated imaging and analysis for zebrafish chemical screens.

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Un neuronale et astrocytes de co-culture de dosage pour l&#39;analyse du contenu haute de la neurotoxicité

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Recherche

Biologie

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

Analyse semi-automatisée Heartbeat optique de petits coeurs

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

La transplantation de blastomères exprimant la GFP pour l&#39;imagerie en direct sur le développement de la rétine et du cerveau dans les embryons de poissons zèbres chimériques

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

Glycanes imagerie dans embryons de poissons zèbres par marquage métabolique et Chimie Cliquez Bioorthogonal

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

Quantitatifs et automatisé à haut débit du génome à l&#39;échelle écrans ARNi dans C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Suivi cellule à l&#39;aide protéines photoconvertible cours de développement poisson zèbre

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Imagerie semi-automatique de la fluorescence spécifique de tissu dans embryons de poissons zèbres

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

Open Source Content Haute Analyse Utilisant automatisé Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Analyse de la fonction des cellules bêta à l’aide d’imagerie calcique résolution unicellulaire dans les îlots de poisson-zèbre

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Automated Detection and Analysis of Exocytosis

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic events in cell culture. To perform an unbiased, efficient identification and analysis of such events, a computer-vision based approach was developed and implemented in MATLAB. The analysis pipeline consists of a cell segmentation and exocytic-event identification algorithm. The computer-vision approach includes tools for investigating multiple parameters of single events, including the half-life of fluorescence decay and peak &#916;F/F, as well as whole-cell analysis of the frequency of exocytosis. These and other parameters of fusion are used in a classification approach to distinguish distinct fusion modes. Here a newly built GUI performs the analysis pipeline from start to finish. Further adaptation of Ripley&#39;s K function in R Studio is used to distinguish between clustered, dispersed, or random occurrence of fusion events in both space and time.

We developed automated computer vision software to detect exocytic events marked by pH-sensitive fluorescent probes. Here, we demonstrate the use of a graphical user interface and RStudio to detect fusion events, analyze and display spatiotemporal parameters of fusion, and classify events into distinct fusion modes.

Détection et analyse automatisées de l’exocytose

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic ...