Diese Arbeit wird durch die NICHD / NIH (1R01HD053287) zu Hukriede, NCI / NIH (P01 CA78039), um Vogt und NHLBI / NIH (1R01HL088016) zu Tsang finanziert.

1) Stellen Sie Zebrafisch Paarungen und BCI-Behandlung. <ol><li> Zwei Tage vor der Behandlung, zu identifizieren homozygoten männlichen Tg (dusp6: d2EGFP) pt6 Zebrafisch und Ort individuell in Paarung Tanks 1. Add-Separator und einer Wildtyp-AB * weiblichen Fische und lassen Sie bei der Paarung Käfigen übernachten. </li><li> Am nächsten Morgen nehmen Sie die Trennzeichen und sammeln Embryonen nach 1 Stunde. -Embryonen zu 100mm Gerichte mit E3 (5 mM NaCl, 0,17 mM KCl, 0,33 mM CaCl2, 0,33 mM MgSO4), Lösung, Inkubation bei 28 ° C für 6 Stunden. Entfernen Sie leere und unbefruchtete Embryonen, fügen frische E3-Lösung und Inkubation über Nacht. </li><li> Sortieren transgenen Embryonen für ähnliche Stufe (24hpf) und einheitliche GFP-Expression. Array Embryonen einzeln in jede Vertiefung einer 96-Well-Platte. </li><li> Entfernen Sie überschüssiges E3-Lösung mit der Pipette. Add 200 ul der E3-Lösung mit einem Multi-Kanal-Pipette in jede Vertiefung. </li><li> Fügen Sie die folgenden Verbindungen als aufgelistet: <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> A </td><td> 2μl DMSO </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl DMSO </td></tr><tr ><td> B </td><td> 2μl DMSO </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl DMSO </td></tr><tr ><td> C </td><td> 2μl DMSO </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl DMSO </td></tr><tr ><td> D </td><td> 2μl DMSO </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl DMSO </td></tr><tr ><td> E </td><td> 2μl DMSO </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl DMSO </td></tr><tr ><td> F </td><td> 2μl DMSO </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl DMSO </td></tr><tr ><td> G </td><td> 2μl DMSO </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mM) </td><td> 2μl DMSO </td></tr><tr ><td> H </td><td> 2μl DMSO </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl DMSO </td></tr></tbody></table> Endkonzentrationen sind 1 &amp;mgr;, 5 um, 10 um, 20 um, 25 um BCI in 1% DMSO. Abdeckplatte in Alufolie und inkubieren für 6 Stunden bei 28 ° C. </li></ol> 2) Automated Imaging von 96-Well-Platten und-analyse. <ol><li> Add 10 &amp;mgr; l der Tricaine (0,61 mm) in jede Vertiefung zu 30hpf Embryonen zu betäuben. </li><li> Lastplatte in Molecular Devices ImageXpress Ultra. </li><li> Öffnen Metaxpress Software. Wählen Sie eine Konfiguration mit 4-fach Objektiv und maximale Durchmesser der Blende, konfigurieren Laserautofokus auf Plattenboden erkennen und bestimmen optimale Z-Ebene in Regionen von Interesse zu erkennen. Erwerben Sie Bilder auf Anregung / Emissionswellenlängen 488/525 nm (GFP). Set Scan-Bereich als eine einzige Stelle 2000x2000 Pixel (4 x 4 mm) ohne Binning. Konfigurieren Laserleistung und gewinnen, so dass hellsten Regionen in positive Kontrolle Abbildungen müssen nicht sättigen. Überprüfen Sie die Bildaufnahme in mehreren positiven und negativen Kontroll-Wells. </li><li> Starten Platte zu scannen. Das Gerät wird automatisch erwerben Bildern aus allen Vertiefungen und archivieren sie in einer SQL Server-Datenbank. </li><li> Bilder hochladen in Definiens Developer mit dem Cellenger application module und ein Import-Filter, der Bild-Format und Datei-Struktur, die durch die Molecular Devices ImageXpress Ultra-Plattform generiert erkennt. </li><li> Prozess Bilder mit einer speziell entworfenen Cognition Network Technology Algorithmus (auch als einen Regelsatz). Der Algorithmus, den wir Sitte auf einer kürzlich veröffentlichten Regelsatz 2-Basis entwickelt wurde, ist entworfen, um automatisch zu identifizieren Embryo, Eigelb, und Kopf, und die Helligkeit und den Bereich der GFP-exprimierenden Kopf Strukturen zu quantifizieren. Wells, in denen kein Embryo nachgewiesen ist oder wo der Kopf kann nicht eindeutig zugeordnet werden (z. B. mit hoher Verbindung Toxizität oder unscharfe Bilder) werden aus der Analyse ausgeschlossen. Konfigurieren Sie den Algorithmus mit mehreren negativen Kontrolle Bilder (Fahrzeug behandelt) und der positiven Kontrolle Bilder (BCI) behandelt, so dass Kopf und Eigelb richtig in beide identifiziert. Setzen Sie ein &quot;Kopf-Strukturen&quot; Nachweisgrenze als Vielfaches der mittleren Eigelb Intensität bis zur Erkennung von hellen Kopf Strukturen entspricht die visuelle Beobachtung. Definieren Sie Parameter für den Export. In diesem Beispiel wurde die informative Parameter die gesamte integrierte Helligkeit des GFP-positive Regionen innerhalb des Kopfes, nachfolgend als &quot;totalen Kopf Strukturen Helligkeit&quot; (Abbildung 1). <img src="/files/ftp_upload/1900/1900fig2.jpg" alt="Abbildung 1" /> Abbildung 1 Graph depicitng die dosisabhängige Wirkung von BCI auf GFP-Expression in transgenen Embryos. </li><li> Führen Sie den Algorithmus, der auf allen Platten Bilder mit der integrierten Job-Scheduler. Die Definiens-Software verwendet verteiltes Rechnen auf mehreren Prozessoren (&quot;Engines&quot;), wodurch die gleichzeitige Analyse mehrerer Bilder. Der Algorithmus berechnet numerische Maßnahmen auf die Ergebnisse der Bildanalyse, die in der Datenbank gespeichert sind zusammen mit einer Kopie des zu bearbeitenden Bildes mit der Analyse angewendet wird. </li></ol> Repräsentative Ergebnisse: Nachdem alle Bilder analysiert werden, können die Daten innerhalb Cellenger visualisiert oder exportiert Software von Drittanbietern (Excel, GraphPad Prism) zur weiteren Analyse und grafische Darstellung. Abbildung 2 zeigt archivierte Bilder scannen aus einem Fahrzeug behandelt und gut, mit und ohne CNT-Analyse angewendet BCI-behandelt. Abbildung 1 Dokumente eine dosisabhängige Wirkung von BCI auf GFP Expression des Reportergens in Tg (dusp6: d2EGFP) pt6 Embryonen. Die Konzentration erforderlich, um eine halbmaximale Reaktion (EC50) zu entlocken war etwa 12 um. <img src="/files/ftp_upload/1900/1900fig1.jpg" alt="Abbildung 2" /> Abbildung 2 Automatisierte Bilderfassung und Analyse von transgenen Zebrafisch-Embryos. Pt6 Embryo 30hpf: (A) DMSO Fahrzeug Tg (d2EGFP dusp6) behandelt. (B) Transgene Embryonen mit 20 um BCI zeigen behandelt steigt in die GFP-Expression. (C) Bild von Embryos aus (A) nach dem Ausführen Definiens Regelsatz auf GFP-Expression in den Kopf zu finden. Orange Flecken bezeichnen, wo die Software GFP Intensität Maßnahmen. (D) Bild von Embryos aus (B) nach dem Ausführen Definiens Regelsatz auf GFP-Expression in den Kopf zu finden. Orange Flecken bezeichnen, wo die Software GFP Intensität Maßnahmen. Beachten Sie, dass der Regelsatz in der Lage, einen erweiterten Bereich der GFP-Expression in den behandelten Embryo zu finden war.

<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>

Ein wesentlicher Faktor, der den Durchsatz von Zebrafisch-chemischen Bildschirmen ist der Mangel an Methoden zur systematischen Prozess 96-Well-Platten für die Bildgebung und Analyse. Weil Bilder von vielzelligen Organismen komplexer, geringem Kontrast sind und heterogener Natur, nicht vorhandenes Bild-Analyse-Algorithmen zur Detektion und Quantifizierung spezifischer Strukturen innerhalb des Organismus. Die meisten veröffentlichten Zebrafisch chemische Bildschirme sind daher mit visuellen Prüfung der einzelnen Vertiefungen durch engagierte Mitarbeiter während des gesamten Verfahrens. Normalerweise verhindert die Erzeugung von numerischen Anzeigen und eine vollständige Archivierung der Bildschirm Bilder und Daten. Darüber hinaus entfällt die manuelle Auswertung einige wesentliche Vorteile von image-based Screening, das heißt die Fähigkeit, retrospektive Analysen von Screen-Performance durchzuführen, um phänotypische Veränderungen, die nicht der primäre Fokus des Screening-Kampagne (zB Toxizität oder Entwicklungsstörungen) zu untersuchen und Kreuz -Referenz-Screening-Daten mit Vergangenheit oder Zukunft Bildschirme mit der gleichen Verbindung Bibliothek. In diesem Bericht haben wir Highlight Methodik zur automatisierten Abbildung und Analyse von Zebrafisch-Embryonen in Mikrotiterplatten ohne Eingreifen des Benutzers. Wir automatisierte Bildaufnahme auf einem ImageXpress Ultra-Laser-Scanning-konfokale (Molecular Devices, Sunnyvale, CA), um Bild Tg (dusp6: d2EGFP) pt6 Embryonen in 96-Well-Platten und entwickelte eine Bildanalyse-Algorithmus auf Definiens Cognition Network Technology, die quantifiziert GFP basiert Ausdruck in den Köpfen der transgenen Embryos. Die Methode geliefert abgestuften Antworten und quantifiziert die in-vivo-Aktivität eines kleinen Moleküls Aktivator von FGF-Signalisierung. Ähnliche Ergebnisse wurden mit der Epifluoreszenz-basierte ArrayScan II (Cellomics Inc., Pittsburgh PA) erhalten zu dokumentieren, dass es möglich ist, automatisierte Bildaufnahme von Zebrafisch-Embryonen auf eine Vielzahl von kommerziell erhältlichen Platereader 2 getroffen haben. Die Entwicklung von Methoden zur automatischen Analyse vielzelligen Organismus Bildern eliminiert die Notwendigkeit für die visuelle Scoring durch einen menschlichen Beobachter und aktiviert die Generierung und Archivierung von numerischen Daten und Bilder für retrospektive Analysen und Vergleiche mit Zukunft Bildschirme.

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<td>Tricaine (MS222)</td>
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development of automated imaging and analysis for zebrafish chemical screens.

Wir demonstrieren die Anwendung der Image-basierten High-Content-Screening (HCS)-Methode, um kleine Moleküle, die Modulation der FGF / RAS / MAPK-Weg kann in Zebrafisch-Embryonen zu identifizieren. Der Zebrafisch Embryo ist ein ideales System für In-vivo-High-Content-chemischen Bildschirme. Die 1-Tage alten Embryo ist ca. 1mm im Durchmesser und lässt sich einfach in 96-Well-Platten, ein Standardformat für das Hochdurchsatz-Screening angeordnet werden. Während der ersten Tage der Entwicklung, sind Embryonen transparent mit den meisten der wichtigsten Organe vorhanden, so dass Visualisierung von Gewebe-Bildung während der Embryogenese. Die vollständige Automatisierung der Zebrafisch-chemischen Bildschirmen ist immer noch eine Herausforderung, aber vor allem in der Entwicklung von automatisierten Bildaufnahme und-analyse. Wir zuvor generierte eine transgene Reporter Linie, grün fluoreszierende Protein (GFP) bringt unter der Kontrolle von FGF-Aktivität und demonstriert deren Nutzen in der chemischen-Bildschirme<sup&gt; 1</sup&gt;. Um festzustellen, Methodik für hohen Durchsatz gesamten Organismus Bildschirme, entwickelten wir ein System für die automatisierte Abbildung und Analyse von Zebrafisch-Embryonen bei 24-48 Stunden nach der Befruchtung (HPF) in 96-Well-Platten<sup&gt; 2</sup&gt;. In diesem Video stellen wir die Verfahren für Anordnen transgenen Embryonen in Mikrotiterplatten bei 24hpf und die Zugabe eines kleinen Moleküls (BCI), dass FGF-Signale hyperactivates<sup&gt; 3</sup&gt;. Die Platten werden für 6 Stunden durch die Zugabe von Tricaine Larven vor Automated Imaging auf einem Molecular Devices ImageXpress Ultra-Laser-Scanning-konfokale HCS Leser betäuben gefolgt inkubiert. Die Bilder werden von Definiens Developer-Software mit einer Cognition Network Technology Algorithmus, die wir entwickelt, um nachzuweisen und zu quantifizieren Expression von GFP in den Köpfen der transgenen Embryos verarbeitet. In diesem Beispiel heben wir die Fähigkeit des Algorithmus, um eine dosisabhängige Wirkung von BCI auf GFP Reportergen-Expression in den behandelten Embryonen zu messen.

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.

Wir berichten über die Entwicklung eines Systems zur automatisierten Abbildung und Analyse von Zebrafisch-transgenen Embryos in Mikrotiterplatten. Dies demonstriert die Fähigkeit, dosisabhängige Wirkung eines kleinen Moleküls, BCI, auf Fibroblast Growth Factor Expression des Reportergens zu messen und Technologie für den Aufbau High-Throughput-Zebrafisch chemische Bildschirme.

Entwicklung von automatisierten Imaging und Analyse für Zebrafisch chemische Bildschirme.

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 Aufrufe.  University of Pittsburgh Drug Discovery Institute. Wir berichten über die Entwicklung eines Systems zur automatisierten Abbildung und Analyse von Zebrafisch-transgenen Embryos in Mikrotiterplatten. Dies demonstriert die Fähigkeit, dosisabhängige Wirkung eines kleinen Moleküls, BCI, auf Fibroblast Growth Factor Expression des Reportergens zu messen und Technologie für den Aufbau High-Throughput-Zebrafisch chemische Bildschirme.

Serie: 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.

Eine Neuronale und Astrocyte Co-Kultur-Assay für High Content Analyse der Neurotoxizität

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

Forschung

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.

Semi-automatische optische Heartbeat-Analyse von kleinen Herzen

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.

Die Transplantation von GFP-exprimierenden Blastomeren für Live Imaging von Retinal und Entwicklung des Gehirns in Chimäre Zebrafischembryonen

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.

Imaging Glykane in Zebrafisch-Embryonen durch metabolische Markierung und Bioorthogonale Click-Chemie

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.

Quantitative und automatisierte Hochdurchsatz-genomweiten RNAi-Screens in 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.

Handy-Tracking Mit Photoconvertible Proteine ​​während der Zebrafisch-Entwicklung

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.

Semi-Automated Imaging von Gewebespezifische Fluoreszenz-in Zebrafischembryonen

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 High Content Analyse Unter Verwendung Automatische Fluorescence Lifetime Imaging Mikroskopie

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 der Beta-Zellfunktion mit einzelligen Auflösung Kalzium Imaging im Zebrafisch Inselchen

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.

Automatisierte Erkennung und Analyse von Exozytose

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