Questo lavoro è finanziato dal NICHD / NIH (1R01HD053287) per Hukriede, NCI / NIH (P01 CA78039) per Vogt, e NHLBI / NIH (1R01HL088016) per Tsang.

1) Impostare accoppiamenti zebrafish e trattamento BCI. <ol><li> Due giorni prima del trattamento, identificare omozigoti maschi Tg (dusp6: d2eGFP) PT6 zebrafish e luogo singolarmente in vasche di accoppiamento 1. Aggiungi separatore e un tipo di pesce selvatico AB * femminile e lasciare in gabbie di accoppiamento durante la notte. </li><li> La mattina seguente togliere il separatore e raccogliere embrioni dopo 1 ora. Trasferimento degli embrioni a 100 mm con piatti E3 (5 mM NaCl, 0,17 mM KCl, 0,33 mM CaCl2, 0,33 mM MgSO4), soluzione e incubare a 28 ° C per 6 ore. Rimuovere morto e non fecondate embrioni, aggiungere soluzione fresca E3, e incubare una notte. </li><li> Ordina embrioni transgenici per equivalente stadio (24hpf) e di espressione GFP uniforme. Embrioni Array individualmente in ciascun pozzetto di una piastra da 96 pozzetti. </li><li> Rimuovere l&#39;eccesso di soluzione E3 con una micropipetta. Aggiungere 200μl di soluzione E3 con un pipettatore multicanale in ciascun pozzetto. </li><li> Aggiungere i seguenti composti elencati: <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 (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </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 (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </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 (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </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 (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </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 (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </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 (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </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 (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </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 (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mm) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </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> Concentrazioni finali sono 1μM, 5 micron, 10μM, 20μM, BCI 25 micron in 1% di DMSO. Piastra di copertura in fogli di alluminio e incubare per 6 ore a 28 ° C. </li></ol> 2) imaging automatico di piastre a 96 pozzetti e analisi. <ol><li> Aggiungere 10μl di tricaine (0.61mM) in ciascun pozzetto per anestetizzare gli embrioni 30hpf. </li><li> Piatto di carico in Molecular Devices ImageXpress Ultra. </li><li> Aprire Metaxpress software. Scegliere una configurazione con un obiettivo 4x e diametro massimo foro stenopeico, configurare laser messa a fuoco automatica per rilevare fondo piatto, e determinare ottimale Z-piano per rilevare le regioni di interesse. Acquisire immagini a eccitazione / emissione di lunghezze d&#39;onda di 488/525 nm (GFP). Impostare la scansione area come un unico sito di 2000x2000 pixel (4 x 4 mm) senza binning. Configurare potenza del laser e guadagno in modo che le regioni più luminose nelle immagini di controllo positivo non saturare. Confermare l&#39;acquisizione corretta di immagini in diversi pozzetti di controllo positivi e negativi. </li><li> Avviare la scansione piatto. Lo strumento automaticamente acquisire immagini da tutti i pozzetti e archiviarli in un database SQL Server. </li><li> Caricare immagini in Developer Definiens utilizzando il modulo applicativo Cellenger e un filtro di importazione che riconosce il formato di immagine e la struttura del file generato dalla piattaforma Molecular Devices ImageXpress Ultra. </li><li> Elaborare le immagini con un algoritmo progettato Technology Network Cognizione (designato anche un insieme di regole). L&#39;algoritmo che abbiamo sviluppato in base personalizzati su un insieme di regole recentemente pubblicato 2, è stato progettato per identificare automaticamente embrione, tuorlo, e la testa, e di quantificare la luminosità e la zona di GFP che esprimono le strutture testa. Wells in cui viene rilevato nessun embrione o in cui la testa non può essere assegnato senza ambiguità (ad esempio, con tossicità composto alta o immagini sfocate) sono esclusi dall&#39;analisi. Configurare l&#39;algoritmo utilizzando diverse immagini di controllo negativo (veicolo trattati) e le immagini di controllo positivo (BCI trattati) in modo tale che la testa e tuorlo sono identificati correttamente in entrambi. Impostare una &quot;testa di strutture&quot; soglia di rilevamento come multipli di intensità medio tuorlo fino rilevamento di strutture testa brillanti partite osservazione visiva. Definire i parametri per l&#39;esportazione. In questo esempio, il parametro più informativo è stata la luminosità totale integrata di GFP-positive regioni all&#39;interno della testa, di seguito definito &quot;totale luminosità testa strutture&quot; (Figura 1). <img src="/files/ftp_upload/1900/1900fig2.jpg" alt="figura 1" /> Figura 1 Grafico depicitng gli effetti dose-dipendente della BCI sull&#39;espressione GFP negli embrioni transgenici. </li><li> Eseguire l&#39;algoritmo su tutte le immagini utilizzando la piastra integrata job scheduler. Il software Definiens utilizza il calcolo distribuito su diversi processori (&quot;motori&quot;), che consente l&#39;analisi simultanea di più immagini. L&#39;algoritmo calcola le misure numeriche sulla base dei risultati della analisi delle immagini, che vengono salvati nel database insieme ad una copia dell&#39;immagine trattati, con l&#39;analisi applicata. </li></ol> Rappresentante dei risultati: Dopo tutte le immagini sono analizzate, i dati possono essere visualizzati all&#39;interno Cellenger o esportati a software di terze parti (Excel, GraphPad Prism) per ulteriori analisi e di rappresentazione grafica. La figura 2 mostra le immagini archiviate scansione da un veicolo e un trattato BCI-trattati bene, con e senza CNT analisi applicata. La figura 1 documenta un effetto dose-dipendente della BCI sull&#39;espressione genica giornalista GFP nel Tg (dusp6: d2EGFP) PT6 embrioni. La concentrazione di richieste per provocare una risposta mezzo massimo (EC50) è stato di circa 12 micron. <img src="/files/ftp_upload/1900/1900fig1.jpg" alt="figura 2" /> Figura 2 Immagine di acquisizione automatica e l&#39;analisi di embrioni di zebrafish transgenico. (A) veicolo DMSO trattati Tg (dusp6: d2eGFP) PT6 embrione a 30hpf. (B) gli embrioni transgenici trattati con spettacolo 20μM BCI aumenti di espressione GFP. (C) Immagine di embrione da (A) dopo l&#39;esecuzione Definiens insieme di regole per trovare espressione GFP nella testa. Macchie arancione indicano dove il software misura l&#39;intensità GFP. (D) Immagine di embrione da (B) dopo l&#39;esecuzione Definiens insieme di regole per trovare espressione GFP nella testa. Macchie arancione indicano dove il software misura l&#39;intensità GFP. Si noti che l&#39;insieme di regole è stata in grado di trovare uno spazio allargato di espressione GFP nell&#39;embrione trattati.

<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 fattore importante che limita la velocità di schermi chimici zebrafish è la mancanza di una metodologia per elaborare sistematicamente le piastre a 96 pozzetti per l&#39;imaging e analisi. Poiché le immagini degli organismi multicellulari sono complessi, a basso contrasto, e di natura eterogenea, esistenti algoritmi di analisi delle immagini non riescono a individuare e quantificare le strutture specifiche all&#39;interno dell&#39;organismo. La maggior parte pubblicati schermi chimici zebrafish quindi coinvolgere l&#39;esame visivo di singoli pozzi da parte di personale dedicato tutta la procedura. Questo impedisce che di solito la generazione di letture numeriche e una completa archiviazione delle immagini dello schermo e dati. Inoltre, la valutazione manuale eliminates alcuni vantaggi chiave di image-based di screening, cioè la capacità di condurre analisi retrospettive di prestazioni dello schermo, per esaminare i cambiamenti fenotipici che non erano l&#39;obiettivo primario della campagna di screening (per esempio, la tossicità o difetti di sviluppo) e di attraversare di riferimento dei dati di screening con schermi passate o future utilizzando la stessa libreria composto. In questo rapporto si evidenzia la metodologia per l&#39;imaging e l&#39;analisi automatizzata di embrioni di zebrafish in piastre a pozzetti multipli senza l&#39;intervento dell&#39;utente. Noi la cattura delle immagini automatico su un laser a scansione confocale ImageXpress Ultra lettore (Molecular Devices, Sunnyvale, CA) per l&#39;immagine Tg (dusp6: d2EGFP) PT6 embrioni in 96 pozzetti e sviluppato un algoritmo di analisi di immagine basato sulla tecnologia Cognizione Definiens &#39;di rete che GFP quantificati espressione nelle teste degli embrioni transgenici. Il metodo consegnato risposte classificato e quantificato in vivo di una piccola molecola attivatore di FGF segnalazione. Risultati simili sono stati ottenuti con l&#39;epifluorescenza a base ArrayScan II (Cellomics Inc., Pittsburgh PA) documenta che è possibile implementare la cattura automatica delle immagini di embrioni di zebrafish su una varietà di lettori di piastre in commercio 2. Lo sviluppo della metodologia per analizzare automaticamente le immagini organismo pluricellulare eliminato la necessità di segnare visivo da un osservatore umano e ha permesso la generazione e l&#39;archiviazione di dati numerici e immagini per l&#39;analisi retrospettiva e confronti con schermi futuro.

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

Dimostriamo l&#39;applicazione di image-based di alto contenuto di screening (HCS) metodologia per identificare piccole molecole in grado di modulare l&#39;FGF / RAS / MAPK in embrioni di zebrafish. L&#39;embrione di zebrafish è un sistema ideale per in vivo di alto contenuto schermi chimici. L&#39;1-day embrione è di circa 1 mm di diametro e può essere facilmente disposte in piastre a 96 pozzetti, un formato standard per lo screening ad alto rendimento. Durante il primo giorno di sviluppo, gli embrioni sono trasparenti con la maggior parte dei principali organi presenti, permettendo così la visualizzazione della formazione di tessuto durante l&#39;embriogenesi. La completa automazione degli schermi chimici zebrafish è ancora una sfida, tuttavia, in particolare nello sviluppo di acquisizione delle immagini e analisi automatizzate. Abbiamo generato in precedenza una linea giornalista transgenico che esprime la proteina fluorescente verde (GFP) sotto il controllo di attività FGF e dimostrato la loro utilità negli schermi chimici<sup&gt; 1</sup&gt;. Per stabilire la metodologia per schermi ad alta velocità intero organismo, abbiamo sviluppato un sistema automatizzato per l&#39;imaging e l&#39;analisi di embrioni di zebrafish a 24-48 ore dopo la fecondazione (HPF) in piastre da 96 pozzetti<sup&gt; 2</sup&gt;. In questo video possiamo evidenziare le procedure per arraying embrioni transgenici in piastre a pozzetti multipli a 24hpf e l&#39;aggiunta di una piccola molecola (BCI) che hyperactivates FGF segnalazione<sup&gt; 3</sup&gt;. Le piastre vengono incubate per 6 ore seguita dall&#39;aggiunta di tricaine per anestetizzare le larve prima di imaging automatizzato su un Molecular Devices ImageXpress Ultra scansione laser confocale HCS lettore. Le immagini vengono elaborate da un software Developer Definiens utilizzando un algoritmo di rete Cognition La tecnologia che abbiamo sviluppato per individuare e quantificare l&#39;espressione di GFP nelle teste degli embrioni transgenici. In questo esempio si segnala la capacità dell&#39;algoritmo di misura dose-dipendente della BCI effetti sull&#39;espressione dei geni GFP giornalista negli embrioni trattati.

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.

Riportiamo lo sviluppo di un sistema automatizzato per l&#39;imaging e l&#39;analisi di zebrafish embrioni transgenici in piastre a pozzetti multipli. Questo dimostra la capacità di misurare gli effetti dose-dipendente di una piccola molecola, BCI, sull&#39;espressione del gene del fattore di crescita dei fibroblasti giornalista e fornire tecnologia per stabilire high-throughput schermi chimici zebrafish.

Sviluppo di imaging automatizzati e analisi chimiche per gli schermi zebrafish.

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 Views.  University of Pittsburgh Drug Discovery Institute. Riportiamo lo sviluppo di un sistema automatizzato per l'imaging e l'analisi di zebrafish embrioni transgenici in piastre a pozzetti multipli. Questo dimostra la capacità di misurare gli effetti dose-dipendente di una piccola molecola, BCI, sull'espressione del gene del fattore di crescita dei fibroblasti giornalista e fornire tecnologia per stabilire high-throughput schermi chimici zebrafish.

Video: 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 e astrociti Co-Cultura Assay per l&#39;analisi dei contenuti elevati di neurotossicità

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

Ricerca

Biologia

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.

Semiautomatici Analisi Heartbeat ottica di piccoli cuori

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.

Il trapianto di GFP che esprimono blastomeri per dell&#39;immagine in tempo reale dello sviluppo della retina e del cervello in embrioni di zebrafish Chimerico

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.

Glicani Imaging in embrioni di zebrafish con marcatura metabolica e Chimica Clicca 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.

Quantitative e automatica High-throughput Genome-wide Schermi RNAi 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.

Cella di rilevamento utilizzando proteine ​​Photoconvertible durante lo sviluppo Zebrafish

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.

Imaging semi-automatico di fluorescenza tessuto-specifica in embrioni di zebrafish

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 alto contenuto di analisi Utilizzando Automated 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.

Analisi della funzione Beta-cellulare usando la formazione immagine del calcio cella singola risoluzione in Zebrafish isolotti

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.

Rilevamento e analisi automatizzati dell'esocitosi

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