Este trabajo está financiado por el NICHD / NIH (1R01HD053287) para Hukriede, NCI / NIH (P01 CA78039) a Vogt, y el NHLBI / NIH (1R01HL088016) a Tsang.

1) Establecer los apareamientos pez cebra y el tratamiento de la ICC. <ol><li> Dos días antes del tratamiento, identificar los homocigotos masculinos Tg (dusp6: d2eGFP) pt6 pez cebra y el lugar de forma individual en un tanques de apareamiento. Añadir separador y un tipo de peces silvestres AB * mujer y dejar en las jaulas de apareamiento durante la noche. </li><li> A la mañana siguiente quitar el separador, y recoger los embriones después de 1 hora. La transferencia de embriones a los platos de 100 mm con E3 (5 mM NaCl, 0,17 mM KCl, 0,33 mM CaCl2, 0,33 mM MgSO4), la solución y se incuba a 28 ° C durante 6 horas. Eliminar embriones muertos y sin fertilizar, agregue la solución E3 fresca, y se incuba durante la noche. </li><li> Ordenar embriones transgénicos para la etapa similar (24hpf) y la expresión de GFP uniforme. Embriones matriz de forma individual en cada pocillo de una placa de 96 pocillos. </li><li> Retire el exceso de solución E3 con una micropipeta. Agregar 200μl de la solución de E3 con una pipeta de canales múltiples en cada pozo. </li><li> Añadir los siguientes compuestos como se indica: <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 (1 mM) </td><td> 2μl BCl (2 mM) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2 mM) </td><td> 2μl BCl (2.5mm) </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 (1 mM) </td><td> 2μl BCl (2 mM) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2 mM) </td><td> 2μl BCl (2.5mm) </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 (1 mM) </td><td> 2μl BCl (2 mM) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2 mM) </td><td> 2μl BCl (2.5mm) </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 (1 mM) </td><td> 2μl BCl (2 mM) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2 mM) </td><td> 2μl BCl (2.5mm) </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 (1 mM) </td><td> 2μl BCl (2 mM) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2 mM) </td><td> 2μl BCl (2.5mm) </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 (1 mM) </td><td> 2μl BCl (2 mM) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2 mM) </td><td> 2μl BCl (2.5mm) </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 (1 mM) </td><td> 2μl BCl (2 mM) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2 mM) </td><td> 2μl BCl (2,5 millones deM) </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 (1 mM) </td><td> 2μl BCl (2 mM) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0,1 mm) </td><td> 2μl BCl (0,5 mm) </td><td> 2μl BCl (1 mM) </td><td> 2μl BCl (2 mM) </td><td> 2μl BCl (2.5mm) </td><td> 2μl DMSO </td></tr></tbody></table> Las concentraciones finales son 1μM, 5μM, 10μM, 20μM, 25μM BCI en DMSO al 1%. Cubrir la placa con papel de aluminio e incubar durante 6 horas a 28 ° C. </li></ol> 2) automática de imágenes de placas de 96 pozos y el análisis. <ol><li> Añadir 10μl de tricaína (0.61mM) a cada pocillo para anestesiar a los embriones 30hpf. </li><li> Plato de carga en dispositivos ultra Imagexpress molecular. </li><li> Abra el software Metaxpress. Elija una configuración con un objetivo de 4x y el diámetro del agujero de alfiler máximo, configurar láser de enfoque automático para detectar la placa inferior, y determinar óptimo plano Z para detectar regiones de interés. Adquirir imágenes en longitudes de onda de excitación / emisión de 488/525 nm (GFP). Establecer como zona de exploración de un sitio único de 2000x2000 pixeles (4 x 4 mm), sin hurgar en la basura. Configure la potencia del láser y la ganancia de tal manera que las regiones brillantes en las imágenes de control positivo no sature. Confirmar la adquisición de una imagen adecuada en varios pocillos de control positivo y negativo. </li><li> Iniciar la exploración de la placa. El instrumento automáticamente obtener imágenes de todos los pozos y los archivan en una base de datos del servidor SQL. </li><li> Cargar las imágenes en Definiens desarrollador utilizando el módulo de la aplicación Cellenger y un filtro de importación, que reconoce el formato de imagen y estructura de los archivos generados por la plataforma de dispositivos moleculares Imagexpress Ultra. </li><li> Procesar imágenes con un algoritmo diseñado a medida Cognition Network Technology (también conocida como un conjunto de reglas). El algoritmo, que a medida desarrollado en base a un conjunto de reglas ha publicado recientemente dos, está diseñado para identificar de forma automática embrión, la yema, y la cabeza, y para cuantificar el brillo y el área de las buenas prácticas agrarias que expresan las estructuras de la cabeza. Pozos en los que se detecta ningún embrión o cuando la cabeza no puede ser claramente asignado (por ejemplo, con la toxicidad de los compuestos de alta o fuera de foco imágenes) se excluyeron del análisis. Configure el algoritmo utilizando varias imágenes de control negativo (tratado con vehículo) y una imagen positiva de control (BCI tratados) de tal manera que la cabeza y la yema estén correctamente identificados en ambos. Establecer una &quot;cabeza de las estructuras&quot; umbral de detección como múltiplos de la intensidad media de la yema hasta la detección de las estructuras de la cabeza brillante coincide con la observación visual. Definir los parámetros para la exportación. En este ejemplo, el parámetro más informativo fue el brillo total integrado de GFP-positivas regiones dentro de la cabeza, en adelante denominado &quot;el brillo total de la cabeza las estructuras&quot; (Figura 1). <img src="/files/ftp_upload/1900/1900fig2.jpg" alt="la figura 1" /> Figura 1 Gráfico depicitng los efectos dependientes de la dosis de BCI en la expresión de GFP en embriones transgénicos. </li><li> Ejecutar el algoritmo en todas las imágenes de placas utilizando el planificador de tareas integrado. El software utiliza Definiens computación distribuida en varios procesadores (&quot;motores&quot;), lo que permite el análisis simultáneo de múltiples imágenes. El algoritmo calcula las medidas numéricas sobre la base de los resultados de los análisis de imágenes, que se guardan en la base de datos junto con una copia de la imagen procesada, con el análisis aplicado. </li></ol> Los resultados representativos: Después de todas las imágenes son analizadas, los datos pueden ser visualizados en Cellenger o exportarse a software de terceros (Excel, GraphPad Prism) para su posterior análisis y representación gráfica. La figura 2 muestra archivar imágenes escaneadas de un tratado con el vehículo y BCI-tratado bien, con y sin el análisis aplicado de la CNT. Figura 1 Los documentos de un efecto dosis-dependiente de BCI en la expresión del gen GFP reportero en Tg (dusp6: d2EGFP) pt6 embriones. La concentración requerida para obtener una respuesta media máxima (EC50) fue de aproximadamente 12 micras. <img src="/files/ftp_upload/1900/1900fig1.jpg" alt="la figura 2" /> Figura 2 Captura automática de imágenes y análisis de los embriones de pez cebra transgénica. (A) del vehículo DMSO tratados Tg (dusp6: d2eGFP) pt6 embrión en 30hpf. (B) los embriones transgénicos tratados con 20μM BCI mostrar aumentos en la expresión de GFP. (C) Imagen de un embrión a partir de (A) después de ejecutar Definiens conjunto de reglas para encontrar la expresión de GFP en la cabeza. Manchas de color naranja denotan que el software mide la intensidad de las buenas prácticas agrarias. (D) Imagen de un embrión a partir de (B) después de ejecutar Definiens conjunto de reglas para encontrar la expresión de GFP en la cabeza. Manchas de color naranja denotan que el software mide la intensidad de las buenas prácticas agrarias. Tenga en cuenta que el conjunto de reglas fue capaz de encontrar un área más amplia de la expresión de GFP en el embrión tratado.

<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 factor importante que limita el rendimiento de las pantallas de química pez cebra es la falta de una metodología sistemática para procesar placas de 96 pozos de imágenes y análisis. Como las imágenes de los organismos multicelulares complejos están contraste, baja, y de naturaleza heterogénea, los algoritmos de análisis de imagen no detectar y cuantificar estructuras específicas dentro del organismo. La mayoría de las pantallas de química publicado pez cebra por lo tanto, conlleve el examen visual de los pozos individuales por parte del personal dedicado durante todo el procedimiento. Esto por lo general evita la generación de lecturas numéricas y un completo archivo de imágenes de la pantalla y los datos. Por otra parte, la evaluación manual elimina varias ventajas claves de la imagen basada en detección, es decir, la capacidad de realizar análisis retrospectivos de rendimiento de la pantalla, para examinar los cambios fenotípicos que no eran el foco principal de la campaña de detección (por ejemplo, toxicidad o defectos en el desarrollo) y la cruz de referencia los datos del cribado con pantallas pasado o el futuro uso de la biblioteca de un mismo compuesto. En este informe se destaca la metodología para la imagen automatizada y el análisis de embriones de pez cebra en placas de pocillos múltiples, sin intervención del usuario. Hemos automatizado de captura de imágenes en un láser confocal de barrido ultra ImageXpress lector (Molecular Devices, Sunnyvale, CA) a la imagen de Tg (dusp6: d2EGFP) pt6 embriones en 96 placas y desarrollado un algoritmo de análisis de imagen basado en la tecnología Definiens &#39;Cognición de red que las buenas prácticas agrarias cuantificados expresión en las cabezas de los embriones transgénicos. El método de entrega respuestas graduadas y cuantificar la actividad in vivo de una pequeña molécula activadora de la señalización de FGF. Se obtuvieron resultados similares con la II epifluorescencia basado ArrayScan (Cellomics Inc., Pittsburgh PA) la documentación que es posible llevar a cabo la captura automática de imágenes de embriones de pez cebra en una variedad de lectores de placas disponibles en el mercado 2. El desarrollo de la metodología para analizar automáticamente las imágenes multicelulares organismo elimina la necesidad de puntuación visual por un observador humano y ha permitido la generación y archivo de los datos numéricos e imágenes para el análisis retrospectivo y comparaciones con las pantallas en el futuro.

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development of automated imaging and analysis for zebrafish chemical screens.

Se demuestra la aplicación de la imagen basada en el contenido de alta selección (HCS) metodología para identificar pequeñas moléculas que pueden modular la vía FGF / RAS / MAPK en embriones de pez cebra. El embrión de pez cebra es un sistema ideal para las pantallas en vivo químicos de alto contenido. El embrión de 1 día es de aproximadamente 1 mm de diámetro y puede ser fácilmente dispuestos en placas de 96 pozos, un formato estándar para la detección de alto rendimiento. Durante el primer día de desarrollo, los embriones son transparentes, con la mayor parte de los órganos actuales más importantes, lo que permite la visualización de la formación de tejidos durante la embriogénesis. La automatización completa de las pantallas de química pez cebra sigue siendo un reto, sin embargo, particularmente en el desarrollo de la adquisición automática de imágenes y análisis. Anteriormente hemos generado una línea reportero transgénico que expresa la proteína verde fluorescente (GFP) bajo el control de la actividad de FGF y ha demostrado su utilidad en las pantallas de productos químicos<sup&gt; 1</sup&gt;. Para establecer la metodología para las pantallas de alto rendimiento todo el organismo, hemos desarrollado un sistema automatizado de imágenes y análisis de los embriones de pez cebra a las 24-48 horas posteriores a la fecundación (HPF) en placas de 96 pozos<sup&gt; 2</sup&gt;. En este video podemos destacar los procedimientos para arraying embriones transgénicos en placas multipocillo en 24hpf y la adición de una molécula pequeña (BCI) que hyperactivates FGF señalización<sup&gt; 3</sup&gt;. Las placas se incuban durante 6 horas, seguido por la adición de tricaína para anestesiar a las larvas antes de la imagen automatizada en un Molecular Devices ImageXpress ultra láser confocal de barrido HCS lector. Las imágenes son procesadas por el software de desarrollo Definiens utilizando una red de Cognición algoritmo de la tecnología que hemos desarrollado para detectar y cuantificar la expresión de GFP en las cabezas de los embriones transgénicos. En este ejemplo podemos destacar la capacidad del algoritmo para medir efectos dosis-dependientes de BCI en la expresión del gen GFP reportero en los embriones tratados.

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.

Se presenta el desarrollo de un sistema automatizado de imágenes y análisis de pez cebra embriones transgénicos en placas de pocillos múltiples. Esto demuestra la capacidad de medir los efectos dependientes de la dosis de una molécula pequeña, BCI, el crecimiento de fibroblastos la expresión del gen del factor periodista y suministrar tecnología para el establecimiento de alto rendimiento de las pantallas de química de pez cebra.

Desarrollo de la imagen y el análisis automatizado de pantallas de química de pez cebra.

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 vistas.  University of Pittsburgh Drug Discovery Institute. Se presenta el desarrollo de un sistema automatizado de imágenes y análisis de pez cebra embriones transgénicos en placas de pocillos múltiples. Esto demuestra la capacidad de medir los efectos dependientes de la dosis de una molécula pequeña, BCI, el crecimiento de fibroblastos la expresión del gen del factor periodista y suministrar tecnología para el establecimiento de alto rendimiento de las pantallas de química de pez cebra.

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.

A neuronales y astrocitos Co-cultivo de ensayo para el análisis de alto contenido de neurotoxicidad

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

Investigación

Biología

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-automatizado de análisis óptico latido de los corazones pequeños

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.

El trasplante de las buenas prácticas agrarias-que expresa Blastómeros de imágenes en vivo del desarrollo de la retina y el cerebro en embriones de pez cebra quiméricos

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.

Glicanos imágenes en embriones de pez cebra mediante marcado metabólico y Química Haga clic 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.

Cuantitativos y automatizada de alto rendimiento del genoma completo de RNAi pantallas en 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.

Celular seguimiento mediante fotoconvertible proteínas durante el desarrollo de pez cebra

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-automatizado de fluorescencia específica de tejido en embriones de pez cebra

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 de análisis de contenidos de alta Utilizando microscopía de fluorescencia automatizado de imágenes por vida

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.

Análisis de la función de célula Beta usando proyección de imagen de resolución sola célula calcio en islotes de pez cebra

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.

Detección y análisis automatizados de exocitosis

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