Este trabajo fue apoyado por la Sociedad Americana del Cáncer (PF-07-103-01-CSM), los Institutos Nacionales de Salud (R01 GM61275 y CA042014 P30), la Sociedad de Leucemia y Linfoma, y ​​la Fundación de Cáncer Huntsman.

siRNA transfección y la preparación de células <ol><li> Prepare un 4-así Labtek II cubre objetos de cámara mediante la adición de 0,5 ml de fibronectina (10μg/mL) a cada pozo que se utilizará. Incubar durante 10-15 minutos a temperatura ambiente. </li><li> Prepare siRNA / Lipofectamine RNAiMAX (Invitrogen) de los complejos de transfección invertir de acuerdo a las instrucciones del fabricante s. Use la cantidad recomendada para un bien de un plato de 24 pozos en cada cámara para ser utilizado (100μL total). Un producto análogo / protocolo de transfección de siRNA puede ser sustituido. </li><li> Retire la fibronectina de los pozos de la cámara cubre objetos. Añadir 100μL de siRNA / Lipofectamine complejos RNAiMAX a cada uno, así que se utilizará. </li><li> Alícuota de 20.000 histona H2B-mCherry células que expresan (en 500μL) por pocillo que contiene la mezcla de transfección. </li><li> Permitir que las células se incuba durante ~ 24 horas antes de volver a la mezcla de transfección con 1 ml de medio de cultivo celular normal. </li><li> Incubar las células de un adicional de 24 horas antes de la adquisición de la imagen (48 horas después de la transfección). </li></ol> Microscopio de instalación y adquisición de imágenes <ol start="7"><li> Hay tres partes principales en la configuración de la adquisición de imágenes: 1) una célula de la incubadora que cabe en la platina del microscopio y mantiene un ambiente húmedo constante de 37 º C y 5% de CO 2, 2) un microscopio invertido equipado con una etapa automática, persianas automatizadas de fluorescencia y campo claro, y las ruedas de filtros automáticos, y 3) un paquete de software que integra y controla la etapa, persianas, y las ruedas de filtros. En nuestra configuración de imagen, se utiliza una medida de células incubadora de OKO de laboratorio que se pueden utilizar con la cámara Labtek II cubre objetos. La etapa, persianas, y las ruedas de filtros son controlados por el software Metamorph Sugerencia:. El Laboratorio de OKO etapa superior del sistema incubadora incluye adaptadores para una variedad de diferentes placas, plato, o el tamaño de cubreobjetos. </li><li> La posición de la cámara Labtek II cubre objetos en la incubadora por la apertura de la tapa de la pre-calentado y equilibrado incubadora, colocar el cubre cámara en el adaptador, y manteniendo en su lugar con plastilina. Deje la tapa de la cámara cubre objetos para que el medio de cultivo celular no se seque. Cierre la tapa y la posición de la totalidad de la incubadora en la platina del microscopio, lo que garantiza que la incubadora se mantiene firmemente en su lugar. </li><li> Utilizando Metamorph, configurar la prueba mediante la opción &quot;Aplicaciones / Adquisición multidimensionales&quot; en la barra de menús del software. Con ello se abre una ventana en la que puede seleccionar la frecuencia y duración de la intervalo, el tiempo de exposición para cada color (si se hacen ajustes múltiples), el número y ubicación de las posiciones de la etapa, y el número de z-secciones (si ) deseado. . Seleccionar dónde guardar los archivos de datos Sugerencia: Si se utiliza otro software de Metamorph, las funciones pueden variar ligeramente, pero el concepto general es el mismo. Micromanager, una fuente abierta ImageJ software basado en la adquisición, que es compatible con la mayoría de configuraciones de hardware, es una excelente alternativa. </li><li> . A los efectos de este protocolo, vamos a adquirir imágenes en dos longitudes de onda (claro y mCherry) cada 15 minutos durante 8 horas a 15 posiciones de escena Sugerencia: Si mejor el tiempo de resolución es deseado, entonces se puede acortar el intervalo de tiempo entre la adquisición, sin embargo , menos puestos de fase se puede utilizar, y por lo tanto menor cantidad de células mitóticas estará disponible para su análisis. Es importante cuando se consideran los intervalos de tiempo para tener en cuenta el tiempo necesario para que la rueda de filtros para cambiar entre los conjuntos de filtros. </li><li> Determinar el tiempo de exposición óptimo para cada longitud de onda, en primer lugar se centra en un campo de las células utilizando iluminación campo claro. En el software, ajustar la función de enfoque automático sólo para el canal de campo claro (esto permitirá que el software para el enfoque automático en cada posición antes de cada etapa de la adquisición) Sugerencia:. Utilizamos un 40x seco de contraste de fase objetivo de dar una excelente resolución, sin necesidad de aceite . Otros objetivos pueden ser utilizados en función de la resolución óptica deseada. </li><li> Cambiar a la longitud de onda mCherry y complemento de una imagen mediante una exposición de 50 ms. Ajustar el tiempo de exposición al mínimo necesario para ver una buena imagen. Es esencial para limitar la cantidad de exposición a la luz para asegurar la viabilidad celular sostenido. Como regla general, esto puede lograrse mejor mediante el uso de un filtro de densidad neutral (para reducir el flujo de luz de excitación) y un tiempo de exposición de la cámara ya no por el aumento de la fuerza de la iluminación. Repita este procedimiento para cualquier longitud de onda adicional que se utilizará Sugerencia:. Para determinar la viabilidad a largo plazo de células en una exposición dada por su experiencia, usted puede ver el número total de imágenes que una célula pueda sobrevivir en el timelapse deseado mediante una adaptación provisional de la separación punto de tiempo de minutos a segundos (es decir, 10 min. se convierte en 10 segundos). Si las células sobrevivir 100-200 exposures (o el número apropiado), entonces la exposición está muy bien. Si no es así, reducir su luz de excitación, el tiempo de exposición, o el número total de imágenes para simular el experimento completo. </li><li> A continuación, seleccionar y marcar un número adecuado de puestos de fase de modo que usted pueda lograr un muestreo adecuado de la población de células. Metamorph registrará las posiciones y volverá allí para cada adquisición Sugerencia:. Yo generalmente tratar de encontrar posiciones en las que hay un montón de células de la imagen, pero no tantos que son demasiado densos. Además, es importante seleccionar posiciones lo suficiente para que usted tendrá una buena oportunidad de observar un gran número de células progresando a través de la mitosis. </li><li> Después de todo intervalo, el tiempo de exposición de longitud de onda, y la posición de la etapa se han registrado, seleccione el botón &quot;Preview&quot; en la pantalla para determinar si el software está controlando el equipo adecuado. </li><li> Una vez que esté satisfecho, seleccione la opción &quot;Adquirir&quot; botón en la pantalla para comenzar la adquisición. A continuación, sentarse y dejar el ordenador y el microscopio para hacer el trabajo Sugerencia:. Observar la automatización a través de al menos una ronda completa de adquisición para asegurarse de que todo funciona correctamente. </li></ol> Procesamiento de imágenes y análisis <ol start="16"><li> Después de la adquisición ha terminado, el siguiente paso es transferir los datos de imagen en una forma que es más fácil de manejar. A los efectos de este protocolo, primero voy a demostrar cómo generar películas con Metamorph, entonces cómo generar montajes de imágenes utilizando el programa ImageJ. Ya sea en formato será útil para propósitos de cuantificación. </li><li> El primer paso es revisar los datos. Para ello, seleccione &quot;Aplicaciones / Revisión de datos multidimensionales&quot; en la barra de menú. Seleccione la ubicación de sus archivos, a continuación, seleccione &quot;Ver&quot;. Esto le permitirá revisar la pila de imágenes de cada posición de la etapa de forma individual. Seleccione la posición de la etapa deseada, y luego &quot;Cargar imágenes&quot;. Usted será capaz de avanzar a través de los datos de cuadro por cuadro. </li><li> Para los puestos de fase en la que las células pasan por mitosis (según lo determinado por el ADN y la morfología celular), puede hacer que las películas y montajes utilizando Metamorph, o con otros programas como ImageJ (disponible de forma gratuita a través de los NIH). </li><li> Para hacer una película en Metamorph, seleccione &quot;Proceso / Guardar película&quot; en la barra de menú. Usted será capaz de seleccionar los cuadros de uso, velocidad y tipo de archivo de la película. A continuación, seleccione &quot;Guardar película&quot;. </li><li> Para hacer un montaje, ahorro de la pila de la imagen como un archivo. STK, que puede ser utilizado en otros programas como ImageJ. </li><li> Abra el archivo en ImageJ y avanzar en los marcos hasta que encuentre una célula pasa por mitosis. . Recortar el área alrededor de la celda y seleccione &quot;Imagen / Duplicar&quot; Sugerencia: Asegúrese de que duplicar todos los fotogramas. </li><li> Para hacer un montaje, seleccione &quot;Imagen / pilas / Hacer montaje&quot; en la barra de menú. Aquí puede especificar los fotogramas que desee incluir, el tamaño y la forma del montaje, y si desea que las fronteras entre los cuadros. Seleccione estas y pulse &quot;Aceptar&quot;. .. Guarde el montaje como un archivo tif y no cualquier otro tratamiento o ImageJ o Adobe Photoshop Consejo: cambia los archivos de montaje desde el formato de 16-bit utilizada por Metamorph en formato de 8-bits de procesamiento de imágenes más fácil. </li><li> Para calcular el tiempo en las diferentes fases de la mitosis, determinar t = 0 (primer signo de la condensación del ADN o el redondeo de células), cuente el número de fotogramas hasta que los cromosomas se alinean en el ecuador (el tiempo en la profase / prometafase), marcos hasta que los cromosomas comienzan a segregar (tiempo en la metafase), y los marcos hasta que las células comienzan a aplanarse (anafase / telofase / citocinesis). El tiempo calculado en cada etapa de mitosis depende del intervalo de tiempo entre cada fotograma. </li></ol> Resultados representante <a href="http://www.jove.com/files/ftp_upload/1878/1878fig1.jpg"><img src="/files/ftp_upload/1878/1878fig1tmb.jpg" border="0" /></a> La Figura 1 ilustra los resultados de un experimento de transfección de control típico de siRNA utilizando una línea celular HeLa que expresan la histona H2B-mCherry. Este método ha sido utilizado de manera efectiva para cuantificar el tiempo mitótico, revelando un inesperado papel para el nucleoporin Nup153 en el momento de finales de la mitosis 1.

<ol>
	<li>Mackay, D. R., Elgort, S. W., Ullman, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nucleoporin+Nup153+has+separable+roles+in+both+early+mitotic+progression+and+the+resolution+of+mitosis.">The nucleoporin Nup153 has separable roles in both early mitotic progression and the resolution of mitosis.</a> Mol Biol Cell. 20, 1652-1660 (2009).</li><li>Khodjakov, A., Rieder, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+division+process+in+living+tissue+culture+cells.">Imaging the division process in living tissue culture cells.</a> Methods. 38, 2-16 (2006).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat Methods. 2, 905-909 (2005).</li><li>Meraldi, P., Draviam, V. M., Sorger, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+and+checkpoints+in+the+regulation+of+mitotic+progression.">Timing and checkpoints in the regulation of mitotic progression.</a> Dev Cell. 7, 45-60 (2004).</li><li>Mora-Bermudez, F., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+structural+dynamics+of+chromosomes+in+living+cells+by+fluorescence+microscopy.">Measuring structural dynamics of chromosomes in living cells by fluorescence microscopy.</a> Methods. 41, 158-167 (2007).</li><li>Prosser, S. L., Fry, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+imaging+of+the+centrosome+cycle+in+mammalian+cells.">Fluorescence imaging of the centrosome cycle in mammalian cells.</a> Methods Mol Biol. 545, 165-183 (2009).</li><li>Malureanu, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BubR1+N+terminus+acts+as+a+soluble+inhibitor+of+cyclin+B+degradation+by+APC/C(Cdc20)+in+interphase.">BubR1 N terminus acts as a soluble inhibitor of cyclin B degradation by APC/C(Cdc20) in interphase.</a> Dev Cell. 16, 118-131 (2009).</li><li>Anderson, D. J., Hetzer, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reshaping+of+the+endoplasmic+reticulum+limits+the+rate+for+nuclear+envelope+formation.">Reshaping of the endoplasmic reticulum limits the rate for nuclear envelope formation.</a> J Cell Biol. 182, 911-924 (2008).</li><li>Beaudouin, J., Gerlich, D., Daigle, N., Eils, R., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+envelope+breakdown+proceeds+by+microtubule-induced+tearing+of+the+lamina.">Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina.</a> Cell. 108, 83-96 (2002).</li><li>Marcus, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+spindle+behavior+using+confocal+microscopy.">Visualization of spindle behavior using confocal microscopy.</a> Methods Mol Med. 137, 125-137 (2007).</li><li>Steigemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aurora+B-mediated+abscission+checkpoint+protects+against+tetraploidization.">Aurora B-mediated abscission checkpoint protects against tetraploidization.</a> Cell. 136, 473-484 (2009).</li><li>Draviam, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+genomic+screen+identifies+a+role+for+TAO1+kinase+in+spindle-checkpoint+signalling.">A functional genomic screen identifies a role for TAO1 kinase in spindle-checkpoint signalling.</a> Nat Cell Biol. 9, 556-564 (2007).</li><li>Neumann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+RNAi+screening+by+time-lapse+imaging+of+live+human+cells.">High-throughput RNAi screening by time-lapse imaging of live human cells.</a> Nat Methods. 3, 385-390 (2006).</li><li>Stegmeier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaphase+initiation+is+regulated+by+antagonistic+ubiquitination+and+deubiquitination+activities.">Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities.</a> Nature. 446, 876-881 (2007).</li></ol>

Con los últimos avances en imagen y tecnología de la proteína fluorescente, imágenes en vivo se ha convertido en un aspecto rutinario de análisis celular 2. Una variedad de buenas prácticas agrarias (y otras variantes de color 3)-etiquetados proteínas están ampliamente disponibles y relativamente fácil de construir y se puede utilizar para rastrear una serie de características, incluyendo la dinámica de división celular del ADN / cromosoma 4, 5, 6 centrosoma la duplicación, la dinámica de la ciclina B 7, desglose envoltura nuclear y 8 de montaje, 9, 10 la formación del huso mitótico, y las distintas etapas de la citocinesis 11. Proteínas etiquetadas se pueden utilizar solos o en combinación, cuando los espectros de excitación / emisión de etiquetas fluorescentes son compatibles, lo que permite la coordinación entre los eventos específicos que deben evaluarse. Si mayor resolución espacial y / o temporal es / son deseados, la microscopía confocal de barrido láser de exploración si, disco giratorio, o la resonancia se puede utilizar, y la lista de opciones sofisticadas de microscopía cada vez con mayor resolución sigue creciendo. Imágenes en vivo de la mitosis en células de mamíferos también ha sido adaptado para su uso en alto rendimiento y pantallas de RNAi pequeña molécula 12-14. Así, mientras que los experimentos iniciales s un uso de esta técnica puede ser relativamente simple, las posibilidades de construir en esta estrategia son extensos.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Lipofectamine RNAiMAX</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13778-075</td>
<td>&nbsp;</td>
<tr>
<td>Lab Tek II Chambered Coverglass (4-well)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>12-565-337</td>
<td>Additional chamber sizes are available</td>
</tr>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F1141</td>
<td>&nbsp;</td>
<tr>
<td>Stage-top cell incubator</td>
<td>&nbsp;</td>
<td>OKO Lab</td>
<td>&nbsp;</td>
<td>Can use any appropriate chamber</td>
</tr>
<tr>
<td>Automated inverted fluorescence microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Can use any appropriate microscope</td>
</tr>
<tr>
<td>Software package</td>
<td>&nbsp;</td>
<td>Metamorph</td>
<td>&nbsp;</td>
<td>Can use any appropriate software</td>
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		</div>

time-lapse imaging of mitosis after sirna transfection

Cambios en la organización celular y la dinámica de los cromosomas que ocurren durante la mitosis están estrechamente coordinadas para garantizar la herencia exacta del contenido genómico y celular. Hallmark eventos de la mitosis, como el movimiento de los cromosomas, pueden ser fácilmente seguidos en forma de células individuales con time-lapse microscopía de fluorescencia de las líneas de células de mamíferos que expresen proteínas GFP-etiquetados. En combinación con RNAi basado en el agotamiento, esto puede ser un poderoso método para localizar la etapa (s) de la mitosis, donde los defectos se producen después de los niveles de una proteína en particular se han reducido. En este protocolo, se presenta un método básico para evaluar el efecto del agotamiento de una proteína de regulación potencial mitótico en el momento de la mitosis. Las células son transfectadas con siRNA, colocado en una cámara de incubación etapa superior, y la imagen usando un microscopio de fluorescencia automatizado. Se describe cómo utilizar el software para crear una experiencia de lapso de tiempo, la forma de procesar las secuencias de imágenes para que sea imagen fija o montajes de películas, y la forma de cuantificar y analizar el calendario de las etapas de mitosis con una línea celular que expresa mCherry- etiquetados histona H2B. Finalmente, se discuten aspectos importantes para el diseño de un experimento de time-lapse. Esta estrategia se complementa con otros enfoques y ofrece las ventajas de 1) la sensibilidad a los cambios en la cinética de que no pueda ser observado cuando se mira en las células como la población y el análisis de 2) de la mitosis sin la necesidad de sincronizar el ciclo celular utilizando tratamientos farmacológicos. La información visual de los experimentos de imagen, no sólo se permite la sub-etapas de la mitosis a ser evaluados, pero también puede dar una idea inesperada que no se desprende del análisis del ciclo celular por FACS.

Watch this Scientific Journal Video about Time-lapse Imaging of Mitosis After siRNA Transfection at JoVE.com

Time-lapse Imaging of Mitosis After siRNA Transfection

Aquí se describe un protocolo básico de la imagen y cuantificar el tiempo mitótico de las células de mamíferos viven de cultivo de tejidos después de la transfección de siRNA.

Time-lapse de imágenes de mitosis después de la transfección de siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

time-lapse-imaging-of-mitosis-after-sirna-transfection

Research

JoVE Journal

Biology

16.9K vistas.  University of Utah. Aquí se describe un protocolo básico de la imagen y cuantificar el tiempo mitótico de las células de mamíferos viven de cultivo de tejidos después de la transfección de siRNA.

Video: Time-lapse Imaging of Mitosis After siRNA Transfection

Monitoring Actin Disassembly with Time-lapse Microscopy

Okay, so what I'd like to do today is to show you how to construct a flow chamber, a fusion chamber for imaging on an upright microscope. What then I then do is I create two spaces using film. So what I do is I take the para film, I cut it into small strips.Next I'm going to, this will basically form the channels for the, the walls, and I dry them with a tilted air. I put the cover slip on top of the para, And here's the finished. Then I heat it on a heat block to melt the param and basically create a seal.So what this is, is the, the cover glass here, and then on top of the cover glass is two layers of phim as you see right here. And then on top of this, again, there's a, it's a 22 by two two mil meter cover slip. This one right here, which I pressed against the param to fall the seal.Okay, so the liquid goes in in one direction here and I can suck it out with a filter paper here in this direction. And I'll show you how to do that in a bit. So what I like to do is I like to put it in a humidified chamber, which is very simple.Take two kind of elevated strips and have a filter of paper underneath. And to keep it noisy as I, I put the cover, cover slip on the flow chamber on. Now the way you introduce a solution to the chamber is that you just put the pipet tip at the entrance of the chamber and you just split.Now what I do is I incubate the chamber with the lid closed for 10 minutes. What I usually do to suck the liquid out from the other side is to use it, get a filter paper and cut it into thin strips like this. And I take one of these thin strips and I simultaneously apply the solution into the entrance while putting the filter paper at the exit to suck the exchange, the solution.So you'll see the liquid flowing through the solution. And then I wait for five minutes for the mid close. Now what I'm ready to do is to actually put that the actin it and the actin, I actually have to polymerize, I polymerize inside the flow chamber and it's just the same procedures before.So because like this, and then now that the action is polymerized, I wash out all the un excess un polymerized action using a large volume of just buffer. So here I have about a hundred microliters and it's a few reaction chamber volumes worth of buffer. Just, you know, when the filter paper becomes saturated, I have to change the direction.Okay?And so I'm ready to do imaging with this. So perfusion chamber with actin attached to it. Now I take the slide, mount it under the microscope, and since I'm imaging with an oil objective, I'm gonna put some immersion oil on the top surface of the cover, grass touch, such so assess solution or protein or anything on acting inside.So what I do is I can actually exchange the solution side while I'm doing the time lapse imaging. As such.

Monitoreo de actina desmontaje con la Microscopía de Time-lapse

Investigación

Biología

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

De dos fotones axotomía y de lapso de tiempo de imagen confocal en embriones de pez cebra en vivo

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

Multicolor Time-lapse de imágenes de pez cebra transgénico: Visualización de células madre retinianas activado por la ablación selectiva de células neuronales

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Time-lapse microscopía de embriogénesis temprana en Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Análisis cuantitativo de la migración desordenada de las células mediante microscopía de Time-lapse vídeo

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

A largo plazo, de alta resolución de imagen confocal Time Lapse de<em&gt; Cotiledones Arabidopsis</em&gt; Epidermis durante la germinación

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

-Alta resolución de imagen de lapso de tiempo y análisis automatizado de los microtúbulos en la dinámica de estar umbilical humana Las células endoteliales de vena

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

Un método versátil para el montaje de Arabidopsis las hojas para la proyección de imagen de Time-lapse Intravital

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

Etiquetado específico de nucleoides mitocondriales para microscopía de iluminación estructurada de lapso de tiempo

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

LarvaSPA, un método para montar larvas de Drosophila para imágenes de lapso de tiempo a largo plazo

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because ...