Este trabalho foi financiado pela American Cancer Society (PF-07-103-01-CSM), o National Institutes of Health (R01 e P30 GM61275 CA042014), a Leucemia and Lymphoma Society, eo Huntsman Cancer Foundation.

transfecção siRNA e preparação celular <ol><li> Prepare um 4-bem LabTek II lamela chambered adicionando 0,5 mL de fibronectina (10μg/mL) a cada poço que será usado. Incubar por 10-15 minutos em temperatura ambiente. </li><li> Prepare siRNA / Lipofectamine RNAiMAX (Invitrogen) complexos para reverter transfecção de acordo com as instruções do fabricante s. Use a quantidade recomendada para um poço de um prato de 24 poços por câmara a ser utilizada (100μL total). Um produto análogo / protocolo de transfecção siRNA pode ser substituído. </li><li> Remova a fibronectina dos poços da lamela câmaras. Adicionar 100μL de siRNA / Lipofectamine complexos RNAiMAX a cada poço que será usado. </li><li> Alíquota de 20 mil histona H2B-mCherry células que expressam (em 500μL) por poço contendo mistura de transfecção. </li><li> Permitir que as células incubar por ~ 24 horas antes de substituir a mistura de transfecção com 1 mL de meio de cultura de células regulares. </li><li> Células incubar mais 24 horas antes da aquisição da imagem (48 horas pós-transfecção). </li></ol> Configuração do microscópio e de aquisição de imagem <ol start="7"><li> Há três partes principais para a configuração de aquisição de imagem: 1) uma célula incubadora que se encaixa no palco microscópio e mantém um ambiente úmido constante de 37 ° C, e 5% CO 2, 2) um microscópio invertido equipado com um palco automatizado, automatizado obturadores de fluorescência e campo claro, e rodas de filtros automáticos e 3) um pacote de software que integra e controla o palco, persianas, e rodas de filtros. Em nossa configuração de imagem, usamos um celular personalizado incubadora de OKO Lab que pode ser usado com o II LabTek chambered lamínula. O palco, persianas, e rodas de filtros são controladas pelo software Metamorph Dica:. O Laboratório OKO fase superior do sistema de incubadora inclui adaptadores para uma variedade de placas diferentes, prato, ou tamanhos lamela. </li><li> Posição do LabTek II lamela chambered na incubadora, abrindo a tampa da incubadora pré-aquecido e equilibrado, colocando a lamela chambered no adaptador, e segurando no lugar com massa de modelar. Deixar a tampa do lamela câmaras para que o meio de cultura celular não vai secar. Feche a tampa e posicione a incubadora inteira no palco microscópio, assegurando que a incubadora é mantida firmemente no lugar. </li><li> Usando Metamorph, configure seu experimento, selecionando &quot;Apps / Aquisição Multidimensional&quot; na barra de menus do software. Isso fará com que uma janela na qual você pode selecionar a freqüência ea duração do timelapse, o tempo de exposição para cada cor (se fazendo várias configurações), o número ea localização das posições de estágio, eo número de z-seções (se ) desejado. Selecione onde deseja salvar os arquivos de dados Dica:. Se estiver usando outro software que não Metamorph, as funções podem ser ligeiramente diferentes, mas o conceito geral é o mesmo. Micromanager, um software de código aberto baseado em aquisição ImageJ que seja compatível com a maioria das configurações de hardware, é uma excelente alternativa. </li><li> . Para efeitos do presente protocolo, iremos adquirir imagens em dois comprimentos de onda (claro e mCherry) a cada 15 minutos para 8 horas a 15 posições estágio Dica: Se a resolução melhor momento é desejada, então você pode encurtar o intervalo de tempo entre as aquisições, no entanto , as posições menos estágio pode ser usado, e células mitóticas, assim, menos estarão disponíveis para análise. É importante quando considerando os intervalos de tempo para ter em conta o tempo necessário para a roda de filtros para alternar entre conjuntos de filtros. </li><li> Determine o tempo de exposição ideal para cada comprimento de onda pela primeira foco em um campo de células com uma iluminação de campo claro. No software, ajustar a função de focagem automática apenas para o canal de campo claro (isso vai permitir que o software de focagem automática em cada posição palco antes de cada aquisição) Dica:. Usamos um 40x objetivo de contraste de fase e seco para dar excelente resolução, sem a necessidade de petróleo . Outros objetivos podem ser usados ​​dependendo da resolução óptica desejada. </li><li> Mude para o comprimento de onda mCherry e encaixe uma imagem usando a 50 msec exposição. Ajustar o tempo de exposição ao mínimo necessário para ver uma boa imagem. É essencial para limitar a quantidade de exposição à luz para garantir a viabilidade celular sustentável. Como regra geral, esta pode ser alcançada usando um filtro de densidade neutra (para reduzir o fluxo de luz de excitação) e um maior tempo de exposição da câmera ao invés de aumentar a força de iluminação. Repita este procedimento para qualquer comprimentos de onda adicionais para serem usados ​​Dica:. Para determinar a longo prazo a viabilidade celular em uma determinada exposição para a sua experiência, você pode visualizar o número total de imagens de uma célula pode sobreviver ao longo dos timelapse desejado adaptando temporariamente o espaçamento timepoint de minutos para segundos (ou seja, 10 min. torna-se 10 segundos). Se suas células sobrevivem 100-200 exposições (ou onúmero apropriado), então sua exposição é muito bem. Se não, reduzir a sua luz de excitação, tempo de exposição, ou o número total de imagens para simular sua experiência completa. </li><li> Em seguida, selecione e marca um número adequado de posições estágio para que você possa obter uma amostragem adequada da população celular. Metamorph irá gravar as posições e vai voltar lá para cada aquisição Dica:. Eu geralmente tentar encontrar posições em que há uma abundância de células a imagem, mas não tantos que eles são muito densos. Além disso, é importante selecionar posições suficiente para que você terá uma boa chance de observar um grande número de células progredindo através de mitose. </li><li> Afinal timelapse, tempo de exposição de comprimento de onda, e informações sobre a posição estágio foram gravadas, selecione a opção &quot;Preview&quot; botão na tela para determinar se o software está controlando o equipamento adequadamente. </li><li> Quando estiver satisfeito, selecione a opção &quot;Adquirir&quot; botão na tela para começar a aquisição. Depois sentar e permitir que o computador e microscópio para fazer o trabalho Sugestão:. Observe a automação através de pelo menos uma rodada cheia de aquisição para garantir que tudo está funcionando corretamente. </li></ol> Processamento de imagem e análise <ol start="16"><li> Após a aquisição for concluída, o próximo passo é transferir os dados de imagem em uma forma que é mais fácil de manusear. Para efeitos do presente protocolo, vou demonstrar primeiro como gerar filmes com Metamorph, então, como para gerar montagens de imagens utilizando o programa ImageJ. O formato será útil para fins de quantificação. </li><li> O primeiro passo é rever os dados. Para fazer isso, selecione &quot;Dados de Aplicativos / Revisão Multidimensional&quot; na barra de menu. Selecione o local de seus arquivos, em seguida, selecione &quot;View&quot;. Isto irá permitir que você reveja a pilha de imagens de cada posição etapa individualmente. Selecione a posição estágio desejado, depois em &quot;Load Images&quot;. Você será capaz de avançar através dos dados de quadro a quadro. </li><li> Para essas posições estágio em que as células passam por mitose (conforme determinado pelo DNA e morfologia celular), você pode fazer filmes e montagens usando Metamorph, ou com outros programas como o ImageJ (disponível gratuitamente através do NIH). </li><li> Para fazer um filme em Metamorph, selecione &quot;Processo / Save filme&quot; na barra de menu. Você será capaz de selecionar quais quadros de usar, a velocidade e tipo de arquivo do filme. Em seguida, selecione &quot;Salvar Filme&quot;. </li><li> Para fazer uma montagem, eu salvo a pilha imagem como um arquivo stk., Que pode ser usado em outros programas como o ImageJ. </li><li> Abra o arquivo no ImageJ e avançar os quadros até encontrar uma célula passando por mitose. . Recortar a área ao redor da célula e selecione &quot;Image / Duplicate&quot; Dica: Certifique-se de duplicar todos os quadros. </li><li> Para fazer uma montagem, selecione &quot;Image / Pilhas / Make Montage&quot; na barra de menu. Aqui você pode especificar os quadros que você deseja incluir, o tamanho ea forma da montagem, e se você quer fronteiras entre os frames. Selecione essas e clique em &quot;Ok&quot;. .. Salvar a montagem como um arquivo tif e qualquer outro tratamento em qualquer ImageJ ou Adobe Photoshop Dica: Alterar arquivos montagem do formato de 16 bits usado por Metamorph a 8 bits formato para o processamento de imagem mais fácil. </li><li> Para calcular o tempo em diferentes estágios de mitose, determinar t = 0 (primeiro sinal de condensação de DNA ou arredondamento das células), contar o número de quadros até cromossomos estão alinhados no equador (tempo em prófase / prometáfase), até quadros cromossomos começam a segregar (tempo em metáfase) e quadros até que as células começam a se achatar (anáfase / telófase / citocinese). O tempo calculado em cada fase mitótica depende do intervalo de tempo entre cada quadro. </li></ol> Resultados representante <img src="/files/ftp_upload/1878/1878fig1tmb.jpg" border="0" /> A figura 1 ilustra os resultados de um experimento típico transfecção controle siRNA usando uma linhagem de células HeLa expressar histona H2B-mCherry. Este método tem sido utilizado de forma eficaz para quantificar tempo mitótico, revelando um inesperado papel para o Nup153 nucleoporin no sincronismo da mitose final 1. Por favor, <a href="http://www.jove.com/files/ftp_upload/1878/1878fig1.jpg">clique aqui</a> para ver uma versão ampliada da figura 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>

Com os recentes avanços na tecnologia de imagem e proteína fluorescente, imagens ao vivo tornou-se um aspecto de análise de rotina celular 2. Uma variedade de GFP (e suas variantes outra cor 3)-tagged proteínas são amplamente disponíveis e relativamente fáceis de construir e pode ser usado para controlar um número de marcas, incluindo a divisão celular DNA / dinâmica cromossomo 4, 5, 6 duplicação centrossoma, ciclina B dinâmica 7, desagregação envelope nuclear e remontagem 8, 9, a formação do fuso mitótico 10, e vários estágios de citocinese 11. Tagged proteínas pode ser usado sozinho ou em combinação, quando os espectros de excitação / emissão de etiquetas fluorescentes são compatíveis, permitindo a coordenação entre os eventos específicos a serem avaliados. Se a resolução espacial maior e / ou temporal é / são desejada, microscopia confocal de varredura se varredura a laser, disco giratório, ou ressonância pode ser utilizado, bem como a lista de opções sofisticadas de microscopia, com cada vez maior a resolução continua a crescer. Imagens ao vivo da mitose em células de mamíferos também foi adaptado para uso em alto rendimento RNAi e telas de pequena molécula 12-14. Assim, enquanto uma experimentos iniciais s usando esta técnica pode ser relativamente simples, as possibilidades de construção desta estratégia são extensas.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>
</tr>		</tbody>
		</table>
		</div>

time-lapse imaging of mitosis after sirna transfection

Mudanças na organização celular e dinâmica cromossomo que ocorrem durante a mitose são fortemente coordenada para garantir a herança precisa do conteúdo genômico e celular. Hallmark eventos da mitose, como o movimento de cromossomos, podem ser facilmente rastreados em uma base de células individuais usando lapso de tempo de microscopia de fluorescência de linhas de células de mamíferos expressar específicas proteínas GFP. Em combinação com RNAi baseado esgotamento, este pode ser um método poderoso para identificar o estágio (s) da mitose, onde os defeitos ocorrem após os níveis de uma proteína particular, têm sido reduzidos. Neste protocolo, apresentamos um método básico para avaliar o efeito de esgotar uma proteína mitótica potencial regulatório sobre o momento da mitose. Células são transfectadas com siRNA, colocados em uma câmara de incubação em estágio superior, e fotografada usando um microscópio de fluorescência automatizado. Descrevemos como usar o software para criar uma experiência de lapso de tempo, como o processo de sequências de imagens para fazer qualquer imagem estática-montagens ou filmes, e como quantificar e analisar o timing das etapas mitóticas usando uma linha de células expressando-mCherry tagged H2B histonas. Finalmente, discute considerações importantes para a concepção de um experimento de lapso de tempo. Esta estratégia é complementar a outras abordagens e oferece as vantagens de 1) sensibilidade a mudanças na cinética que pode não ser observada quando se olha para as células de uma população e 2) Análise da mitose sem a necessidade de sincronizar o ciclo celular utilizando tratamentos medicamentosos. A informação visual a partir de experimentos de imagem, não só permite que as fases sub-da mitose de ser avaliado, mas também pode fornecer uma visão inesperada que não seria aparente a partir da análise do 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

Aqui nós descrevemos um protocolo básico para a imagem e quantificar o tempo de mitose de células vivas de tecidos de mamíferos cultura após transfecção siRNA.

Lapso de tempo de imagens de Mitose Depois de Transfection 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 visualizações.  University of Utah. Aqui nós descrevemos um protocolo básico para a imagem e quantificar o tempo de mitose de células vivas de tecidos de mamíferos cultura após transfecção siRNA.

Vídeo: 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.

Monitoramento Actina Desmontagem com a Time-lapse Microscopia

Biologia

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.

Dois fótons axotomia e lapso de tempo de imagem confocal em embriões de peixe-zebra ao 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 imagem em tempo-lapso de Transgênicos Zebrafish: Visualizando Células-Tronco da retina Ativado por Ablação célula-alvo Neuronal

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.

Lapso de tempo Microscopia de embriogênese inicial em 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.

Análise Quantitativa de Migração aleatória de células usando microscopia de lapso de tempo 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.

Longo prazo, de alta resolução de imagem Lapse Confocal Tempo de<em&gt; Cotilédone Arabidopsis</em&gt; Epiderme durante a germinação

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.

De alta resolução de imagem Time-lapse e análise automatizada de microtúbulos Dynamics em Viver umbilical humano Células Endoteliais da Veia

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.

Um método versátil para a montagem de Arabidopsis deixa para Intravital imagem de lapso de tempo

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.

Rotulagem específica de nucleóides mitocondriais para microscopia estruturada de iluminação com lapso de tempo

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, um método para montagem de larva drosophila para imagem de lapso de tempo de longo prazo

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