Questo lavoro è stato sostenuto dalla American Cancer Society (PF-07-103-01-CSM), il National Institutes of Health (R01 GM61275 e P30 CA042014), la Leukemia and Lymphoma Society, e la Huntsman Cancer Foundation.

trasfezione di siRNA e la preparazione delle cellule <ol><li> Preparare un 4-ben LabTek II coprioggetti incamerata con l&#39;aggiunta di 0,5 ml di fibronectina (10μg/mL) in ciascun pozzetto che verrà utilizzato. Incubare per 10-15 minuti a temperatura ambiente. </li><li> Preparare siRNA / Lipofectamine RNAiMAX (Invitrogen) complessi di trasfezione inverso secondo le istruzioni del produttore s. Usare le quantità raccomandate di un pozzetto di un piatto ben 24 per camera da utilizzare (100μL totale). Un prodotto analogo / protocollo per la trasfezione di siRNA possono essere sostituiti. </li><li> Rimuovere la fibronectina dai pozzi del coprioggetti camerati. Aggiungere 100μL di siRNA / Lipofectamine complessi RNAiMAX in ogni pozzetto che verrà utilizzato. </li><li> Aliquota 20.000 istone H2B-mCherry cellule che esprimono (in 500μL) per pozzetto contenente miscela di trasfezione. </li><li> Consentono alle cellule ad incubare per ~ 24 ore prima della sostituzione miscela trasfezione con 1 ml di medium di coltura delle cellule normali. </li><li> Incubare cellule ulteriori 24 ore prima acquisizione delle immagini (48 ore post-trasfezione). </li></ol> Microscopio setup e acquisizione delle immagini <ol start="7"><li> Ci sono tre parti principali per la configurazione di acquisizione delle immagini: 1) una cella incubatore che si adatta in scena microscopio e mantiene un ambiente umido costante di 37 ° C, e il 5% di CO 2; 2) un microscopio invertito dotato di uno stadio automatizzato, automatizzato persiane fluorescenza e chiaro, e automatizzato ruote portafiltri e 3) un pacchetto software che integra e controlla il palco, persiane, e le ruote filtro. Nella nostra configurazione di imaging, si usa una cella personalizzata incubatore da OKO laboratorio che possono essere utilizzati con il II LabTek chambered coprioggetti. Il palco, persiane, e le ruote filtro sono controllate dal software Metamorph. Suggerimento: Il Laboratorio OKO stadio-top sistema incubatore comprende adattatori per una varietà di differenti piatto, piatto, o dimensioni coprioggetto. </li><li> Posizionare il LabTek II coprioggetti camerata in dell&#39;incubatore aprendo il coperchio del pre-riscaldato ed equilibrato incubatore, posizionando il coprioggetti camerata in l&#39;adattatore, e tenendo in posizione con plastilina. Lasciare il coperchio sul coprioggetti incamerata in modo che il terreno di coltura cellulare non si secchino. Chiudere il coperchio e posizionare il tutto incubatore sul palco microscopio, assicurando che l&#39;incubatore si tiene saldamente in posizione. </li><li> Utilizzando Metamorph, istituito l&#39;esperimento selezionando &quot;Applicazioni / Acquisizione multidimensionali&quot; nella barra dei menu del software. Si aprirà una finestra nella quale è possibile selezionare la frequenza e la lunghezza del Timelapse, il tempo di esposizione per ogni colore (se fare le regolazioni multiple), il numero e la localizzazione delle posizioni palco, e il numero di z-sezioni (se ) desiderata. . Scegliere dove salvare i file di dati Suggerimento: se usate un software diverso Metamorph, le funzioni possono essere leggermente diversi, ma il concetto generale è lo stesso. Micromanager, un open source ImageJ basato su software di acquisizione che è compatibile con gran parte delle configurazioni hardware, è un&#39;ottima alternativa. </li><li> . Ai fini di questo protocollo, che acquisirà le immagini a due lunghezze d&#39;onda (campo chiaro e mCherry) ogni 15 minuti per 8 ore a 15 posizioni fase Suggerimento: Se la risoluzione del momento migliore è desiderato, quindi si può ridurre l&#39;intervallo di tempo tra le acquisizioni, ma , le posizioni fase meno può essere utilizzato, e quindi un minor numero di cellule mitotiche saranno disponibili per l&#39;analisi. E &#39;importante quando si considera intervalli di tempo di prendere in considerazione il tempo necessario per la ruota filtro per passare da un set di filtri. </li><li> Determinare il momento ottimale di esposizione per ogni lunghezza d&#39;onda dapprima concentrandosi su un campo di cellule con illuminazione campo chiaro. Nel software, regolare la funzione di messa a fuoco automatica solo per il channel campo chiaro (questo permetterà al software di messa a fuoco automatica ad ogni posizione stadio prima di ogni acquisto). Suggerimento: Si utilizza un 40x a contrasto di fase obiettivo di dare resolution eccellente senza la necessità di petrolio . Altri obiettivi possono essere utilizzati a seconda della risoluzione ottica desiderata. </li><li> Passare alla lunghezza d&#39;onda mCherry e scattare un&#39;immagine utilizzando un 50 esposizione msec. Regolare il tempo di esposizione al minimo indispensabile per vedere una buona immagine. È essenziale per limitare la quantità di esposizione alla luce per garantire la vitalità delle cellule sostenuta. Come regola generale, questo può essere realizzato utilizzando un filtro a densità neutra (per ridurre il flusso luminoso eccitazione) e più a lungo un tempo di esposizione della fotocamera, piuttosto che aumentando la resistenza illuminazione. Ripetere questa procedura per ogni lunghezza d&#39;onda aggiuntive da utilizzare. Suggerimento: Per determinare la redditività a lungo termine delle cellule in una data esposizione per l&#39;esperimento, è possibile visualizzare in anteprima il numero totale di immagini una cella può sopravvivere a Timelapse desiderato modificando temporaneamente la spaziatura timepoint da minuti a secondi (cioè, 10 min. diventa 10 secondi). Se le cellule sopravvivono 100-200 esposizioni (o ilnumero appropriato), quindi l&#39;esposizione è bene. In caso contrario, ridurre la luce di eccitazione, tempo di esposizione, o il numero totale di immagini per simulare l&#39;esperimento completo. </li><li> Poi, selezionare e segnare un numero adeguato di posizioni fase in modo che si può ottenere adeguato campionamento della popolazione di cellule. Metamorph registrerà le posizioni e farvi ritorno per ogni acquisizione. Suggerimento: io in genere cercano di trovare posizioni in cui ci sono molte cellule di immagine, ma non così tanti che sono troppo densi. Inoltre, è importante scegliere posizioni abbastanza in modo che si hanno buone possibilità di osservare un gran numero di cellule progredendo attraverso la mitosi. </li><li> Dopo tutto Timelapse, tempo di esposizione lunghezza d&#39;onda, e la posizione informazione fase sono stati registrati, selezionare il pulsante &quot;Anteprima&quot; sullo schermo per determinare se il software è il controllo delle attrezzature in modo appropriato. </li><li> Una volta soddisfatti, selezionare il pulsante &quot;Acquisisci&quot; sullo schermo per iniziare l&#39;acquisizione. Poi sedersi e consentire al computer e il microscopio per fare il lavoro. Suggerimento: Osservare l&#39;automazione attraverso almeno un giro completo di acquisizione per garantire che tutto funzioni correttamente. </li></ol> Elaborazione delle immagini e analisi <ol start="16"><li> Dopo l&#39;acquisizione è terminata, il passo successivo è quello di trasferire i dati immagine in una forma che è più facile da gestire. Ai fini di questo protocollo, voglio dimostrare prima come generare filmati utilizzando Metamorph, poi come generare montaggi immagine utilizzando il programma ImageJ. Sia in formato sarà utile ai fini di quantificazione. </li><li> Il primo passo è quello di rivedere i dati. Per farlo, selezionare &quot;Applicazioni / Rassegna dati multidimensionali&quot; nella barra dei menu. Selezionare la posizione del file, quindi selezionare &quot;View&quot;. Questo vi permetterà di rivedere la pila di immagini da ogni posizione stadio individualmente. Selezionare la posizione di fase desiderata, quindi &quot;Carica Immagini&quot;. Sarete in grado di avanzare attraverso i dati fotogramma per fotogramma. </li><li> Per quelle posizioni fase in cui le cellule passano attraverso la mitosi (come determinato da entrambi i DNA e morfologia cellulare), è possibile realizzare filmati e montaggi utilizzando Metamorph, o con altri programmi come ImageJ (disponibile gratuitamente attraverso NIH). </li><li> Per fare un film in Metamorph, selezionare &quot;Processo / Save film&quot; nella barra dei menu. Sarete in grado di selezionare i fotogrammi da utilizzare, la velocità e il tipo di file del film. Quindi selezionare &quot;Salva filmato&quot;. </li><li> Per fare un montaggio, risparmio il serie di immagini come file. Stk, che possono essere utilizzati in altri programmi come ImageJ. </li><li> Aprire il file in ImageJ e far avanzare i telai fino a trovare una cellula passa attraverso la mitosi. . Raccolto l&#39;area intorno alla cella e selezionare &quot;Immagine / Duplica&quot; Suggerimento: assicurarsi di duplicare tutti i fotogrammi. </li><li> Per fare un montaggio, selezionare &quot;Immagine / stack / Fai Montage&quot;, nella barra dei menu. Qui è possibile specificare quali fotogrammi che si desidera includere, la dimensione e la forma del montaggio, e se si desidera confini tra i fotogrammi. Selezionare queste e clicca su &quot;Ok&quot;. .. Salvare il montaggio come file tif e fare ogni ulteriore elaborazione sia in ImageJ o Adobe Photoshop Suggerimento: Modificare i file di montaggio da 16-bit formato utilizzato da Metamorph a 8-bit per l&#39;elaborazione delle immagini più facile. </li><li> Per calcolare il tempo in diverse fasi della mitosi, determinare t = 0 (primo segno di condensa DNA o arrotondamento delle cellule), contare il numero di fotogrammi fino cromosomi si allineano all&#39;equatore (tempo in profase / prometafase), cornici fino cromosomi cominciano a segregare (tempo in metafase), e le cornici fino cellule cominciano ad appiattirsi (anafase / telofase / citodieresi). Il tempo calcolato in ogni fase mitotica dipende dall&#39;intervallo di tempo tra ogni fotogramma. </li></ol> Rappresentante Risultati <img src="/files/ftp_upload/1878/1878fig1tmb.jpg" border="0" /> La figura 1 illustra i risultati di un tipico esperimento di trasfezione di siRNA di controllo utilizzando una linea cellulare HeLa che esprimono istone H2B-mCherry. Questo metodo di è stato effettivamente utilizzato per quantificare tempi mitotica, rivelando un ruolo inaspettato per la nucleoporin Nup153 nei tempi della tarda mitosi 1. Si prega di <a href="http://www.jove.com/files/ftp_upload/1878/1878fig1.jpg">cliccare qui</a> per vedere una versione più grande della 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>

Con i recenti progressi nel campo dell&#39;imaging e della tecnologia proteina fluorescente, immagini dal vivo è diventato un aspetto di routine di analisi cellulare 2. Una varietà di GFP (e altre varianti di colore 3)-tagged proteine ​​sono ampiamente disponibili e relativamente facile da realizzare e può essere usato per tenere traccia di un certo numero di caratteristiche tra cui la divisione cellulare DNA / dinamiche cromosoma 4, 5, centrosoma duplicazione 6, ciclina dinamiche B 7, ripartizione involucro nucleare e rimontaggio 8, 9, fuso mitotico formazione di 10, e varie fasi di citocinesi 11. Proteine ​​tag possono essere usati da soli o in combinazione, quando l&#39;eccitazione / emissione spettri di tag fluorescenti sono compatibili, permettendo il coordinamento tra gli eventi specifici da valutare. Se la risoluzione spaziale maggiore e / o temporale è / sono desiderati, se microscopia confocale a scansione laser scanning, disco rotante, o risonanza può essere utilizzata, e l&#39;elenco delle opzioni di microscopia sofisticate con sempre maggiore risoluzione continua a crescere. Immagini dal vivo della mitosi nelle cellule di mammifero è stato adattato per l&#39;uso in high-throughput RNAi e gli schermi di piccole molecole 12-14. Così, mentre gli esperimenti iniziale di un s utilizzando questa tecnica può essere relativamente semplice, le possibilità di costruire su questa strategia sono molteplici.

<!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

Cambiamenti nell&#39;organizzazione del cellulare e della dinamica dei cromosomi che si verificano durante la mitosi sono strettamente coordinato per garantire eredità accurata del contenuto genomica e cellulare. Hallmark eventi della mitosi, come il movimento dei cromosomi, può essere facilmente monitorati su base singola cella utilizzando time-lapse microscopia a fluorescenza di linee cellulari di mammiferi che esprimono specifiche proteine ​​GFP-tagged. In combinazione con base di RNAi esaurimento, questo può essere un metodo efficace per individuare la fase di (s) della mitosi in cui i difetti si verificano dopo i livelli di una proteina particolare sono stati abbassati. In questo protocollo, vi presentiamo un metodo di base per valutare l&#39;effetto di impoverire una proteina potenziale mitotico regolamentazione sui tempi della mitosi. Le cellule sono transfettate con siRNA, posto in un palco-top camera di incubazione, e fotografato con un microscopio a fluorescenza automatizzato. Abbiamo descritto come utilizzare il software per impostare un time-lapse experiment, come elaborare il sequenze di immagini per dare sia di immagini fisse montaggi o film, e come quantificare e analizzare i tempi di stadi mitotico si usa una cella-line expressing mCherry- tag istone H2B. Infine, si discute considerazioni importanti per la progettazione di un time-lapse esperimento. Questa strategia è complementare ad altri approcci e offre i vantaggi di 1) sensibilità ai cambiamenti di cinetica che potrebbe non essere osservati quando si guarda cellule come una popolazione e 2) l&#39;analisi della mitosi, senza la necessità di sincronizzare il ciclo cellulare con trattamenti farmacologici. Le informazioni visive dagli esperimenti di imaging, non solo permette la sub-fasi della mitosi da valutare, ma può anche fornire indicazioni inaspettati che non sarebbe evidente dall&#39;analisi del ciclo cellulare da 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

Qui si descrive un protocollo di base per l&#39;immagine e quantificare i tempi mitotico delle cellule dei tessuti dei mammiferi vivono cultura dopo trasfezione di siRNA.

Time-lapse imaging di mitosi dopo la trasfezione di 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 Views.  University of Utah. Qui si descrive un protocollo di base per l'immagine e quantificare i tempi mitotico delle cellule dei tessuti dei mammiferi vivono cultura dopo trasfezione di 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.

Monitoraggio Actina Smontaggio con Time-lapse Microscopia

Ricerca

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.

Due fotoni assotomia e time-lapse confocale in embrioni di zebrafish 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 imaging di transgenici Zebrafish: Visualizzare cellule staminali della retina Attivato da mirata ablazione delle cellule neuronali

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.

Microscopia time-lapse di Embriogenesi All&#39;inizio del 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.

Analisi quantitativa della migrazione casuale di celle usando Time-lapse Microscopia Video

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 lungo termine, ad alta risoluzione confocale Lapse Imaging Tempo di<em&gt; Cotyledon Arabidopsis</emEpidermide&gt; durante la germinazione

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.

Ad alta risoluzione Time-lapse imaging e analisi automatizzata dei microtubuli Dynamics in Living ombelicale umano cellule endoteliali 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 metodo Versatile per il montaggio di Arabidopsis lascia per l'Imaging di time-lapse videomicroscopia

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.

Etichettatura specifica dei nucleoidi mitocondriali per la microscopia dell'illuminazione strutturata time-lapse

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 metodo per montare la larva della Drosophila per l'imaging time-Lapse a lungo termine

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