Vorremmo esprimere il nostro apprezzamento per l&#39;ingresso e commenti dei membri del laboratorio Padilla. Ceppi di nematodi in questo lavoro sono stati forniti dalla genetica Caenorhabditis Center, che è finanziato dalla National NIH Center for Research (NCRR). Prendiamo atto e ringraziamo il Dott. Lon Turnbull per l&#39;assistenza tecnica con la microscopia confocale. Questo lavoro è stato sostenuto da un finanziamento della National Science Foundation (NSF-IOS, CARRIERA) per PAP

1. Preparazione del campione <ol><li> Produrre o ottenere adeguata transgenici C. elegans ceppo di interesse utilizzando metodologie transgeniche o da Caenorhabditis Genetica Image Center (CGC) o un collega. In questo caso stiamo usando ceppo TH32 (pie-1 :: TBG-1 :: GFP; pie-1 :: GFP :: H2B) 5 per visualizzare i cromosomi e centrosomi come marcatori per la divisione cellulare. </li><li> Genera una popolazione sincronizzato utilizzando uno dei tre metodi: 1) si disintegrano adulti gravide in soluzione di ipoclorito e utilizzare gli embrioni restanti 6, 2) raccogliere adulti gravide su un piatto semi e farli posare embrioni per 1-2 ore, o 3) raccogliere L4 larve da una popolazione mista e passaggio ad una nuova piastra testa di serie. </li><li> Grow nematodi a 20 ° C fino all&#39;età adulta gravido giovani, che avrà circa 96 ore dalla schiusa, 72 ore da larve L1 o 24 ore dopo molt L4. Gli embrioni (2-20 celle) in utero contengono blastomeri grandi e nuclei che sono ottimali per imagmodifiche zione sub-cellulari e sub-nucleare, rispettivamente. Questo metodo fornisce un mezzo per generare una popolazione di giovani adulti che contengono un numero abbondante di embrioni precoci. </li></ol> 2. Preparazione Far scorrere e anossia Camera Set Up <ol><li> Effettuare una camera umida per contenere vetrini preparati mettendo un tovagliolo di carta umido all&#39;interno di un grande piatto Petri o contenitore coperto con un coperchio di dimensioni sufficienti. </li><li> Preparare fuso agarosio al 2% in acqua deionizzata H 2 O per riscaldamento composto in vetro con bagno forno a microonde o in acqua. </li><li> Mettere 1-3 gocce di caldo agarosio su un vetrino da microscopio pulito di vetro. Porre immediatamente una seconda slitta invertito perpendicolarmente sulla cima della goccia agarosio (s), premere leggermente in modo che la pastiglia risultante sarà sottile e privo di bolle d&#39;aria. </li><li> Attendere che si raffreddi e lentamente smontare i due vetrini che lasciano intatto il pad agarosio su una delle diapositive. È importante assicurare il pad agarosio è sufficientemente sottile da non interfenuovamente con la capacità di concentrare il microscopio tardi. Capacità di visualizzare campione dipende anche la distanza ottica specifica dell&#39;obiettivo in uso, che può variare tra microscopi. </li><li> Utilizzare una lama di rasoio pulito per modellare pad agarosio in una piccola piazza. Memorizzare nella camera umida preparata fino al momento dell&#39;uso. </li><li> Accuratamente, utilizzando la stessa lama di rasoio pulito, prendere il bordo del pad agarosio e trasferirlo dal vetrino su un tondo 25 millimetri micro coprioggetti, evitando l&#39;accumulo di bolle d&#39;aria sotto il pad. Il copri tutto selezionato da utilizzare si inserisce in modo appropriato nella microincubator. </li><li> Aggiungere una goccia di anestetico (0,5% tricaine, 0,05% in Tetramisole M9 buffer) sul pad agarosio per uso come anestetico. Scegli vermi 5-10 e posto nella goccia di anestetico. Lasciare 1-3 min per i vermi a cessare il movimento. </li><li> Il razionale per osservare embrioni all&#39;interno dell&#39;utero adulto, invece di embrioni sezionato, è ridurre al minimo il potenziale di embrione desiccazione e movimento a causa del flusso di gas di azoto attraverso l&#39;embrione. </li><li> Immediatamente aggiungere una goccia di olio Halocarbon sopra i vermi per evitare che i vermi di disidratazione il gas viene fatto fluire attraverso la camera. Dal momento che l&#39;olio è viscoso Halocarbon si può usare una punta della pipetta o la punta di una vite senza fine sceglie di raccogliere l&#39;olio e caduta sui vermi. </li><li> Una volta che il coverglass circolare contenente i vermi è pronto, le due metà del Leiden chiuso perfusione microincubator sono separati, e poi attentamente coverglass è posto verticalmente sull&#39;anello inferiore. I due lati della camera vengono poi chiusi ermeticamente insieme. </li><li> La camera è quindi collegato al serbatoio del gas azoto attraverso tubi di plastica flessibile e collocato nel punto appropriato sulla scena microscopio (Figura 1C). In questo momento il tubo e microincubator devono essere attentamente controllati per eventuali perdite assicurando il fissaggio sicuro dei tubi e dei tappi lungo il lato della camera. Si può alcon tanta leggerezza diffondere una piccola quantità di acqua e sapone sul tubo alla ricerca di bolle, che andranno a formare, se è presente una perdita. </li></ol> 3. Microscopia in anossia <ol><li> Individuare gli animali a basso ingrandimento, poi, passare al più alto ingrandimento appropriato (64x per questa applicazione) ai tessuti immagine o le cellule di interesse come blastomeri embrionali o ovociti negli adulti. Accendere il laser 488 nm per visualizzare il segnale GFP e proteina di fusione GFP individuare nella cella (s) di interesse. </li><li> Per visualizzare l&#39;arresto al determinata fase di arresto del ciclo cellulare (ad es profase tardi) identificano un blastomero in una fase preliminare di divisione cellulare (ad es interfase in ritardo o profase precoce). Marcatori cellulari, come la posizione centriolo, avvio della condensazione cromosomica, la posizione dei cromosomi e la presenza o l&#39;assenza della busta nucleare permetterà di individuare fasi del ciclo cellulare (Figura 2A). </li><li> La camera viene poi perfusi con gas azoto (99,999%N 2; &lt;2 ppm O 2). In questo caso si utilizza una pressione di 6 psi, tuttavia la pressione può essere necessario regolare e ottimizzato a seconda della dimensione di ciascun microincubator e diametro del tubo in uso. Continuare a monitorare la blastomero (s) come azoto riempie la camera, la regolazione del piano focale come necessario. </li><li> A questo punto, time-lapse imaging è iniziata. Imaging è condotto per il tempo necessario per catturare fenomeno di interesse, in questo caso e arresto profase aggancio dei cromosomi alla membrana interna nucleare. Le immagini sono presi una volta ogni 10 secondi per non più di 30 min. Le frequenze di cattura di immagini e impostazioni quali il guadagno e la potenza del laser deve essere regolato secondo necessità. </li><li> Il tempo in cui gli embrioni rispondere all&#39;ambiente anossico può variare a seconda dello stadio del ciclo cellulare in seguito all&#39;esposizione anossia. Tipicamente anossia arresto indotto profase si verifica entro 20-30 minuti di inizio flusso di gas di azoto. Abbiamo osservato anossia-indotta del ciclo cellulare occu arrestoRing in &lt;20 min. Le immagini possono essere ottenute durante questo lasso di tempo, o le immagini fisse possono essere isolati in seguito da un time-lapse serie. Un&#39;ulteriore opzione è quella di utilizzare la microscopia video per visualizzare strutture subcellulari in embrioni. </li><li> Per documentare recupero dalla anossia indotto stato arrestato, il gas viene spento e la camera viene permesso di tornare normossia per diffusione durante la registrazione di un time-lapse serie. Ripresa della progressione del ciclo cellulare e lo scollegamento dei cromosomi si verifica in genere entro 5-20 minuti di trasformare l&#39;azoto off. </li><li> Si noti che in esperimenti separati un indicatore anossia, resazurina, è stato posto in flusso continuo microcamera, è stato determinato che la camera diventa anossica in media di circa 19 min dopo il flusso del gas di azoto è iniziata. </li><li> Le immagini ei video sono trattati con Imaris, Immagine a J o Photoshop e importati in QuickTime per la visualizzazione. </li></ol>

<ol>
	<li>Powell-Coffman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+signaling+and+resistance+in+C.+elegans.">Hypoxia signaling and resistance in C. elegans.</a> Trends Endocrinol. Metab. 21 (10), 435-440 (2010).</li><li>Padilla, P. A., Ladage, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suspended+animation,+diapause+and+quiescence:+Arresting+the+cell+cycle+in+C.+elegans.">Suspended animation, diapause and quiescence: Arresting the cell cycle in C. elegans.</a> Cell Cycle. 11, (2012).</li><li>Hu, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dauer.">Dauer.</a> WormBook. , 1-19 (2007).</li><li>Zhou, K. I., Pincus, Z., Slack, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longevity+and+stress+in+Caenorhabditis+elegans.">Longevity and stress in Caenorhabditis elegans.</a> Aging (Albany NY). 3, 733-753 (2011).</li><li>Schmidt, D. J., Rose, D. J., Saxton, W. M., Strome, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+cytoplasmic+dynein+heavy+chain+in+Caenorhabditis+elegans+with+fast-acting+temperature-sensitive+mutations.">Functional analysis of cytoplasmic dynein heavy chain in Caenorhabditis elegans with fast-acting temperature-sensitive mutations.</a> Mol. Biol. Cell. 16, 1200-1212 (2005).</li><li>Sulston, J., Hodgkin, J., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Nematode Caenorhabditis elegans. , 587-606 (1988).</li><li>Padilla, P. A., Nystul, T. G., Zager, R. A., Johnson, A. C., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dephosphorylation+of+Cell+Cycle-regulated+Proteins+Correlates+with+Anoxia-induced+Suspended+Animation+in+Caenorhabditis+elegans.">Dephosphorylation of Cell Cycle-regulated Proteins Correlates with Anoxia-induced Suspended Animation in Caenorhabditis elegans.</a> Mol. Biol Cell. 13, 1473-1483 (2002).</li><li>Hajeri, V. A., Trejo, J., Padilla, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+sub-nuclear+changes+in+Caenorhabditis+elegans+embryos+exposed+to+brief,+intermediate+and+long-term+anoxia+to+analyze+anoxia-induced+cell+cycle+arrest.">Characterization of sub-nuclear changes in Caenorhabditis elegans embryos exposed to brief, intermediate and long-term anoxia to analyze anoxia-induced cell cycle arrest.</a> BMC Cell Biol. 6, 1471-2121 (2005).</li><li>Padilla, P. A., Goy, J. M., Hajeri, P. A., Padilla, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anoxia. , (2012).</li><li>Hajeri, V. A., Little, B. A., Ladage, M. L., Padilla, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NPP-16/Nup50+function+and+CDK-1+inactivation+are+associated+with+anoxia-induced+prophase+arrest+in+Caenorhabditis+elegans.">NPP-16/Nup50 function and CDK-1 inactivation are associated with anoxia-induced prophase arrest in Caenorhabditis elegans.</a> Mol. Biol. Cell. 21, 712-724 (2010).</li><li>Nystul, T. G., Goldmark, J. P., Padilla, P. A., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suspended+animation+in+C.+elegans+requires+the+spindle+checkpoint.">Suspended animation in C. elegans requires the spindle checkpoint.</a> Science. 302, 1038-1041 (2003).</li><li>Margalit, A., Vlcek, S., Gruenbaum, Y., Foisner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+and+making+of+the+nuclear+envelope.">Breaking and making of the nuclear envelope.</a> J. Cell Biochem. 95, 454-465 (2005).</li><li>Maddox, A. S., Maddox, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+of+cellular+processes+in+Caenorhabditis+elegans.">High-resolution imaging of cellular processes in Caenorhabditis elegans.</a> Methods Cell Biol. 107, 1-34 (2012).</li><li>Miller, D. L., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+are+protected+from+lethal+hypoxia+by+an+embryonic+diapause.">C. elegans are protected from lethal hypoxia by an embryonic diapause.</a> Curr. Biol. 19, 1233-1237 (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

Privazione di ossigeno e Suspended Animation Sebbene l&#39;esposizione alla carenza di ossigeno grave può essere fatale per alcuni organismi, alcuni organismi sono in grado di sopravvivere l&#39;esposizione a anossia. Nel caso di C. elegans, la sopravvivenza di esposizione anossia dipende stadio di sviluppo e la risposta alla anossia è entrata in uno stato di animazione sospesa reversibile in cui sono stati arrestati una serie di processi biologici osservabili. Processi di divisione ...

<table border="1"><tbody><tr><td> Reagenti / Accessori </td><td></td><td></td><td> Composizione </td></tr><tr><td> Soluzione di ipoclorito di </td><td></td><td></td><td> 0,7 g KOH, 12 ml NaOCl 5%, portare a 50 ml con DDH 2 0 </td></tr><tr><td> M9 tampone </td><td></td><td></td><td> 3 g KH 2 PO 4, 11 g di Na 2 HPO 4, 5 g NaCl, 1 ml (1 M) MgSO 4 per 1 L DDH 2 O </td></tr><tr><td> Vetro microscopio diapositive </td><td> Fisher Scientific </td><td> 12-550-343 </td><td> 3 &quot;x1&quot; x1.0 millimetro </td></tr><tr><td> Turno micro coprioggetti </td><td> Scienze della microscopia elettronica </td><td> 72223-01 </td><td> 25 mm Diametro </td></tr><tr><td> Halocarbon olio 700 </td><td> Sigma </td><td> H8898-100ml </td><td></td></tr><tr><td> Anestetico </td><td></td><td></td><td> 0,5% TricAine, 0,05% Tetramisole </td></tr><tr><td> Leiden chiuso perfusione Microincubator </td><td> Harvard Apparatus </td><td> 650041 </td><td></td></tr><tr><td> UHP azoto </td><td> Calgaz (Air Liquide) </td><td></td><td> &gt; 99,9990% N 2 &lt;2 ppm O 2 </td></tr><tr><td> Tubo in plastica </td><td> VWR </td><td> 89068-468 </td><td> 0,062 &quot;ID x 0.125&quot; OD </td></tr><tr><td> Spinning microscopio confocale disco </td><td> McBain Sistemi </td><td></td><td> Zeiss microscopio ottico invertito, sistema di illuminazione epifluorescenza, CSU-10 Yokogawa scanner confocale, Hamamatsu moltiplicatore elettronico CCD. </td></tr></tbody></table>

use of time lapse microscopy to visualize anoxia-induced suspended animation in c. elegans embryos

Elegans Caenorhabdits è stato ampiamente utilizzato nello studio della resistenza alle sollecitazioni, che è facilitato dalla trasparenza degli stadi adulti ed embrioni nonché dalla disponibilità di mutanti genetici e ceppi transgenici che esprimono una miriade di proteine ​​di fusione 1-4. Inoltre, i processi dinamici come la divisione cellulare può essere visualizzato utilizzando proteine ​​del reporter fluorescente etichettati. Lo studio della mitosi può essere facilitata attraverso l&#39;utilizzo di time-lapse esperimenti in vari sistemi, compresi gli organismi intatti e di conseguenza l&#39;inizio C. embrione elegans è adatto per questo studio. Presentato qui è una tecnica con la quale immagini in vivo di strutture subcellulari in risposta a anossica (99.999% N 2; &lt;2 ppm O 2) lo stress è possibile con un semplice flusso di gas attraverso la configurazione di un potente microscopio. Una camera microincubation viene utilizzato in combinazione con un flusso di azoto e gas attraverso un microscopio confocale disco rotanteper creare un ambiente controllato in cui gli animali possono essere esposte in vivo. Utilizzando GFP-tubulina gamma etichettato e istoni, la dinamica e arresto della divisione cellulare può essere monitorato prima, durante e dopo l&#39;esposizione ad un ambiente privo di ossigeno. I risultati di questa tecnica sono ad alta risoluzione, video e immagini dettagliate delle strutture cellulari all&#39;interno blastomeri di embrioni esposti a deprivazione di ossigeno.

C. elegans embrioni esposti a grave carenza di ossigeno (anossia) sono in grado di sopravvivere con l&#39;arresto di processi biologici tra cui lo sviluppo e la divisione cellulare 7. L&#39;anossia arresto indotto di divisione cellulare può essere monitorata, ad un livello sub-cellulare, utilizzando una camera microincubation in combinazione con flusso di azoto e gas attraverso un microscopio confocale disco rotante per creare un ambiente controllato in cui gli animali possono essere esposte in ...

Watch this Scientific Journal Video about Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos at JoVE.com

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Qui descritto è un<em&gt; In vivo</em&gt; Tecnica di immagine strutture subcellulari in animali esposti ad anossia utilizzando un flusso di gas attraverso la camera microincubation in congiunzione con un microscopio confocale disco rotante. Questo metodo è semplice e flessibile abbastanza per soddisfare una varietà di parametri sperimentali e sistemi modello.

L&#39;uso della microscopia Time lapse di visualizzare anossia indotta Suspended Animation in<em&gt; C. elegans</em&gt; Embrioni

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

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Research

JoVE Journal

Biology

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

11.5K Views.  University of North Texas. Qui descritto è un In vivo Tecnica di immagine strutture subcellulari in animali esposti ad anossia utilizzando un flusso di gas attraverso la camera microincubation in congiunzione con un microscopio confocale disco rotante. Questo metodo è semplice e flessibile abbastanza per soddisfare una varietà di parametri sperimentali e sistemi modello.

Video: Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Monitoring Actin Disassembly with Time-lapse Microscopy

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.  ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

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 ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Imaging C. elegans Gli embrioni utilizzando un microscopio epifluorescente e Software Open Source

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Determinare profili di espressione genetica in C. elegans Utilizzo Microarray e Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

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 ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi di screening per identificare fenotipi postembrionale in C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

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 ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Visualizzazione di sviluppo craniofacciale nei SOX10: Kaede transgenici Zebrafish line utilizzando Time-lapse Microscopia confocale

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Metodi di valutazione subcellulari Compartimenti di muscoli in<em&gt; C. elegans</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...