Gli autori ringraziano Robert Kelsh per gentile condividere il zebrafish reagente promotore SOX10.

1. Embrione Collection per Fotoconversione <ol><li> Impostare almeno dieci coppie di SOX10: Kaede zebrafish transgenico tra il 5 e 6:00 di sera. </li><li> La mattina successiva, tirare divisori e raccogliere le uova intorno a mezzogiorno. Trasferirli su piastre di Petri e metterli in 28,5 ° C incubatore. </li><li> A circa 24 ore dopo la fecondazione, pulire i piatti Petri, eliminando gli embrioni morti. Controllare lo stadio di sviluppo degli embrioni 13 usando la microscopia campo di luce e ogni microscopio a fluorescenza con una maggiore proteina fluorescente verde (EGFP) del filtro. Kaede sarà visibile attraverso questo filtro. </li><li> Quando gli embrioni raggiungono il 60 trasferimento hpf cinque embrioni fluorescenti vivaci in una capsula di Petri separata e dechorionate se necessario. </li></ol> 2. Montaggio di embrioni per Fotoconversione <ol><li> Montare l&#39;embrione utilizzando un microscopio fluorescente normale. Montaggio di embrioni su microscopio confocale è estremamente impegnativo. </li><li> Utilizzare supportoacqua ard E3 o preparare E3 fresco con la seguente ricetta: Aggiungere il seguente per litro di acqua distillata dH acqua 2 O aggiungere il seguente rendere 1x E3L: 0.292 g 5.0 mM NaCl 0,013 g 0.17mm KCl 0.044 g 0,33 mm CaCl 2 (essiccante, 96%, il reagente ACS) 0,081 g 0,33 mM MgSO4 (anhydrated) Facoltativo: aggiungere 200 ml di 0,05% di blu di metilene a 1X E3 come fungicida e mescolate. Piano mantiene a temperatura ambiente per 1 settimana. </li><li> Utilizzare tricaine standard o preparare tricaine usando seguente ricetta: Per 5 L di 0,2% tricaine mescolare i seguenti ingredienti: 45 ml Tris-Cl (1M pH9.5) 5 LH 2 O 10 g tricaine </li><li> Preparare &#39;succo film&#39; (50 ml E3, soluzione tricaine 0,015%), con l&#39;aggiunta di 3,75 ml di 0,2% tricaine a 46.25 ml E3. </li><li> Preparare agarosio mescolando 46.25 ml di acqua E3 a 46,35 mg basso punto di fusione (LMP) agarosio in un pallone di vetro da 50 ml e sciogliere le particelle solide da forno a microonde. Quindi aggiungere3,73 ml 0,2% tricaine di agarosio e mettere boccetta di vetro con agarosio in 50 ° C a bagnomaria fino a quando utilizzo. </li></ol> 3. Preparazione e Montaggio di Embryo <ol><li> Anestetizzare gli embrioni con una soluzione tricaine E3/0.015% made in fase 1.1.2 e trasferimento di embrioni di diapositiva da camera. </li><li> Immergetevi in ​​eccesso E3 con il tessuto carta e la messa agarosio su embrione. </li><li> Posizione embrione dorsale perfettamente con punte microloader. Assicurarsi che l&#39;intero embrione non galleggia sopra l&#39;agarosio, ma spingere il corpo verso il basso. Poi versare la soluzione tricaine E3/0.015% rispetto dell&#39;embrione agarosio incorporato fino a camera è completamente riempita di fluido. </li></ol> 4. Regolazione delle impostazioni del software sul microscopio confocale <ol><li> Utilizzare 20X obiettivo dell&#39;aria immersible (apertura numerica 0,75; dimensione pinhole 20) per mettere a fuoco l&#39;embrione. Quindi premere il pulsante della luce L100 sul microscopio e selezionare i canali nel software. Controls/A1settings aprire le icone di visualizzazione / acquisizione e premetek il pulsante di configurazione confocale. Avanti fare clic su &#39;auto&#39; e seleziona seguenti canali prima di chiudere nuovamente la finestra: Ch1: DAPI canale 403.4 nm (lunghezze d&#39;onda tra 350-410 nm possono essere utilizzati) CH2: 488,0 nm Ch3: 561,9 nm </li></ol> 5. Fotoconversione <ol><li> Spegnere Ch1 e 3 e accendere Ch2 prima di fare clic sul pulsante Rimuovi blocco e la scansione. </li><li> Focus su cellule di interesse per fotoconversione, che è meglio farlo partendo da 1frame/sec e alta tensione (HV) 200 e poi andare nella fotogrammi / sec e abbassando la HV di conseguenza fino a raggiungere 1/32 fotogrammi / sec e HV intorno 100-120, a seconda del rumore di fondo. </li><li> Quando l&#39;embrione è a fuoco, guardare sul lato destro dello schermo, afferrare il bordo verde e renderlo più piccolo modo che si inserisce l&#39;area di interesse per fotoconversione e fare clic destro con il mouse. Più piccolo è meglio perché la fotoconversione è irreversibile. </li><li> Girare fotogrammi / sec a 1 e HV 200 e colpirescansione. Una immagine ingrandita dell&#39;area di fotoconversione è ora visibile. </li><li> Cellule Photoconvert girando sul canale 1 (403.4 nm). Esporre al laser photoconverting ovunque 5-60 secondi, a seconda delle dimensioni della regione fino Kaede verde come si è visto attraverso il canale GFP è quasi assente. </li><li> Quando il segnale Kaede verde è quasi assente, girare Ch1 off. Girare Ch2 e 3. Quindi fare clic a destra sulla finestra sul campo area di scansione A1 e premere &#39;ritorno alla dimensione originale&#39; per verificare se sono stati photoconverted cellule desiderate. </li></ol> 6. Fare un Z-stack <ol><li> Trovare l&#39;icona &#39;cattura Z-serie&#39; nella barra dei menu e impostare i limiti superiore e inferiore della Z-stack prima di avviarlo. Imposta LUT. </li></ol> 7. Software-Settings Time-lapse <ol><li> Vai al box A1Simple GUI e selezionare le impostazioni seguenti: 1/32 fotogrammi / sec dimensioni 1024 mediamente 4 o 8X seconda del numero di Z-stack che deve essere presa in un loop </li><li> Vai a:controlli view / acquisizione / ND acquisizione sequenza e telaio tempo impostato (8-30min), fare clic su continua e fare clic su Z-stack. Imposta frontiere come provato prima e iniziare film. </li><li> Continuare ad aggiungere E3/0.015% soluzione tricaine diapositiva camera tutto il giorno. Di notte, riempire camera completamente. Che durerà fino al mattino successivo. </li><li> La mattina seguente, riempire soluzione tricaine E3/0.015% e verificare se embrione è ancora vivo. Perdita di fluorescenza indica morte. Continua ricariche e controlli per tutta la giornata. </li><li> Quando la struttura è terminata formatura, o l&#39;embrione è morto, finire il film. </li></ol> 8. Elaborazione post-produzione <ol><li> Fare clic su &#39;show proiezione massima intensita&#39; sulla barra degli strumenti, quindi fare clic destro sull&#39;immagine e creare un nuovo documento. Il video è ora visibile nella migliore qualità possibile. Vai a eliminare i fotogrammi che non sono necessari e regolare LUT. Infine salvare come. Avi. Questo formato sarà bene per Windows. Per Mac software scaricare to convertire. mov o. wmv. </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breakthroughs+in+the+genetics+of+orofacial+clefting.">Breakthroughs in the genetics of orofacial clefting.</a> Trends Mol. Med. 17 (12), 725-733 (2011).</li><li>Kimmel, C. B., Miller, C. T., Moens, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+and+morphogenesis+of+the+zebrafish+larval+head+skeleton.">Specification and morphogenesis of the zebrafish larval head skeleton.</a> Dev. Biol. 233 (2), 239-257 (2001).</li><li>Dougherty, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+Fate+Map+of+First+Pharyngeal+Arch+Structures+in+the+sox10:+kaede+Zebrafish+Transgenic+Model.">Embryonic Fate Map of First Pharyngeal Arch Structures in the sox10: kaede Zebrafish Transgenic Model.</a> J. Craniofac. Surg. 23 (5), 1333-1337 (2012).</li><li>Trainor, P. A., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hox+genes,+neural+crest+cells+and+branchial+arch+patterning.">Hox genes, neural crest cells and branchial arch patterning.</a> Curr. Opin. Cell Biol. 13 (6), 698-705 (2001).</li><li>Schilling, T. F., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segment+and+cell+type+lineage+restrictions+during+pharyngeal+arch+development+in+the+zebrafish+embryo.">Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo.</a> Development. 120 (3), 483-494 (1994).</li><li>Swartz, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+a+palatogenic+gene+program+in+zebrafish.">Examination of a palatogenic gene program in zebrafish.</a> Dev. Dyn. 240 (9), 2204-2220 (2011).</li><li>McCollum, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+toxicity+screening+in+zebrafish.+Birth+Defects+Res.">Developmental toxicity screening in zebrafish. Birth Defects Res.</a> C. Embryo Today. 93 (2), 67-114 (2011).</li><li>Wada, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+is+required+for+cranial+neural+crest+morphogenesis+and+chondrogenesis+at+the+midline+in+the+zebrafish+skull.">Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull.</a> Development. 132 (17), 3977-3988 (2005).</li><li>Eberhart, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Hedgehog+signaling+from+neural+to+oral+epithelium+organizes+anterior+craniofacial+development.">Early Hedgehog signaling from neural to oral epithelium organizes anterior craniofacial development.</a> Development. 133 (6), 1069-1077 (2006).</li><li>Ando, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc. Natl. Acad. Sci. U S A. 99 (20), 12651-12656 (2002).</li><li>Kimmel, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203 (3), 253-310 (1993).</li><li>Kawakami, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish-secreted+matrix+protein+you/scube2+is+implicated+in+long-range+regulation+of+hedgehog+signaling.">The zebrafish-secreted matrix protein you/scube2 is implicated in long-range regulation of hedgehog signaling.</a> Curr. Biol. 15 (5), 480-488 (2005).</li><li>Lombardo, V. A., Sporbert, A., Abdelilah-Seyfried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+tracking+using+photoconvertible+proteins+during+zebrafish+development.">Cell tracking using photoconvertible proteins during zebrafish development.</a> J. Vis. Exp. (67), e4350 (2012).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Qui è illustrato un nuovo metodo per la visualizzazione di sviluppo craniofacciale nel modello zebrafish. I SOX10: Kaede transgenico linea zebrafish è stato usato con successo per descrivere il modello migratorio di CNCC in dettaglio viene utilizzato come organismo modello 5. Precedenti studi hanno utilizzato i luoghi di interesse lordi come l&#39;occhio alle cellule bersaglio e si sono affidati a iniezione Kaede mRNA, saggi fotoconversione o in gabbia fluoresceina destrano per ...

Schisi oro-facciali rappresentano la deformità cranio-facciale più diffusa, con 1/700-1, 000 consegne interessati 1. Turbativa del primo embrionale sviluppo craniofacciale può portare alla formazione di labio-palatoschisi (CL / P). Mentre cause di schisi sindromica sono stati ampiamente dimostrato, le basi genetiche ed epigenetiche delle forme non sindromiche di clefting orofacciale devono ancora essere scoperti 2-4. Per comprendere l&#39;eziologia e la patogenesi di queste malformazioni, è necessario chiarire lo sviluppo di strutture craniofacciali su base cellulare. In tutte le specie di vertebrati le cellule della cresta neurale cranica (CNCC) migrano dal tubo neurale dorsale per popolare gli archi faringei, che contribuiranno alla formazione di strutture oro-facciale. Turbativa di sviluppo embrionale cresta neurale precoce può portare alla formazione di malformazioni cranio-facciali tra cui CL / P 5-7. In annunciodizione di somiglianze strutturali tra zebrafish e mammiferi sviluppo craniofacciale (CNCCs risiedono in regioni omologhe), la rete di regolamentazione gene è altamente conservata. È stato anche dimostrato che CNCCs evolve allo stesso modo tra specie amniote e zebrafish 8, rendendo il zebrafish un potente organismo per lo studio di base dello sviluppo e genetica di CL / P. Ha molti vantaggi, tra cui piccole dimensioni, rapida ed ex-utero sviluppo embrionale, e di alti tassi di allevamento. Inoltre, l&#39;embrione è otticamente trasparente, rendendolo suscettibile di osservazione di eventi evolutivi complessi sotto il microscopio 9. Si tratta di un modello animale ideale per lo studio della migrazione e differenziazione delle cellule della cresta neurale cranica. Ampliando precedentemente lavoro pubblicato 8, 10, 11, il modello migratorio di CNCC è stato descritto in dettaglio utilizzando le SOX10: kaede modello transgenico 5. Kaede è una proteina foto-convertibile che turns da verde a rosso dopo l&#39;attivazione di foto e permette di tracciare con precisione CNCCs. Durante questa trasformazione il scheletro peptidico viene scisso, suggerendo che la conversione è stabile, cioè le cellule possono essere monitorate per la loro destinazione finale 12. Linee transgeniche etichettati con Kaede sotto il controllo trascrizionale di SOX10 hanno mostrato che il palato amniote e la piastra etmoide di zebrafish sono formate omologo dalla fusione di protuberanze mascellari bilaterali (MXP) con il rilievo frontonasale (FNP) e che la cucitura di fusione a forma di Y è analogo tra specie. Tra le altre applicazioni, i SOX10: Kaede transgenici modello zebrafish è stato utilizzato per generare video di embrioni di zebrafish a diversi stadi di sviluppo di mostrare formazione delle strutture cranio-facciali normali e anormali. Fotoconversione di gruppi specifici di cellule permette di monitorare il loro sviluppo. Con questo metodo un metodo per creare immagini dal vivo di sviluppare craniofacstrutture IAL di zebrafish viene introdotta, rendendo più facile dimostrare visivamente il processo di sviluppo complesso. Questo protocollo è finalizzato a condividere l&#39;esperienza di generare questi video utilizzando il normale sviluppo della piastra etmoide in SOX10: Kaede zebrafish transgenico come esempio. Questo protocollo può essere ulteriormente applicato a fare video time-lapse di qualsiasi struttura derivata da craniche cellule della cresta neurale in zebrafish.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>sox10: kaede transgenic zebrafish line</td>
			<td>MGH</td>
			<td></td>
			<td>Available via the Liao lab</td>
		</tr>
		<tr>
			<td>Petri dishes 100x15 mm</td>
			<td>BD Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 35 mmx10 mm</td>
			<td>BD Falcon</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure Low melting point (LMP) Agarose</td>
			<td>Invitrogen</td>
			<td>15517022</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Tek 2 Chamber SlideSystem</td>
			<td>LabTek</td>
			<td>154453</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloaders 200/pk</td>
			<td>Fisher</td>
			<td>E5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R Si Confocal Ti series</td>
			<td>Nikon</td>
			<td>No Catalog number</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements Software AR3.2 64-bit</td>
			<td>Nikon</td>
			<td>No Catalog number</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of craniofacial development in the sox10: kaede transgenic zebrafish line using time-lapse confocal microscopy

Vertebrato palatogenesis è un processo di sviluppo altamente coreografica e complesso, che coinvolge la migrazione di cranica cresta neurale (CNC), le cellule, la convergenza e l&#39;estensione delle protuberanze facciali, e la maturazione dello scheletro cranio-facciale. Per studiare il contributo della cresta neurale cranica a specifiche regioni del palato zebrafish una SOX10: Kaede linea zebrafish transgenico è stato generato. Sox10 prevede la restrizione lignaggio della proteina Kaede giornalista alla cresta neurale, rendendo in tal modo la cella etichettare un processo più preciso di tintura tradizionale o giornalista iniezione di mRNA. Kaede è una proteina foto-convertibile che passa dal verde al rosso dopo l&#39;attivazione foto e permette di seguire con precisione le cellule. I SOX10: Kaede linea transgenica è stato utilizzato per eseguire l&#39;analisi lignaggio di delineare popolazioni di cellule del CNC che danno luogo a mascellare contro elementi mandibolari e illustrano omologia di protuberanze facciali per amnioti. Questo protocollo è descritto il passaggios per generare un time-lapse video live di una SOX10: Kaede zebrafish. Sviluppo della piastra etmoide servirà come esempio pratico. Questo protocollo può essere applicato a una registrazione confocale time-lapse di qualsiasi Kaede o simile proteina reporter fotoconvertibile in zebrafish transgenico. Inoltre, può essere utilizzato per catturare non solo normale, ma anche anormale sviluppo di strutture craniofacciali nei mutanti di zebrafish.

Nel SOX10: CNCCs Kaede linea transgenica, migratori e post-migratori sono etichettati fluorescente verde. Le cellule CNCC etichettati con Kaede fluorescente verde ricapitolano endogena espressione SOX10 mRNA 5. Tra le altre applicazioni, questo modello animale è stato usato per visualizzare meglio lo sviluppo di CNCC strutture craniofacciali dipendenti. Il normale sviluppo di strutture specifiche e anche lo sviluppo patologico di malformazioni cranio-facciali, in particolare il l...

Watch this Scientific Journal Video about Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy at JoVE.com

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

Visualizzazione dei dati sperimentali è diventata un elemento chiave per la presentazione dei risultati alla comunità scientifica. Generazione di live time-lapse di embrioni in crescita contribuisce a una migliore presentazione e la comprensione dei processi di sviluppo complessi. Questo protocollo è una guida passo-passo per l&#39;etichettatura cellulare tramite fotoconversione di proteine ​​Kaede 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, ...

visualization-craniofacial-development-sox10-kaede-transgenic

Research

JoVE Journal

Biology

12.7K Views.  Massachusetts General Hospital.  Visualizzazione dei dati sperimentali è diventata un elemento chiave per la presentazione dei risultati alla comunità scientifica. Generazione di live time-lapse di embrioni in crescita contribuisce a una migliore presentazione e la comprensione dei processi di sviluppo complessi. Questo protocollo è una guida passo-passo per l'etichettatura cellulare tramite fotoconversione di proteine ​​Kaede in zebrafish. 

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

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.  ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Vivere Imaging of the Brain Zebrafish embrionali da microscopia confocale

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

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 ...

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 ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Elettroporazione di Mesenchima craniofacciale

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

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 ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Analizzando craniofacciale morfogenesi in Zebrafish Uso 4D Microscopia confocale

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...