Questa ricerca è stata resa possibile da una carriera premio National Science Foundation (MCB-0746066, Caratterizzazione di attivatori trascrizionali che regolano il comportamento emergente) per RDW Siamo grati a LJ Shimkus, BS Goldman, G. Suen, M. Singer, LG Welch, KA Murphy, e HG Taylor per utili discussioni e commenti sul manoscritto.

Forniture necessarie: <ul><li> Klett metro </li><li> Pipetta e suggerimenti </li><li> Microprovette 2,5 ml </li><li> Microcentrifuga </li><li> CTTYE media: <ul><li> 1,0% Casitone (Laboratori Difco), estratto di lievito 0,5% (Difco Laboratories), </li><li> 10,0 mM Tris-HCl (pH 8,0), 1,0 mM KH 2 PO 4, 8.0 mM MgSO 4 </li></ul></li><li> TPM media: <ul><li> 10,0 mM Tris-HCl (pH 8,0), 1,0 mM KH 2 PO 4, 8.0 mM MgSO 4 </li></ul></li><li> Agarosio </li><li> Bagnomaria regolato a 55 ° C </li><li> Vetrini per microscopio </li><li> Coprioggetto di vetro di grandi dimensioni </li><li> 2 x 2 cm, 0.5 mm di spessore guarnizione in gomma di silicone (Grazia Bio-Lab Inc.) </li><li> Parafilm (o nastro etichettatura) </li><li> Forcipe </li><li> Clip legante piccoli </li><li> Micro-campionamento pipetta (Fisher) </li><li> 100 microlitri punta di vetro monouso (Fisher) </li><li> Kimwipes </li></ul> Parte 1: Preparazione cellulare Iniziamo creando un ambiente sterile. <ul><li> Pulire lavoro, indossare guanti, e bruciatore luce. </li></ul><ol><li> Avviare una cultura durante la notte di cellule da inoculare in un pallone contenente brodo CTTYE e incubate al buio a 32 ° C, rimestando vigoroso. </li><li> Una volta che la cultura ha raggiunto una densità di 5 x 10 8 cellule / ml, pipetta da 1 ml di cellule in una provetta 2,5 ml. </li><li> Celle a pellet per centrifugazione per 2 minuti a 16.000 xg (o la velocità max). </li><li> Decantare il surnatante e gettarlo. </li><li> Lavare il pellet con 1 ml di TPM (sale-bilanciato, nutriente senza media). <ul><li> risospendere e vortice </li></ul></li><li> Re-pellet cellule facendo girare per 2 minuti a 16.000 xg (o la velocità max). </li><li> Decantare il surnatante e gettarlo. PUNTO CRITICO </li><li> Completamente risospendere pellet in 100 mezzi TPM microlitri utilizzando una combinazione di pipettaggio e vortex. IMPORTANTE: Questo passaggio garantisce che non ci siano grumi di cellule sinistra nel tubo. Questa operazione potrebbe richiedere un po &#39;. </li><li> Impostare le celle da parte a temperatura ambiente durante la preparazione delle piastre test di monitoraggio. </li></ol> Parte 2: Preparazione Agar <ol><li> Preparare 50 ml di entrambi i TPM (saggio di media) e CTTYE (disco nutritivo) dei media. </li><li> Aggiungere 0,5 g di agarosio ad ogni (agarosio è meno diffrazione di agar). </li><li> Autoclave per la sterilizzazione. </li><li> Dopo la sterilizzazione, mantenere i media a 55 ° C in un incubatore (o bagnomaria), mentre la costruzione del monitoraggio complessi micropiastra. </li></ol> Parte 3: Costruzione del disco Nutritive <ol><li> Inserire una sterile 0,5 mm di spessore guarnizione in gomma siliconica sulla cima di una fiamma sterilizzato vetrino microscopio, formando un piccolo pozzo. </li><li> Pipettare ~ 300 microlitri della 55 ° C CTTYE media / agarosio nel pozzo creato dalla guarnizione della diapositiva e contenente il disco nutritive. I media CTTYE / agarosio deve tumulo alto. </li><li> Posizionare un vetrino sterilizzato fiamma (senza guarnizione) sulla parte superiore del supporto CTTYE / agarosio. IMPORTANTE: Questa diapositive devono essere stabilite con un angolo per evitare la formazione di bolle. </li><li> Una volta che la diapositiva è a posto, bloccare il complesso insieme legante mini clip - uno su ciascun lato. </li><li> Posizionare il complesso tagliato a 4 ° C e lasciare i media CTTYE / agarosio da impostare. Questo richiede di solito circa 5 min. </li></ol> Parte 4: Preparazione del saggio di monitoraggio - Set Up Complessi Piastra Assemblare i componenti, preparare i complessi di diapositive, posizionare il disco nutritive, versare i media TPM / agarosio, complessi piastra separata e secco, le cellule piatto, e assemblare il monitoraggio complessi micropiastra. Assemblare componenti piastra complessi <ol><li> Per ogni complesso, fiamma sterilizzato un vetrino per microscopio </li><li> Posizionare una guarnizione in autoclave sulla parte superiore del vetrino e mettere da parte (sterilizzato verso l&#39;alto). Ciò costituirà la base del test. </li><li> Fiamma sterilizzare una copertura in vetro antiscivolo e posizionarlo in cima ad una seconda diapositiva microscopio vetro avvolto con nastro etichettatura (o Parafilm) e mettere da parte (sterilizzato verso l&#39;alto). Questo viene utilizzato come una diapositiva supporto per mantenere le coprioggetto di cracking. Questa sarà la parte superiore del test. </li><li> Sterilizzate terzo vetrino da microscopio in vetro e mettere da parte (sterilizzato verso l&#39;alto). Questo sarà usato per appiattire l&#39;agarosio. IMPORTANTE: Assicurarsi che le guarnizioni forma un sigillo con il vetro premendolo verso il basso con pinza, altrimenti la media / agarosio potrebbe seccare. </li></ol> P<html> pizzo disco nutritive IMPORTANTE: passaggi da 5 a 10 deve essere fatto per un complesso diapositiva alla volta, altrimenti i media / agarosio potrebbe iniziare a solidificare con conseguente scarsa qualità del film. <ol start="5"><li> Rimuovere il modulo nutritive disco complesso 4 ° C (passo 5 da parte 3) e indagare separatamente le diapositive esporre l&#39;ormai solidificato CTTYE / agarosio. </li><li> Rimuovere un 1 &#39;disco nutritivi&#39; mm di diametro dai media CTTYE / agarosio ben descritta sopra utilizzando una micro-pipetta di campionamento con una punta di 100 microlitri bicchiere usa e getta e metterla nel pozzo creata dalla guarnizione sul vetrino (punto 3) . </li></ol> Versare nelle piastre PUNTO CRITICO <ol start="7"><li> Pipettare ~ 300 microlitri della 55 ° C TPM media / agarosio nel pozzo creato dalla guarnizione sul vetrino e contenente il disco nutritive. I media TPM / agarosio deve tumulo alto. PUNTO CRITICO </li><li> Posizionare il vetrino fiamma sterilizzato senza guarnizione (dal punto 3) sulla parte superiore del supporto TPM / agarosio. IMPORTANTE: Questa diapositive devono essere stabilite con un angolo per evitare la formazione di bolle. </li><li> Una volta che la diapositiva (dal punto 5) è in posizione, bloccare il complesso insieme legante mini clip - uno su ciascun lato. </li><li> Posizionare il complesso tagliato a 4 ° C e lasciare i mezzi di comunicazione / agarosio da impostare. Questo richiede di solito circa 5 min. </li></ol> Lastre separate e asciutto IMPORTANTE: per evitare che i media / agarosio si secchi, i passaggi da 11 a 20 deve essere eseguita solo su un complesso diapositiva alla volta. IMPORTANTE: Per ottenere i migliori risultati, i passaggi da 11 a 13 deve essere eseguita a 4 ° C. <ol start="11"><li> Una volta che i media / agarosio ha creato, rimuovere la clip legante e spremere la fine del complesso per allentare il nastro avvolto diapositive. </li><li> Rimuovere il nastro avvolto diapositive e metterlo in panchina per un ulteriore uso. PUNTO CRITICO </li><li> Utilizzando pinze come un cuneo, separare la polizza di copertura / guarnizione / media / agarosio complesso dalla diapositiva di supporto (senza guarnizione) e scartare scivolo supporto. IMPORTANTE: Non usare un movimento indiscreti per separare la polizza di copertura / complesso guarnizione. Questo potrebbe causare la rottura slittamento copertura e / o il media / agarosio attenersi alla diapositiva supporto. </li><li> Collocare il vetrino / guarnizione / media / agarosio complesso sul nastro avvolto scivolo (lato guarnizione in su) e togliere dal 4 ° C. Inserire questo accanto ad un complesso bruciatore per permettere a tutti evaporare l&#39;umidità visibile dalla recente esposizione media / agarosio superficie - non più di 1 min. IMPORTANTE: Non lasciare che la media / agarosio a secco per troppo tempo in quanto ciò potrebbe influire il M. xanthus sciame comportamento. </li></ol> Piastra di cellule PUNTO CRITICO <ol><li> Una volta che l&#39;umidità in eccesso è evaporata, pipettare 0,5 l di cellule concentrate (punto 8 da parte 1) sul supporto / agarosio circa 1 mm di distanza dal disco di nutrienti. IMPORTANTE: E &#39;estremamente importante assicurarsi che le cellule si depositano spostando la pipetta verso il basso e poi verso l&#39;alto. Questo assicura che lo sciame sarà circolare. IMPORTANTE: E &#39;estremamente importante non toccare la punta della pipetta ai media / agarosio. Questo farà una depressione sulla superficie e cambiare il comportamento del M. xanthus sciame. SUGGERIMENTO: Premere la punta della pipetta prima di avvicinarsi ai media / agarosio. Questo permetterà una goccia di cellule ad apparire sul fondo la punta della pipetta e rendere più facile per depositare le cellule. </li><li> Una volta che le cellule sono depositate, luogo il prossimo complesso al bruciatore per consentire lo spot cellulare a secco - non più di 20 sec. </li></ol> Assemblare test <ol><li> Una volta che lo spot cellulare si è asciugato, allineare la diapositiva / guarnizione complesso (dal punto 1) con la guarnizione sul vetrino / guarnizione / media / agarosio / cellulari complessi (dal punto 13) e premere delicatamente insieme per formare un sigillo. PUNTO CRITICO </li><li> Pulire le superfici del vetrino e coprire scivolare con un Kimwipe per rimuovere i residui lasciati dai Parafilm. Il test completato dovrebbe essere simile a Figura 1. </li><li> Posizionare il complesso scivolo completato la fase di riscaldamento mantenuta a 32 ° C (scivolare verso il basso, coprire scivolare verso l&#39;alto) subito dopo pulire (passo 15) per evitare che forma la formazione di condensa. In caso di formazione di condensa, lasciare riposare complesso scivolare sul palco riscaldato per alcuni secondi prima di avviare il software di acquisizione immagini. </li><li> Scegli l&#39;obiettivo appropriato (2X, 4X o 10X). </li></ol> Parte 5: Preparazione del film t &quot;&gt; PUNTO CRITICO <ol><li> Accendere la fotocamera e il microscopio, e controllare i livelli di luce (assicuratevi che la luce non è troppo intenso). Avviare il computer e aprire il programma di acquisizione delle immagini. </li><li> Fare clic sulla finestra dell&#39;immagine dal vivo e mettere a fuoco il microscopio. L&#39;immagine dovrebbe essere simile a quello visto in Figura 3. Si noti la superficie uniforme agar. </li><li> Avviare l&#39;acquisizione di immagini. PUNTO CRITICO </li><li> Controllare regolarmente la messa a fuoco durante la prima ora, come i media / agar tende a sedimentare causando la messa a fuoco ad andare alla deriva. </li><li> Pulire l&#39;area di lavoro. </li><li> Una volta che l&#39;acquisizione delle immagini è completa, il trasferimento delle immagini acquisite per la conservazione e abbattere il complesso diapositiva immersione in etanolo al 90% durante la notte. </li><li> Guarnizioni autoclave per il riutilizzo. </li></ol><img src="/files/ftp_upload/2038/2038fig1.jpg" alt="Figura 1" /> Figura 1. Cartoon illustrazione del complesso piastra TM. (A) mostra la piastra di base complesso TM in vista esplosa e sezione. (B) mostra l&#39;uso di grandi guarnizioni. <img src="/files/ftp_upload/2038/2038fig2.jpg" alt="Figura 2" /> Figura 2. Grappolo microscopio. Ogni nodo microscopio (nel riquadro) è costituito da un microscopio Nikon E400, obiettivi, uno stadio riscaldato, una fotocamera Insight, e un computer portatile. Ogni nodo è in rete tra loro e collegati ad un computer master controller. Due i nodi sono configurati con capacità di fluorescenza che consistono della sorgente di luce EXFO e due ante Uniblitz. <img src="/files/ftp_upload/2038/2038fig3.jpg" alt="Figura 3" /> Figura 3. Un 20X immagine di apparati di analisi di tracciamento al tempo = 0. Scala grafica, 1 mm. <img src="/files/ftp_upload/2038/2038fig4.jpg" alt="Figura 4" /> Figura 4. Adattabilità del complesso piastra TM. (A) una immagine 100X di M. xanthus scivolando sulla motilità CTTYE a 1,0% agar. (B) un 20X l&#39;immagine di P. aeruginosa spasmi motilità. (C) un 20X immagine S. marcescens brulicante motilità. Entrambi (B) e (C) sono stati analizzati con LB nel 1,0% agar. (D) una immagine 40X di M. smegmatis scorrevole motilità su LB nello 0,5% agar. Questa immagine è stata scattata con la configurazione alternativa test visto in Fig. 1B. Video 1. Un time-lapse video di uno sciame sottoposto al test di rilevamento. Video 2. Un time-lapse video di un M. xanthus sciame dove 1% delle cellule sono marcate. Alternando immagini a contrasto di fase e fluorescenti sono stati catturati e sovrapposti per chiarire la posizione delle cellule fluorescenti all&#39;interno dello sciame. Questo video è stato catturato il CTTYE a 1,0% agar. Video 3. Un time-lapse video di M. xanthus deltaplano motilità. Questo video è stato catturato il CTTYE a 1,0% agar. Video 4. Un time-lapse video di P. aeruginosa spasmi motilità. Questo video è stato catturato su LB nel 1,0% agar. Video 5. Un time-lapse video di S. marcescens brulicante motilità. Questo video è stato catturato su LB nel 1,0% agar. Video 6. Un time-lapse video di M. smegmatis scorrevole motilità. Questo video è stato catturato su LB nello 0,5% agar utilizzando la configurazione alternativa test visto in Fig. 1B.

<ol>
	<li>Taylor, R. G., Welch, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+as+an+emergent+property+of+a+swarm.">Chemotaxis as an emergent property of a swarm.</a> J Bacteriol. 190, 6811-6816 (2008).</li><li>Curtis, P. D., Taylor, R. G., Welch, R. D., Shimkets, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Organization+of+Myxococcus+xanthus+During+Fruiting+Body+Formation.">Spatial Organization of Myxococcus xanthus During Fruiting Body Formation.</a> J Bacteriol. 189, 9126-9130 (2007).</li><li>Mignot, T., Merlie, J. P., Zusman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+pole-to-pole+oscillations+of+a+bacterial+gliding+motility+protein.">Regulated pole-to-pole oscillations of a bacterial gliding motility protein.</a> Science. 310, 855-857 (2005).</li><li>Stoodley, P., Hall-Stoodley, L., Lappin-Scott, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detachment+surface+migration,+and+other+dynamic+behavior+in+bacterial+biofilms+revealed+by+digital+time-lapse+imaging.">Detachment surface migration, and other dynamic behavior in bacterial biofilms revealed by digital time-lapse imaging.</a> Methods Enzymol. 337, 306-319 (2001).</li><li>O'Toole, G., Kaplan, H. B., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+as+microbial+development.">Biofilm formation as microbial development.</a> Annu Rev Microbiol. 54, 49-79 (2000).</li><li>O'Toole, G. A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flagellar+and+twitching+motility+are+necessary+for+Pseudomonas+aeruginosa+biofilm+development.">Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development.</a> Mol Microbiol. 30, 295-304 (1998).</li><li>Dalton, H. M., Poulsen, L. K., Halasz, P., Angles, 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=Substratum-induced+morphological+changes+in+a+marine+bacterium+and+their+relevance+to+biofilm+structure.">Substratum-induced morphological changes in a marine bacterium and their relevance to biofilm structure.</a> J Bacteriol. 176, 6900-6906 (1994).</li><li>Dworkin, M., Eide, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myxococcus+xanthus+does+not+respond+chemotactically+to+moderate+concentration+gradients.">Myxococcus xanthus does not respond chemotactically to moderate concentration gradients.</a> J Bacteriol. 154, 437-442 (1983).</li><li>Dworkin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactic+behavior+of+Myxococcus+xanthus.">Tactic behavior of Myxococcus xanthus.</a> J Bacteriol. 154, 452-459 (1983).</li><li>Wu, S. S., Kaiser, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+expression+of+the+pilA+gene+in+Myxococcus+xanthus.">Regulation of expression of the pilA gene in Myxococcus xanthus.</a> J Bacteriol. 179, 7748-7758 (1997).</li><li>Bustamante, V. H., Mart&#237;nez-Flores, I., Vlamakis, H. C., Zusman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+Frz+signal+transduction+system+of+Myxococcus+xanthus+shows+the+importance+of+the+conserved+C-terminal+region+of+the+cytoplasmic+chemoreceptor+FrzCD+in+sensing+signals.">Analysis of the Frz signal transduction system of Myxococcus xanthus shows the importance of the conserved C-terminal region of the cytoplasmic chemoreceptor FrzCD in sensing signals.</a> Mol Microbiol. 53, 1501-1513 (2004).</li></ol>

Nessun conflitto di interessi dichiarati.

Time-lapse microcinematografia (TM) è diventato un approccio standard allo studio della motilità procarioti 2-7. Tradizionalmente, TM viene eseguita utilizzando stoppini carta da filtro, pastiglie agar sottile, o lastre di agar come substrati 8-11. Questi metodi sono adeguati ed economicamente efficace quando usata per generare sequenze di immagini per le illustrazioni generale del movimento dei batteri. Tuttavia, se le sequenze di immagini deve comportare la generazione di dati riproducibili e q...

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<td>1.0% Casitone</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>0.5% yeast extract</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Micro-sampling pipette</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
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<td>100 &mu;l glass disposable tip</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
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<td>2 x 2 cm, 0.5-mm-thick silicone rubber gasket</td>
<td>&nbsp;</td>
<td>Grace Bio-Lab Inc.</td>
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recording multicellular behavior in myxococcus xanthus biofilms using time-lapse microcinematography

Uno sciame di δ-proteobacterium Myxococcus xanthus contiene milioni di cellule che agiscono come un collettivo, il movimento coordinamento attraverso una serie di segnali per creare complessi, i modelli dinamici come risposta a stimoli ambientali. Questi modelli sono auto-organizzazione ed emergenti, non può essere previsto osservando il comportamento delle singole celle. Utilizzando un time-lapse analisi monitoraggio microcinematografia, abbiamo identificato un modello distinto emergente in M. xanthus chiamato chemiotassi, definito come il movimento diretto di uno sciame di un gradiente di nutrienti verso la sua sorgente 1. Al fine di caratterizzare in modo efficiente chemiotassi tramite time-lapse microcinematografia, abbiamo sviluppato un piatto altamente modificabile complesso (Figura 1) e costruito un cluster di 8 microscopi (Figura 2), ciascuno in grado di catturare video time-lapse. Il test è sufficientemente rigorosa per consentire la replicazione coerente di dati quantificabili, e il video risultante ci permettono di osservare e tracciare i cambiamenti sottili nel comportamento sciame. Una volta catturati, i video vengono trasferiti ad una analisi / storage computer con memoria sufficiente per elaborare e memorizzare migliaia di video. La flessibilità di questa configurazione è dimostrato utile a diversi membri della M. xanthus comunità.

Watch this Scientific Journal Video about Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography at JoVE.com

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

Per studiare<em&gt; Myxococcus xanthus</em&gt; Comportamento sciame, abbiamo progettato un time-lapse protocollo microcinematografia che possono essere modificati per saggi differenti. Impiega le condizioni di crescita standard adattate per la microscopia, e produce risultati riproducibili con l&#39;uso di poco costoso, guarnizioni in silicone riutilizzabile. Abbiamo usato questo metodo per quantificare chemiotassi pluricellulari.

Registrazione Comportamento multicellulari in Myxococcus xanthus Biofilm utilizzando Time-lapse microcinematografia

A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of ...

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Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

12.2K Views.  University of South Carolina (USC). Per studiare Myxococcus xanthus Comportamento sciame, abbiamo progettato un time-lapse protocollo microcinematografia che possono essere modificati per saggi differenti. Impiega le condizioni di crescita standard adattate per la microscopia, e produce risultati riproducibili con l'uso di poco costoso, guarnizioni in silicone riutilizzabile. Abbiamo usato questo metodo per quantificare chemiotassi pluricellulari.

Video: Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

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.  ...

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 ...

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 ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Misurazione quantitativa della Invadopodia-mediata proteolisi della matrice extracellulare in singoli contesti e pluricellulari

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

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

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

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

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

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 ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

L&#39;acquisizione di fluorescenza filmati time-lapse di lievito in erba e Analisi Dinamica singola cella utilizzando innesti

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

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, ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...