Cette recherche a été rendue possible grâce à une bourse de la National Science Foundation carrière (MCB-0746066, Caractérisation des activateurs transcriptionnels qui régulent le comportement émergent) pour RDW Nous sommes reconnaissants à LJ Shimkus, BS Goldman, G. Suen, M. Singer, LG Welch, KA Murphy, et HG Taylor pour des discussions utiles et des commentaires sur le manuscrit.

Fournitures nécessaires: <ul><li> Klett mètres </li><li> Pipette et pointes </li><li> 2,5 ml microtubes </li><li> Microcentrifugeuse </li><li> CTTYE médias: <ul><li> 1,0% casitone (Difco Laboratories), extrait de levure 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 médias: <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> Agarose </li><li> Bain-marie réglé à 55 ° C </li><li> Lames de microscope </li><li> Lamelles de verre de grandes </li><li> 2 x 2 cm, 0,5 mm d&#39;épaisseur de joint en caoutchouc de silicone (Grace Bio-Lab Inc) </li><li> Parafilm (ou bande d&#39;étiquetage) </li><li> Forceps </li><li> Clips liant petites </li><li> Micro-échantillonnage pipette (Fisher) </li><li> 100 ul pointe de verre jetable (Fisher) </li><li> Kimwipes </li></ul> Partie 1: Préparation des cellules Commencez par créer un environnement stérile. <ul><li> Propre espace de travail, gants de Don, et allumer le brûleur. </li></ul><ol><li> Démarrer une culture de nuit de cellules en inoculant dans un flacon contenant un bouillon CTTYE et incubés à l&#39;obscurité à 32 ° C en remuant vigoureusement. </li><li> Une fois que la culture a atteint une densité de 5 x 10 8 cellules / ml, pipeter 1 ml de cellules dans un tube de 2,5 ml. </li><li> Cellules par centrifugation pendant 2 min à 16000 xg (ou vitesse max). </li><li> Décanter et éliminer le surnageant. </li><li> Laver culot de cellules avec 1 ml TPM (sel équilibrés en nutriments médias libres). <ul><li> remettre en suspension et le vortex </li></ul></li><li> Re-pellets cellules par filage pendant 2 min à 16.000 xg (ou vitesse max). </li> &lt;li&gt; Décanter et éliminer le surnageant. ÉTAPE CRITIQUE </li><li> Complètement remis en suspension le culot dans 100 médias TPM ul utilisant une combinaison de pipetage et vortex. IMPORTANT: Cette étape permet de s&#39;assurer qu&#39;il n&#39;y ait pas des amas de cellules restant dans le tube. Cela peut prendre un certain temps. </li><li> Définissez les cellules réserver à température ambiante pendant la préparation des plaques d&#39;essai de suivi. </li></ol> Partie 2: Préparation Agar <ol><li> Préparer 50 ml de fois TPM (analyse des médias) et CTTYE (disque nutritive) des médias. </li><li> Ajouter 0,5 g d&#39;agarose à chacun (agarose est moins diffraction de gélose). </li><li> Autoclave pour stériliser. </li><li> Après stérilisation, de maintenir le milieu à 55 ° C dans un incubateur (ou l&#39;eau du bain) lors de la construction des complexes de suivi de plaque d&#39;essai. </li></ol> Partie 3: Construction de disque Nutritive <ol><li> Placez un stérile 0,5-mm d&#39;épaisseur joint en caoutchouc de silicone sur le dessus d&#39;une flamme-lame de verre de microscope stérilisé, formant ainsi un petit. </li><li> Pipette ~ 300 ul de la 55 ° C CTTYE médias / agarose dans le puits créé par le joint sur la diapositive contenant le disque et nutritive. Les médias CTTYE / agarose devrait monticule. </li><li> Placer une lame de flamme stérilisé (sans joint) au-dessus du support CTTYE / agarose. IMPORTANT: Cette diapositive doit être posé à un angle pour éviter la formation de bulles. </li><li> Une fois que la lame est en place, serrer le complexe avec des clips liant mini - un de chaque côté. </li><li> Placer le complexe coupé à 4 ° C et laisser le support CTTYE / agarose à régler. Cela prend habituellement environ 5 min. </li></ol> Partie 4: Préparation Essai de suivi - Set Up Complexes plaques Assemblez les composants, préparer les complexes de diapositives, placez le disque nutritive, versez les médias TPM / agarose, sepcomplexes plaques arate et sec, les cellules de plaques et assembler le suivi des complexes plaque de dosage. Assembler les composants de plaques complexes <ol><li> Pour chaque complexe, la flamme stérilisé une lame de microscope en verre </li><li> Placez un joint autoclave au-dessus de la lame et mettre de côté (stérilisé vers le haut). C&#39;est ce qui constituera le fond de l&#39;essai. </li><li> Flamme stériliser un couvercle en verre de glisser et de le placer sur une lame de microscope second verre enveloppé d&#39;étiquetage bande (ou Parafilm) et mettre de côté (stérilisé vers le haut). Il est utilisé comme un chariot de support pour maintenir la lamelle de craquage. Cela formera la partie supérieure de l&#39;essai. </li><li> Flamme stériliser une diapositive troisième verre de microscope et mettre de côté (stérilisé vers le haut). Il sera utilisé pour aplatir l&#39;agarose. IMPORTANT: Assurez-vous que les joints de former un joint avec le verre en appuyant dessus avec une pince, sinon les médias / agarose risque de sécher. </li></ol> Pdentelle disque nutritive IMPORTANT: les étapes 5 à 10 doit être fait à un complexe diapositive à la fois, sinon les médias / agarose pourrait commencer à se solidifier pour une qualité vidéo médiocre. <ol start="5"><li> Retirez le disque sous forme nutritive complexe de 4 ° C (étape 5 de la partie 3) et essayer de séparer les diapositives exposant la maintenant solidifié CTTYE / agarose. </li><li> Suppression d&#39;un &quot;disque nutritive &#39;1 mm de diamètre à partir du support CTTYE / agarose bien décrite ci-dessus à l&#39;aide d&#39;une pipette de micro-échantillonnage avec une pointe en verre de 100 ul jetable et placez-le dans le puits créé par le joint sur la lamelle (étape 3 ci-dessus) . </li></ol> Verser dans les boîtes ÉTAPE CRITIQUE <ol start="7"><li> Pipette ~ 300 ul de la 55 ° C TPM médias / agarose dans le puits créé par le joint sur la lamelle et contenant le disque nutritive. Les médias TPM / agarose devrait monticule. CRITIQUE STEP </li><li> Placer la lame flamme stérilisés sans joint (à partir de l&#39;étape 3) sur le dessus du support TPM / agarose. IMPORTANT: Cette diapositive doit être posé à un angle pour éviter la formation de bulles. </li><li> Une fois que le coulisseau (à partir de l&#39;étape 5) est en place, serrer le complexe avec des clips liants pour mini - une de chaque côté. </li><li> Placer le complexe coupé à 4 ° C et laisser le support / agarose à régler. Cela prend habituellement environ 5 min. </li></ol> Plaques séparées et sèche IMPORTANT: Pour éviter des médias / agarose de se dessécher, les étapes 11 à 20 doit être effectuée que sur un complexe diapositive à la fois. IMPORTANT: Pour de meilleurs résultats, les étapes 11 à 13 doivent être effectuées à 4 ° C. <ol start="11"><li> Une fois le support / agarose a pris, retirer les clips liant et presser le bout du complexe de desserrer le ruban gainé glissée. </li><li> Retirez la lame de bande enroulée et le placer sur le banc pour une utilisation ultérieure. ÉTAPE CRITIQUE </li><li> En utilisant une pince comme un coin, séparer la lamelle / joint / support / agarose complexe de la glissière de support (sans joint) et jeter coulisseau de support. IMPORTANT: Ne pas utiliser un mouvement de levier pour séparer la lamelle / complexe d&#39;étanchéité. Cela pourrait entraîner la rupture lamelle et / ou le collage des médias / agarose pour la lame support. </li><li> Placez la lamelle / joint / media / agarose complexe sur la lame de bande enroulée (côté joint haut) et retirer de 4 ° C. Placez ce complexe à côté d&#39;un brûleur pour permettre à tous d&#39;humidité visible s&#39;évaporer de la nouvellement exposée médias / agarose surface - pas plus de 1 min. IMPORTANT: Ne pas laisser sécher les médias / agarose pendant trop longtemps car cela pourrait influer sur la M. xanthus essaim comportement. </li></ol> Cellules de la plaque <pclass = &quot;jove_content&quot;&gt; étape cruciale <ol><li> Une fois l&#39;excès d&#39;humidité se soit évaporée, une pipette 0,5 ul de cellules concentrées (étape 8 de la partie 1) sur les médias environ 1 / agarose mm du disque en éléments nutritifs. IMPORTANT: Il est extrêmement important de s&#39;assurer que les cellules sont déposées en déplaçant la pipette vers le bas puis vers le haut. Cela garantit que l&#39;essaim sera circulaire. IMPORTANT: Il est extrêmement important de ne pas toucher l&#39;embout de la pipette aux médias / agarose. Cela fera une dépression sur la surface et changer le comportement de l&#39;M. xanthus essaim. ASTUCE: Appuyez sur la pointe de la pipette avant d&#39;approcher les médias / agarose. Cela permettra une baisse de cellules à apparaître sur le fond de l&#39;embout de pipette et le rendre plus facile à déposer dans les cellules. </li><li> Une fois que les cellules sont déposées, placez le côté complexe du brûleur pour permettre à l&#39;endroit de la cellule<html> sèche - pas plus de 20 secondes. </li></ol> Assemblez test <ol><li> Une fois l&#39;endroit cellule a séché, alignez le complexe coulissant / joint (de l&#39;étape 1) avec le joint sur la lamelle / joint / media / agarose / cellule complexe (de l&#39;étape 13) et appuyez doucement pour former un joint. ÉTAPE CRITIQUE </li><li> Nettoyer les surfaces de la lame et lamelle avec un Kimwipe pour enlever le résidu laissé par le Parafilm. Le test complet devrait ressembler à la Figure 1. </li><li> Placez le complexe glissière achevée sur la scène chauffée maintenue à 32 ° C (toboggan, couvrir glisser vers le haut) immédiatement après essuyer (étape 15) pour éviter la formation de condensation forme. En cas de condensation, laisser reposer glissement complexe sur la scène chauffée pendant plusieurs secondes avant de démarrer le logiciel d&#39;acquisition d&#39;image. </li><li> Choisissez l&#39;objectif approprié (2X, 4X, 10X ou). </li></ol> Partie 5: Préparation Film t &quot;&gt; étape cruciale <ol><li> Allumez l&#39;appareil photo et d&#39;un microscope, et de vérifier les niveaux de lumière (assurez-vous que la lumière n&#39;est pas trop intense). Démarrez l&#39;ordinateur et ouvrez le programme d&#39;acquisition d&#39;images. </li><li> Cliquez sur la fenêtre d&#39;image en direct et orienter le microscope. L&#39;image devrait ressembler à celui illustré à la figure 3. Notez la surface de la gélose uniforme. </li><li> Démarrer l&#39;acquisition d&#39;images. ÉTAPE CRITIQUE </li><li> Vérifiez régulièrement l&#39;objet au cours de la première heure, comme les médias / agar-agar a tendance à s&#39;installer provoquant la mise au point à la dérive. </li><li> Nettoyer la zone de travail. </li><li> Une fois l&#39;acquisition d&#39;image est terminée, le transfert des images acquises pour le stockage et briser le complexe coulissant par trempage dans de l&#39;éthanol à 90% du jour au lendemain. </li><li> Joints autoclaves pour la réutilisation. </li></ol><img src="/files/ftp_upload/2038/2038fig1.jpg" alt="Figure 1" /> Illustration de bande dessinée la figure 1. Du complexe plaque TM.(A) montre la complexité plaque de base TM en vue éclatée et en coupe. (B) montre l&#39;utilisation de plus gros joints. <img src="/files/ftp_upload/2038/2038fig2.jpg" alt="Figure 2" /> Figure 2. Groupe microscope. Chaque noeud de microscope (encart) est constitué d&#39;un microscope Nikon E400, objectifs, une platine chauffante, un appareil photo Insight, et un ordinateur portable. Chaque noeud est mis en réseau et reliés à un ordinateur de commande maître. Deux des nœuds sont mis en place avec des capacités de fluorescence qui se composent de la source de lumière d&#39;EXFO et deux volets Uniblitz. <img src="/files/ftp_upload/2038/2038fig3.jpg" alt="Figure 3" /> Figure 3. A l&#39;image de l&#39;appareil d&#39;essai 20X suivi au temps = 0. La barre d&#39;échelle, de 1 mm. <img src="/files/ftp_upload/2038/2038fig4.jpg" alt="Figure 4" /> Figure 4. Adaptabilité du complexe plaque TM. (A) d&#39;une image de 100XM. xanthus glisse sur la motilité CTTYE dans la gélose 1,0%. (B) une image à 20X de P. aeruginosa secousses motilité. (C) une image 20X S. marcescens essaimage motilité. Les deux (B) et (C) ont été testées sur LB dans la gélose 1,0%. (D) une image à 40X de M. smegmatis glisser sur la motilité LB agar dans 0,5%. Cette image a été capturée à l&#39;aide de la configuration d&#39;essai de remplacement vu dans la figure 1B. Vidéo 1. Une vidéo time-lapse d&#39;un essaim soumis à l&#39;essai de suivi. Vidéo 2. Une vidéo time-lapse d&#39;un M. xanthus essaim où 1% des cellules sont marquées par fluorescence. Alternant à contraste de phase et des images fluorescentes ont été capturés et recouvert d&#39;élucider la position de cellules fluorescentes dans l&#39;essaim. Cette vidéo a été capturée sur CTTYE dans la gélose 1,0%. Vidéo 3. Une vidéo time-lapse de M. xanthus glisse motilité. Cette vidéo a été capturée sur CTTYE dansAgar 1,0%. Vidéo 4. Une vidéo time-lapse de P. aeruginosa secousses motilité. Cette vidéo a été capturée sur LB dans la gélose 1,0%. Vidéo 5. Une vidéo time-lapse de S. marcescens essaimage motilité. Cette vidéo a été capturée sur LB dans la gélose 1,0%. Vidéo 6. Une vidéo time-lapse de M. smegmatis glisser la motilité. Cette vidéo a été capturée sur LB agar 0,5% en utilisant la configuration d&#39;essai de remplacement vu dans la figure 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>

Time-lapse microcinématographie (TM) est devenu une approche standard à l&#39;étude de la motilité procaryote 2-7. Traditionnellement, TM est effectuée en utilisant des mèches de papier filtre, blocs de gélose ou en plaques minces, en tant que substrats de gélose 8-11. Ces méthodes sont adéquates et rentable lorsqu&#39;il est utilisé pour générer des séquences d&#39;images d&#39;illustrations généraux du mouvement bactérienne. Toutefois, si des séquences d&#39;images doit se tradu...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"><html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en"><div><table border="1"><thead><tr><th> Nom du produit </th><th> Type </th><th> Entreprise </th><th> Numéro de catalogue </th><th> Commenter </th></tr></thead><tbody><tr><td> Casitone 1,0% </td><td></td><td> Difco Laboratories </td><td></td><td></td><tr><td> 0,5% d&#39;extrait de levure </td><td></td><td> Difco Laboratories </td><td></td><td></td><tr><td> Micro-pipette d&#39;échantillonnage </td><td></td><td> Pêcheur </td><td></td><td></td><tr><td> 100 ul pointe de verre jetable </td><td></td><td> Pêcheur </td><td></td><td></td><tr><td> 2 x 2 cm, 0,5 mm d&#39;épaisseur de joint en caoutchouc de silicone </td><td></td><td> Grâce Bio-Lab Inc </td><td></td><td></td></tbody></table></div>

recording multicellular behavior in myxococcus xanthus biofilms using time-lapse microcinematography

Un essaim de la δ-protéobactérie xanthus Myxococcus contient des millions de cellules qui agissent comme un collectif, le mouvement de coordination à travers une série de signaux complexes pour créer des modèles dynamiques, en réponse à des signaux environnementaux. Ces modèles sont auto-organisation et émergence; ils ne peuvent pas être prédits en observant le comportement des cellules individuelles. En utilisant un test time-lapse microcinématographie suivi, nous avons identifié un modèle distinct émergente dans M. xanthus appelé chimiotactisme, défini comme le mouvement dirigé d&#39;un essaim d&#39;un gradient de nutriment vers la source 1. Afin de caractériser efficacement la chimiotaxie par time-lapse microcinématographie, nous avons développé une très plaque modifiables complexe (figure 1) et construit un groupe de 8 microscopes (figure 2), chacune capable de capturer des vidéos time-lapse. Le test est assez rigoureux pour permettre la réplication cohérente des données quantifiables, et le resulting vidéos nous permettent d&#39;observer et de suivre les changements subtils dans le comportement essaim. Une fois capturés, les vidéos sont transférées à un ordinateur d&#39;analyse / de stockage avec assez de mémoire pour traiter et stocker des milliers de vidéos. La flexibilité de cette configuration s&#39;est avéré utile pour plusieurs membres de la M. xanthus communauté.

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

Pour étudier<em&gt; Myxococcus xanthus</emComportement essaim&gt;, nous avons conçu un protocole microcinématographie time-lapse qui peut être modifié pour des analyses différentes. Il emploie des conditions de croissance standard adaptés pour la microscopie, et donne des résultats reproductibles en utilisant des joints en silicone, peu coûteux réutilisables. Nous avons utilisé cette méthode pour quantifier la chimiotaxie multicellulaires.

Enregistrement Comportement multicellulaire en<em&gt; Myxococcus xanthus</em&gt; Les biofilms en utilisant Time-lapse microcinématographie

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

recording-multicellular-behavior-myxococcus-xanthus-biofilms-using

Research

JoVE Journal

Biology

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

12.2K vues.  University of South Carolina (USC). Pour étudier Myxococcus xanthus, nous avons conçu un protocole microcinématographie time-lapse qui peut être modifié pour des analyses différentes. Il emploie des conditions de croissance standard adaptés pour la microscopie, et donne des résultats reproductibles en utilisant des joints en silicone, peu coûteux réutilisables. Nous avons utilisé cette méthode pour quantifier la chimiotaxie multicellulaires.

Vidéo: 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.

Surveillance actine démontage Time-lapse microscopie

Recherche

Biologie

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.

Deux photons axotomie et time-lapse imagerie confocale dans embryons de poissons zèbres vivent

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.

Imagerie time-lapse de la mitose après transfection de 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.

Time-lapse de microscopie du début de l&#39;embryogenèse dans les 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.

Analyse quantitative des migrations au hasard des cellules en utilisant la microscopie vidéo time-lapse

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.

Mesure quantitative de invadopodes la protéolyse matrice extracellulaire dans des contextes simples et multicellulaire

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;utilisation de la microscopie à Time Lapse Visualisez anoxie induite Suspended Animation en<em&gt; C. elegans</em&gt; Embryons

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;acquisition de fluorescence Time-lapse films de levure bourgeonnante et analyse de la dynamique de cellules uniques en utilisant des greffes

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.

Visualisation de développement cranio-facial dans les SOX10: Kaede poisson zèbre transgénique ligne à l&#39;aide Time-lapse de microscopie 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 ...