Nous tenons à exprimer notre reconnaissance pour la contribution et les commentaires des membres du Laboratoire Padilla. Souches de nématodes dans ce travail ont été fournies par le Centre de Caenorhabditis Genetics, qui est financé par le NIH Centre National de Ressources de recherche (NCRR). Nous reconnaissons et remercions le Dr Lon-Turnbull pour l&#39;assistance technique en microscopie confocale. Ce travail a été soutenu par une subvention de la National Science Foundation (NSF-IOS, carrière) au PAP

1. Préparation des échantillons <ol><li> Produire ou obtenir approprié transgénique C. elegans souche d&#39;intérêt en utilisant des méthodes transgéniques ou de Caenorhabditis Genetics Stock Center (CGC) ou d&#39;un collègue. Dans ce cas, nous utilisons souche TH32 (pie-1 :: tbg-1 :: GFP, tarte-1 :: GFP :: H2B) 5 à visualiser les chromosomes et les centrosomes comme marqueurs de la division cellulaire. </li><li> Générer une population synchronisée en utilisant l&#39;une des trois méthodes suivantes: 1) se désintègrent adultes gravides dans une solution d&#39;hypochlorite et d&#39;utiliser des embryons restants 6, 2) choisir adultes gravides ensemencées sur une plaque et laissez-les poser embryons pendant 1-2 heures, ou 3) prendre L4 larves d&#39;une population mixte et de passer à une nouvelle plaque ensemencée. </li><li> Cultivez les nématodes à 20 ° C à l&#39;âge adulte gravide, ce qui prendra environ 96 heures à partir de l&#39;éclosion, les larves de 72 heures à partir de L1 ou L4 24 h après la mue. Embryons (2-20 cellules) in utero contiennent de grandes blastomères et des noyaux qui sont optimales pour imagtion des changements sub-cellulaires et sub-nucléaire, respectivement. Cette méthodologie fournit un moyen pour générer une population de jeunes adultes qui contiennent un nombre abondant d&#39;embryons précoces. </li></ol> 2. Préparation Faites glisser et anoxie Chambre Set Up <ol><li> Faites une chambre humide à tenir lames préparées en plaçant une serviette en papier humide à l&#39;intérieur d&#39;un grand plat de Pétri ou dans le bac recouvert d&#39;un couvercle de taille suffisante. </li><li> Préparer fondu 2% d&#39;agarose dans désionisée H 2 O par chauffage dans de la verrerie mélange par micro-ondes ou bain d&#39;eau. </li><li> Placer 1-3 gouttes d&#39;eau tiède agarose sur une lame de microscope en verre propre. Placer immédiatement une deuxième lame inversée verticalement au-dessus de la chute d&#39;agarose (s), appuyez légèrement de sorte que le coussin résultant sera mince et sans bulles d&#39;air. </li><li> Laisser le temps de refroidir lentement et démonter les deux lames de microscope en laissant intact le tampon d&#39;agarose sur une des diapositives. Il est important d&#39;assurer le pad agarose est assez mince pour ne pas perturre avec la capacité de se concentrer au microscope après. Possibilité de visualiser l&#39;échantillon dépend aussi de la distance optique spécifique de l&#39;objectif utilisé, qui peut varier entre microscopes. </li><li> Utilisez une lame de rasoir propre pour façonner tampon d&#39;agarose dans un petit carré. Conserver dans la chambre humide préparée jusqu&#39;au moment de servir. </li><li> Soigneusement, en utilisant la même lame de rasoir propre, prendre le bord du pad agarose et le transférer de la faire glisser sur un rond de 25 mm lamelle micro, en évitant l&#39;accumulation de bulles d&#39;air sous le clavier. La lamelle ronde choisi d&#39;utiliser s&#39;insère correctement dans le microincubator. </li><li> On ajoute une goutte d&#39;anesthésique (0,5% tricaïne, tétramisole 0,05% en tampon M9) sur le bloc d&#39;agarose destiné à être utilisé comme anesthésique. Choisissez les vers 5-10 et lieu dans la goutte d&#39;anesthésique. Laisser 1-3 min pour les vers à cesser le mouvement. </li><li> La justification de l&#39;observation des embryons dans l&#39;utérus adulte, au lieu d&#39;embryons disséqués, est de minimiser le potentiel de l&#39;embryon desiccation et le mouvement dû à l&#39;écoulement du gaz d&#39;azote dans l&#39;embryon. </li><li> Immédiatement ajouter une goutte d&#39;huile halocarbone au-dessus des vers de terre pour empêcher les vers de déshydratation de gaz s&#39;écoule à travers la chambre. Comme l&#39;huile est visqueuse halocarbures, on peut utiliser un embout de pipette ou de la pointe d&#39;un ver prendre pour recueillir l&#39;huile et déposer sur les vers de terre. </li><li> Une fois la lamelle circulaire contenant les vers est prêt, les deux moitiés de la fermeture Leiden perfusion microincubator sont séparées, et la lamelle est ensuite soigneusement placé à la verticale sur l&#39;anneau inférieur. Les deux côtés de la chambre sont ensuite fermées hermétiquement ensemble. </li><li> La chambre est ensuite reliée au réservoir d&#39;azote gazeux par l&#39;intermédiaire de tubes en matière plastique souple et placé à l&#39;endroit approprié sur la platine du microscope (figure 1C). À ce moment le tube et microincubator doit être soigneusement contrôlée pour les fuites en assurant une fixation sûre de la tuyauterie et des bouchons d&#39;orifice le long du côté de la chambre. On peut ALsi légèrement étaler une petite quantité d&#39;eau savonneuse sur le tube de chercher des bulles, qui formeront si une fuite est présente. </li></ol> 3. Microscopie en anoxie <ol><li> Localiser les animaux à faible grossissement, puis déplacez le grossissement supérieur approprié (64x pour cette application) pour les tissus d&#39;image ou cellules d&#39;intérêt tels que blastomères embryonnaires ou des ovocytes chez les adultes. Activer le laser 488 nm pour afficher signal de la GFP et de localiser la protéine de fusion GFP dans la cellule (s) d&#39;intérêt. </li><li> Pour visualiser l&#39;arrestation au stade précis de l&#39;arrêt du cycle cellulaire (par exemple prophase tardive) d&#39;identifier un blastomère à un stade antérieur de la division cellulaire (par exemple interphase prophase tardive ou précoce). Marqueurs cellulaires tels que la position centriole, l&#39;initiation de la condensation des chromosomes, la localisation chromosomique et la présence ou l&#39;absence de l&#39;enveloppe nucléaire aidera à identifier les étapes du cycle cellulaire (figure 2A). </li><li> La chambre est ensuite perfusé avec de l&#39;azote gazeux (99,999%N 2; &lt;2 ppm O 2). Dans ce cas, nous utilisons une pression de 6 psi, mais la pression peut avoir besoin d&#39;être ajustée et optimisée en fonction de la taille de chaque microincubator et le diamètre du tuyau en cours d&#39;utilisation. Continuer à surveiller le blastomère (s) que l&#39;azote remplit la chambre, le réglage du plan focal selon les besoins. </li><li> À ce stade, imagerie time-lapse a commencé. L&#39;imagerie est réalisée aussi longtemps que nécessaire pour capturer phénomène d&#39;intérêt, dans ce cas prophase arrestation et l&#39;amarrage des chromosomes à la membrane nucléaire interne. Les images sont prises une fois toutes les 10 sec pour une durée de 30 min. Les fréquences de capture d&#39;image et des paramètres tels que le gain et la puissance du laser doit être ajustée si nécessaire. </li><li> Le temps pendant lequel les embryons répondre à l&#39;environnement anoxique peut varier selon le stade du cycle cellulaire lors de l&#39;exposition anoxie. Typiquement arrestation prophase anoxie induite se produit dans les 20-30 min de commencer flux d&#39;azote gazeux. Nous avons observé une anoxie induite occu arrêt du cycle cellulairerring à &lt;20 min. Les images peuvent être obtenus au cours de cette période, ou des images spécifiques peuvent être isolés par la suite d&#39;une série time-lapse. Une autre option est d&#39;utiliser la vidéo-microscopie de visualiser des structures subcellulaires dans les embryons. </li><li> Pour documenter la récupération de l&#39;état d&#39;anoxie induite par arrêté, le gaz est éteint et que la chambre est autorisé à retourner à la normoxie par diffusion lors de l&#39;enregistrement d&#39;une série time-lapse. Reprise de la progression du cycle cellulaire et le désarrimage des chromosomes se produit généralement dans les 5-20 min de transformer l&#39;azote gazeux hors tension. </li><li> Notez que dans des expériences séparées un indicateur anoxie, résazurine, a été placé dans la microchambre accréditives, il a été déterminé que la chambre devient anoxique, en moyenne, d&#39;environ 19 minutes après l&#39;écoulement du gaz d&#39;azote a commencé. </li><li> Images et vidéos sont traitées à l&#39;aide Imaris, Image J ou Photoshop et importés dans QuickTime pour l&#39;affichage. </li></ol>

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

Aucun conflit d&#39;intérêt déclaré.

La privation d&#39;oxygène et de Suspended Animation Bien que l&#39;exposition à la privation d&#39;oxygène graves peuvent être mortels pour certains organismes, certains organismes sont capables de survivre à l&#39;exposition à l&#39;anoxie. Dans le cas de C. elegans, la survie de l&#39;exposition anoxie dépend du stade de développement et de réponse à l&#39;anoxie est entrée dans un état ​​d&#39;animation suspendue réversible dans lequel un certain nombre de processu...

<table border="1"><tbody><tr><td> Réactifs / Équipement </td><td></td><td></td><td> Composition </td></tr><tr><td> Solution d&#39;hypochlorite </td><td></td><td></td><td> 0,7 g de KOH, 12 ml de NaOCl 5%, porter à 50 ml avec de ddH 2 0 </td></tr><tr><td> M9 tampon </td><td></td><td></td><td> 3 g de KH 2 PO 4, 11 g de Na 2 HPO 4, 5 g de NaCl, 1 ml (1 M) MgSO 4 pour 1 L ddH O 2 </td></tr><tr><td> Lames de microscope </td><td> Fisher Scientific </td><td> 12-550-343 </td><td> 3 &quot;x 1&quot; x1.0 mm </td></tr><tr><td> Round micro lamelle </td><td> Sciences microscopie électronique </td><td> 72223-01 </td><td> 25 mm Diamètre </td></tr><tr><td> Huile halocarbone 700 </td><td> Sigma </td><td> H8898-100ml </td><td></td></tr><tr><td> Anesthésique </td><td></td><td></td><td> Trique de 0,5%aine, tétramisole 0,05% </td></tr><tr><td> Leiden perfusion fermé Microincubator </td><td> Harvard Apparatus </td><td> 650041 </td><td></td></tr><tr><td> L&#39;azote UHP </td><td> CALGAZ (Air Liquide) </td><td></td><td> &gt; 99,9990% de N 2 &lt;2 ppm O 2 </td></tr><tr><td> Tubes en plastique </td><td> VWR </td><td> 89068-468 </td><td> 0.062 &quot;ID x 0.125&quot; OD </td></tr><tr><td> Microscope confocal Spinning Disk </td><td> Systèmes McBain </td><td></td><td> Zeiss inversé microscope optique, système d&#39;éclairage à épifluorescence, CSU-10 Yokogawa confocale scanner, Hamamatsu multiplicateur d&#39;électrons caméra CCD. </td></tr></tbody></table>

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

Elegans Caenorhabdits a été largement utilisé dans l&#39;étude de la résistance au stress, ce qui est facilité par la transparence des stades adultes et d&#39;embryons ainsi que par la disponibilité de mutants génétiques et des souches transgéniques exprimant une myriade de 1-4 des protéines de fusion. En outre, les processus dynamiques comme la division cellulaire peut être consulté en utilisant des protéines marquées par fluorescence reporter. L&#39;étude de la mitose peut être facilitée par l&#39;utilisation de time-lapse expériences dans divers systèmes, y compris les organismes intacts; ainsi le début C. embryon elegans est bien adapté pour cette étude. Présentée ici est une technique par laquelle l&#39;imagerie in vivo de structures subcellulaires en réponse à anoxique (99,999% de N 2; &lt;2 ppm 2 O) le stress est possible en utilisant un flux de gaz simple grâce à l&#39;installation sur un microscope très puissant. Une chambre microincubation est utilisé en conjonction avec un débit d&#39;azote gazeux à travers et un microscope confocal à disque rotatifà créer un environnement contrôlé dans lequel les animaux peuvent être imagé in vivo. Utilisant la GFP-tubuline marqués gamma et des histones, la dynamique et arrêt de la division cellulaire peut être surveillé avant, pendant et après l&#39;exposition à un environnement privé d&#39;oxygène. Les résultats de cette technique sont en haute résolution, des vidéos et des images détaillées des structures cellulaires au sein de blastomères d&#39;embryons exposés à la privation d&#39;oxygène.

C. elegans embryons exposés à la privation d&#39;oxygène sévère (anoxie) sont capables de survivre en arrêtant les processus biologiques, y compris le développement et 7 de la division cellulaire. L&#39;arrestation anoxie induite par la division cellulaire peut être contrôlé, à un niveau sub-cellulaire, en utilisant une chambre microincubation en conjonction avec flux d&#39;azote gazeux à travers un microscope et confocale à disque rotatif pour créer un environnement contrôlé dans leq...

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

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

Décrit ici est un<em&gt; In vivo</emTechnique&gt; à l&#39;image des structures sous-cellulaires chez les animaux exposés à une anoxie en utilisant un écoulement de gaz à travers la chambre microincubation en liaison avec un microscope confocal à disque rotatif. Cette méthode est simple et suffisamment souple pour s&#39;adapter à une variété de paramètres expérimentaux et des systèmes modèles.

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 ...

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Research

JoVE Journal

Biology

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

11.5K vues.  University of North Texas. Décrit ici est un In vivo à l'image des structures sous-cellulaires chez les animaux exposés à une anoxie en utilisant un écoulement de gaz à travers la chambre microincubation en liaison avec un microscope confocal à disque rotatif. Cette méthode est simple et suffisamment souple pour s'adapter à une variété de paramètres expérimentaux et des systèmes modèles.

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

Monitoring Actin Disassembly with Time-lapse Microscopy

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 ...

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

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

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

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

Imagerie C. elegans Les embryons en utilisant un microscope à épifluorescence et de logiciels Open Source

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

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

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

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

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

Détermination des profils d&#39;expression génétique dans les C. elegans Utilisation microréseaux et PCR en temps réel

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

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

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 ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

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

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

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

Dépistage ARNi pour identifier les phénotypes postembryonnaires dans C. elegans</em

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

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

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 ...

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, ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

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

Methods to Assess Subcellular Compartments of Muscle in C. elegans

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

Méthodes d&#39;évaluation de Subcellular compartiments de muscle en<em&gt; C. elegans</em

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