Wir möchten unsere Wertschätzung für die Eingabe und Kommentare von Mitgliedern des Padilla Lab vermitteln. Nematode Stämme in dieser Arbeit wurden von der Caenorhabditis Genetics Center, das von der NIH National Center for Research Resources (NCRR) finanziert ist. Wir anerkennen und danken Dr. Lon Turnbull für technische Hilfe bei der konfokalen Mikroskopie. Diese Arbeit wurde durch ein Stipendium der National Science Foundation (NSF-IOS, CAREER) an PAP unterstützt

Ein. Probenvorbereitung <ol><li> Produzieren oder zu erhalten entsprechenden transgenen C. elegans-Stamm von Interesse unter Verwendung transgener Methoden oder von Caenorhabditis Genetics Stock Center (CGC) oder Kollegen. In diesem Fall sind wir mit dem Stamm TH32 (pie-1 :: tbg-1 :: GFP; pie-1 :: GFP :: H2B) 5 bis Chromosomen und Zentrosomen als Marker für die Zellteilung zu visualisieren. </li><li> Generieren Sie eine synchronisierte Bevölkerung mit einem der drei Methoden: 1) zerfallen gravid Erwachsenen in Hypochloritlösung und verwenden restlichen Embryonen 6, 2) holen gravid Erwachsenen auf eine gesetzte Platte und laß ihnen lag Embryonen für 1-2 Stunden, oder 3) Pick L4 Larven von einer gemischten Bevölkerung und Umzug in ein neues seeded Platte. </li><li> Wachsen Nematoden bei 20 ° C bis graviden jungen Erwachsenenalter, die in etwa 96 Stunden aus dem Schlüpfen dauert, 72 h von L1-Larven oder 24 h nach der Häutung L4. Embryonen (2-20 Zellen) in utero enthalten große Blastomeren und Kerne, die optimal für imag sindten sub-zellulären und sub-atomaren Veränderungen sind. Diese Methode bietet eine Möglichkeit, eine Bevölkerung von jungen Erwachsenen, die eine reichliche Anzahl von frühen Embryonen enthalten generieren. </li></ol> 2. Slide Vorbereitung und Anoxia Chamber Set Up <ol><li> Machen Sie eine feuchte Kammer, vorbereitete Folien, indem Sie ein feuchtes Papiertuch in einem großen Petrischale oder bin mit einem Deckel von ausreichender Größe bedeckt halten. </li><li> Planen geschmolzene 2% Agarose in entionisiertem H 2 O durch Erhitzen Mischung in Glaswaren über Mikrowelle oder Wasserbad. </li><li> Platz 1-3 Tropfen warmen Agarose auf einer sauberen Glasobjektträger. Sofort einen zweiten Schieber senkrecht auf der Agarose Drop (s) invertiert, leicht drücken, so dass die resultierende Pad dünn sein und frei von Luftblasen. </li><li> Nehmen Sie sich Zeit zu kühlen und langsam auseinander nehmen die beiden Objektträger Verlassen der Agarose Pad intakt auf einer der Folien. Es ist wichtig sicherzustellen, die Agarose-Pad ist dünn genug, um nicht Störe mit der Fähigkeit, das Mikroskop später konzentrieren. Fähigkeit zur Probe visualisieren ist auch abhängig von der spezifischen optischen Abstand des Objektivs im Gebrauch die zwischen Mikroskopen variieren kann. </li><li> Mit einem sauberen Rasierklinge eine Agarose-Pad in ein kleines Quadrat formen. Bewahren Sie in der vorbereiteten feuchten Kammer bis zum Gebrauch. </li><li> Sorgfältig unter Verwendung der gleichen sauberen Rasierklinge, nehmen die Kante der Agarose Pad und sie von der Folie auf eine runde 25 mm Mikro Abdeckglases, die Vermeidung der Ansammlung von Luftblasen unter dem Pad. Der runde Deckgläser gewählt zu bedienen passt entsprechend in der microincubator. </li><li> Einen Tropfen Anästhetikum (0,5% Tricain, 0,05% Tetramisol in M9-Puffer) auf die Agarose Pad zur Verwendung als Anästhetikum. Wählen 5-10 Würmer und Stelle in den Tropfen Anästhetikum. Lassen Sie 1-3 min für Würmer, um die Bewegung zu beenden. </li><li> Die Begründung für die Beobachtung Embryonen im Uterus erwachsenen, anstatt zerschnitten Embryonen, ist das Potenzial der Embryo desicca minimierention und Bewegung durch die Strömung von Stickstoffgas über den Embryo. </li><li> Sofort einen Tropfen Halocarbonöl auf der Würmer, die Würmer vor Austrocknung zu verhindern, wie Gas durch die Kammer durchströmt wird. Da die Halocarbonöl ist zähflüssig kann man wählen Sie eine Pipettenspitze oder der Spitze eines Wurms nutzen, um Öl-und Drop auf die Würmer zu sammeln. </li><li> Sobald der kreisförmigen Deckglas mit den Würmern wartet schlossen die beiden Hälften des Leiden Perfusion microincubator werden getrennt und die Deckgläser wird dann vorsichtig auf die aufrechte Bodenrings angeordnet. Die beiden Seiten der Kammer werden dann geschlossen fest zusammen. </li><li> Die Kammer wird dann mit dem Stickstoff Gastank über flexible Kunststoffschlauch verbunden und in die entsprechende Stelle auf dem Mikroskoptisch (Abbildung 1C). Zu diesem Zeitpunkt wird der Schlauch und microincubator sollte sorgfältig auf eventuelle Leckagen werden durch eine sichere Befestigung der Schläuche und der Anschluss-Stecker entlang der Seite der Kammer kontrolliert. Eines kann ALso leicht eine geringe Menge Seife Wasser über die Rohrleitung für Luftblasen, die sich bilden, wenn ein Leck vorhanden ist aussehen ausbreiten. </li></ol> 3. Mikroskopie in Anoxia <ol><li> Suchen Tieren bei niedriger Vergrößerung, dann in den entsprechenden höherer Vergrößerung (64x für diese Anwendung) zu Bild Gewebe oder Zellen von Interesse wie embryonale Blastomeren oder Oozyten bei Erwachsenen bewegen. Einschalten des Lasers, um 488 nm GFP-Signal anzuzeigen und zu lokalisieren GFP-Fusionsprotein in der Zelle (n) von Interesse. </li><li> Um der Verhaftung zu bestimmten Stadium des Zellzyklus visualisieren (zB späten Prophase) identifizieren Blastomeren in früheren Phasen der Zellteilung (zB verspätete Interphase oder frühen Prophase). Zellulären Marker wie Centriol Position Einleitung Chromosomenkondensation, Chromosom Lage und Vorhandensein oder Nichtvorhandensein der Kernhülle wird zur Ermittlung Zellzyklusstadium (Abbildung 2A). </li><li> Die Kammer wird dann mit Stickstoffgas (99,999% perfundiertN 2; &lt;2 ppm O 2). In diesem Fall haben wir einen Druck von 6 bar zu verwenden, jedoch Druck eventuell angepasst und optimiert werden je nach Größe der einzelnen microincubator und Durchmesser der Schlauch in Gebrauch ist. Weiterhin die Blastomere (en) als der Stickstoff füllt die Kammer, Einstellen der Brennebene nach Bedarf überwachen. </li><li> An diesem Punkt hat Zeitraffer-Aufnahmen begonnen. Abbildungssystem wird so lange wie notwendig durchgeführt, um interessierende Phänomen erfassen, in diesem Fall Prophase Verhaftung und Andocken von Chromosomen auf der inneren Kernmembran. Die Bilder werden einmal alle 10 sec nicht länger als 30 min genommen. Frequenzen der Bilderfassung und Einstellungen wie Gain und Laserleistung sollte nach Bedarf eingestellt werden. </li><li> Die Zeit, in welcher die Embryonen zu der anoxischen Umgebung reagieren kann je nach Zellzyklusstadium auf Anoxie Belichtung. Typischerweise Anoxie-induzierten Prophase Festnahme erfolgt innerhalb von 20-30 min zu beginnen Stickstoffgasstrom. Wir haben Anoxie-induzierten Zellzyklusarrest Berufs beobachtetrring in &lt;20 min. Bilder können in diesem Zeitraum erhalten werden, oder bestimmte Bilder können später von einem Zeitraffer-Serie werden. Eine weitere Option ist es, Video-Mikroskopie verwenden, um sub-zellulären Strukturen in Embryonen zu visualisieren. </li><li> Um eine Wiederherstellung aus der Anoxie-induzierten arretierten Zustand zu dokumentieren, wird das Gas abgeschaltet und die Kammer erlaubt ist, zu Normoxie durch Diffusion wieder während der Aufzeichnung eines Zeitraffer-Serie. Wiederaufnahme der Zellzyklusprogression und Undocking von Chromosomen tritt normalerweise innerhalb von 5-20 min Drehen des Stickstoffgases aus. </li><li> Beachten Sie, dass in getrennten Experimenten ein Indikator Anoxie, Resazurin, in der Durchflusskammer Mikrokammer platziert wurde, es wurde ermittelt, dass die Kammer anoxischen auf durchschnittlich ca. 19 min wird nach Stickstoffgasstrom wurde gestartet. </li><li> Bilder und Videos werden unter Verwendung Imaris, Image J oder Photoshop importiert und in QuickTime für die Anzeige. </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. 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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>

Keine Interessenskonflikte erklärt.

Sauerstoffmangel und Suspended Animation Obgleich das Engagement in schweren Sauerstoffmangel kann tödlich sein für einige Organismen, sind einige Organismen, um die Exposition gegenüber Anoxie überleben. Im Falle von C. elegans, hängt Überleben von Anoxie Belichtung auf Entwicklungsstadium und ist ansprechend auf Anoxie Eintritt in einen Zustand der reversiblen suspendiert Animation in dem eine Anzahl von beobachtbaren biologische Prozesse werden verhaftet. Zellteilung, Entwickl...

<table border="1"><tbody><tr><td> Reagenzien / Equipment </td><td></td><td></td><td> Zusammensetzung </td></tr><tr><td> Hypochloritlösung </td><td></td><td></td><td> 0,7 g KOH, 12 ml 5% NaOCl, um 50 ml zu bringen mit ddH 2 0 </td></tr><tr><td> M9-Puffer </td><td></td><td></td><td> 3 g KH 2 PO 4, 11 g Na 2 HPO 4, 5 g NaCl, 1 ml (1 M) MgSO 4 pro 1 L ddH 2 O </td></tr><tr><td> Glasobjektträger </td><td> Fisher Scientific </td><td> 12-550-343 </td><td> 3 &quot;x1&quot; x1.0 mm </td></tr><tr><td> Round micro Deckglas </td><td> Electron Microscopy Sciences </td><td> 72223-01 </td><td> 25 mm Durchmesser </td></tr><tr><td> Halocarbonöl 700 </td><td> Sigma </td><td> H8898-100ml </td><td></td></tr><tr><td> Narkose </td><td></td><td></td><td> 0,5% tricaine, 0,05% Tetramisol </td></tr><tr><td> Leiden Closed Perfusion Microincubator </td><td> Harvard Apparatus </td><td> 650041 </td><td></td></tr><tr><td> UHP Stickstoff </td><td> Calgaz (Air Liquide) </td><td></td><td> &gt; 99,9990% N 2 &lt;2 ppm O 2 </td></tr><tr><td> Kunststoffschlauch </td><td> VWR </td><td> 89068-468 </td><td> 0,062 &quot;ID x 0,125&quot; OD </td></tr><tr><td> Spinning-Disk-konfokale Mikroskop </td><td> McBain Systeme </td><td></td><td> Zeiss invertiert optischen Mikroskop, Epifluoreszenz Beleuchtungssystem, CSU-10 Yokogawa konfokalen Scanner, Hamamatsu-Elektronen-Vervielfacher CCD-Kamera. </td></tr></tbody></table>

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

Caenorhabdits elegans wurde ausgiebig in der Studie der Stressresistenz, die durch die Transparenz des adulten Stadien und Embryo als auch durch die Verfügbarkeit von genetischen Mutanten und transgenen Linien Expression einer Vielzahl von Fusionsproteinen 1-4 erleichtert wird. Darüber hinaus können dynamische Prozesse wie Zellteilung Darstellung unter Verwendung von fluoreszierend markierten Reporter-Proteine ​​werden. Das Studium der Mitose kann durch den Einsatz von Zeitrafferexperimente in verschiedenen Systemen einschließlich intakten Organismen erleichtert; somit der frühen C. elegans Embryo wird auch für diese Studie geeignet. Präsentiert hier ist eine Technik, mit der in vivo Bildgebung von sub-zellulären Strukturen in Reaktion auf Sauerstoffmangel (99,999% N 2; &lt;2 ppm O 2) Stress ist möglich, mit einem einfachen Gasfluss durch Setup auf einem High-Power-Mikroskop. Ein microincubation Kammer in Verbindung mit Stickstoffgas durchströmt und einer drehenden Scheibe konfokalen Mikroskop verwendetum eine kontrollierte Umgebung, in der Tiere in vivo abgebildet werden können. Verwendung GFP-markierten Gamma-Tubulin und Histon, kann die Dynamik und Verhaftung der Zellteilung kontrolliert werden, bevor, während und nach der Einwirkung von einer sauerstoffarmen Umgebung. Die Ergebnisse dieser Technik sind hochauflösende, detaillierte Videos und Bilder von zellulären Strukturen innerhalb Blastomeren von Embryonen ausgesetzt Sauerstoffmangel.

C. elegans Embryonen bei starken Sauerstoffmangel (Anoxie) können durch die Verhaftung biologische Prozesse einschließlich Entwicklung und Zellteilung 7 überleben. Die Anoxie induzierte Verhaftung der Zellteilung können überwacht werden, bei einem sub-zellulären Ebene durch Verwendung einer microincubation Kammer in Verbindung mit Stickstoffgas durchströmt und einer drehenden Scheibe konfokalen Mikroskops um eine kontrollierte Umgebung, in der Tiere in vivo abgebildet werden kann erst...

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

Hier wird beschrieben ein<em&gt; In vivo</em&gt; Technik zur Bilder subzellulären Strukturen in Tieren, die mit Anoxie einen Gasstrom durch microincubation Kammer in Verbindung mit einer drehenden Scheibe Konfokalmikroskop. Dieses Verfahren ist einfach und flexibel genug, um eine Vielzahl von experimentellen Parameter und Modellsystemen anzupassen.

Verwendung von Zeitraffer-Mikroskopie zur Anoxie-induzierten Suspended Animation in Visualisieren<em&gt; C. elegans</em&gt; Embryonen

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

use-time-lapse-microscopy-to-visualize-anoxia-induced-suspended

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

11.6K Aufrufe.  University of North Texas. Hier wird beschrieben ein In vivo Technik zur Bilder subzellulären Strukturen in Tieren, die mit Anoxie einen Gasstrom durch microincubation Kammer in Verbindung mit einer drehenden Scheibe Konfokalmikroskop. Dieses Verfahren ist einfach und flexibel genug, um eine Vielzahl von experimentellen Parameter und Modellsystemen anzupassen.

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

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Einfache mikrofluidischen Systemen für<em&gt; In vivo</em&gt; Imaging der<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; Und Zebrafisch

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Forschung

Biotechnik

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Isotrope Licht-Sheet-Mikroskopie und automatisierte Zelllineage-Analysen zu Katalog-Caenorhabditis elegans Embryogenese mit subzellulärer Auflösung

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Entwicklungsbiologie

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

Visualisierung Neuroblast Cytokinesis Während<em&gt; C. elegans</em&gt; Embryogenese

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

Neurowissenschaften

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

Ein Instrument zur Analyse der C. elegans Embryonal development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans has emerged as one of the best models for studying development. From an optical point of view, its size and transparent body make this nematode an ideal specimen for DIC (Differential Interference Contrast or Nomarski) microscopy. This article illustrates a protocol for growing C. elegans nematodes, preparing and mounting their embryos, performing 4D microscopy and cell lineage tracing. The method is based on multifocal time-lapse records of Nomarski images and analysis with specific software. This technique reveals embryonic developmental dynamics at the cellular level. Any embryonic defect in mutants, such as problems in spindle orientation, cell migration, apoptosis or cell fate specification, can be efficiently detected and scored. Virtually every single cell of the embryo can be followed up to the moment the embryo begins to move. Tracing the complete cell lineage of a C. elegans embryo by 4D DIC microscopy is laborious, but the use of specific software greatly facilitates this task. In addition, this technique is easy to implement in the lab. 4D microscopy is a versatile tool and opens the possibility of performing an unparalleled analysis of embryonic development.

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

Here, we present a protocol for preparing and mounting Caenorhabditis elegans embryos, recording development under a 4D microscope and tracing cell lineage.

4D-Mikroskopie: Entschlüsselung der Caenorhabditis elegans Embryonalentwicklung mittels Nomarski-Mikroskopie

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Novel Metrics zur Charakterisierung von Embryonic Dehnung des Nematode<em&gt; Caenorhabditis elegans</em

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

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

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

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

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

Imaging C. elegans Embryos mit einem Epifluoreszenz-Mikroskop und Open Source Software

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

Biologie

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.

Zeitraffer-Mikroskopie der frühen Embryogenese 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 ...

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

Ein einfacher mikrofluidischer Chip für Langzeitwachstum und Bildgebung von Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

Ein Anoxia-Hunger-Modell für Ischämie / Reperfusion in<em&gt; C. elegans</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Verwendung von Zeitraffer-Mikroskopie zur Anoxie-induzierten Suspended Animation in Visualisieren

Zusammenfassung

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