Name: long-term live imaging device for improved experimental manipulation of zebrafish larvae
Uploaded: 2017-10-27T15:00:00
Description: Watch this Scientific Journal Video about Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae at JoVE.com

Forfatterne ønsker å erkjenne primære prosjektfinansiering fra Morgridge Institutt for forskning og laboratoriet for optisk og beregningsorientert instrumentering. Vi erkjenner også finansiering fra NIH # R01GM102924 (AH og KWE). KH, JMS, RS, AH og KWE unnfanget og utformet studien. KH og JMS utført alle eksperimenter med støtte fra DL, KP og RS. KH, JS, RS, AH og KWE bidratt til skriving av manuskriptet.

Merk: base zWEDGI design ble formulert for sebrafisk larver som er 2 til 4 dager etter befruktning (dpf) og følge retningslinjene for University of Wisconsin-Madison forskning dyr ressurssenter.
 1. design og 3D utskrift av muggsopp <ol><li>modell av PDMS komponenten av enheten med ønsket geometrier og attributter i en 3D-modellering programvare 5. Opprette en samling av en tom mold og PDMS og generere en negativ mold for PDMS del ved å opprette et hulrom i mold ...

<ol>
	<li>Yoo, S. K., Freisinger, C. M., LeBert, D. C., Huttenlocher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+redox,+Src+family+kinase,+and+calcium+signaling+integrate+wound+responses+and+tissue+regeneration+in+zebrafish.">Early redox, Src family kinase, and calcium signaling integrate wound responses and tissue regeneration in zebrafish.</a> J. Cell Biology. 199 (2), 225-234 (2012).</li><li>Kawakami, A., Fukazawa, T., Takeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+fin+primordia+of+zebrafish+larvae+regenerate+by+a+similar+growth+control+mechanism+with+adult+regeneration.">Early fin primordia of zebrafish larvae regenerate by a similar growth control mechanism with adult regeneration.</a> Dev. Dynam. 231 (4), 693-699 (2004).</li><li>Konantz, J., Antos, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+genetic+morpholino+approach+using+cardiac+ventricular+injection+to+transfect+multiple+difficult-to-target+tissues+in+the+zebrafish+larva.">Reverse genetic morpholino approach using cardiac ventricular injection to transfect multiple difficult-to-target tissues in the zebrafish larva.</a> JoVE. (88), (2014).</li><li>Hall, C., Flores, M. F., Kamei, M., Crosier, K., Crosier, P., Sampath, K., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+Innate+Immune+Cell+Behavior+During+Normal+Development,+Wound+Healing+and+Infection.">Live Imaging Innate Immune Cell Behavior During Normal Development, Wound Healing and Infection.</a> Live Imaging in Zebrafish: Insights into Development and Disease. , (2010).</li><li>Huemer, K., Squirrell, J. M., Swader, R., LeBert, D. C., Huttenlocher, A., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=zWEDGI:+Wounding+and+Entrapment+Device+for+Imaging+Live+Zebrafish+Larvae.">zWEDGI: Wounding and Entrapment Device for Imaging Live Zebrafish Larvae.</a> Zebrafish. , (2016).</li><li>Lisse, T. S., Brochu, E. A., Rieger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+tissue+repair+in+zebrafish+larvae+with+time-lapse+brightfield+stereomicroscopy.">Capturing tissue repair in zebrafish larvae with time-lapse brightfield stereomicroscopy.</a> JoVE. (95), (2015).</li><li>Kamei, M., Isogai, S., Pan, W., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+blood+vessels+in+the+zebrafish.">Imaging blood vessels in the zebrafish.</a> Methods Cell Biol. 100, 27-54 (2010).</li><li>Graeden, E., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+the+zebrafish+embryonic+brain+by+confocal+microscopy.">Live imaging of the zebrafish embryonic brain by confocal microscopy.</a> JoVE. (26), (2009).</li><li>Petzold, A. M., Bedell, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCORE+imaging:+specimen+in+a+corrected+optical+rotational+enclosure.">SCORE imaging: specimen in a corrected optical rotational enclosure.</a> Zebrafish. 7 (2), 149-154 (2010).</li><li>Macdonald, N. 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Formålet med zWEDGI enheten er å fange 3D tidsforløp imaging stabilisere og orientere fisken innen de små arbeider fra et høyoppløselig mikroskop mål. Mens møte disse design spesifikasjoner, er det også en forbedring over tradisjonelle agar-baserte forberedelse til live bildebehandling. Det er tre viktige trinn (nedenfor) i produksjon av zWEDGI, som, hvis det ikke gjøres riktig, kan resultere i defekt enheter:
PDMS forberedelse (figur 5A)
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Regenerativ kapasitet sebrafisk Larvene Danio rerio gjøre dem en ideell modell organisme for å undersøke såret respons samt healing og gjenvekst1,2,3,4. Tilgang til en rekke transgene sebrafisk linjer og sebrafisks anatomiske transparency ytterligere forsterke deres nytte for i vivo studier av såret svar arrangementer samt langsiktige regenerativ prosesser4. Studiet av disse biologiske prosesser med høy oppløsning time-lapse fluorescens mikroskopi derfor krever en live bildebehandling sebrafisk enhet som gir høy stabilitet og minimal bevegelse av sebrafisk Larven samtidig levedyktighet. Det er viktig at enheten gir effektiv såret mens healing og gjenfødelse oppstår upåvirket av enheten.
Live bildebehandling stabilisering standardmetoden for innebygging Larven i agarose under live bildebehandling begrenser veksten og sår gjenfødelse5 og kan øke dødelighet siden Larvene begynner å vise tegn på cardiac stress og vev nekrose etter fire timer4. Derfor fjerning av agarose fra regioner av interesse er ofte nødvendig å tillate normal utvikling og regeneration6, utsette Larvene til potensielle skader som agarose er kuttet bort. Videre med agarose innebygging teknikk, må brukeren orientere Larvene på kort tid før agarose stivner5,6,7. Raskt manipulere Larven ikke bare krever dyktighet av brukeren, også risiko skade Larven. Selv om metoder for å stabilisere Larven for live bildebehandling har blitt beskrevet for å omgå disse ulempene, som ridged agar brønner3 eller divets8, bruk av silikon vakuum fett for å lage en tenkelig kammer med PVC rør eller andre materialer6og roterende rør9, mange av disse metodene er arbeidskraft intensiv, rotete, ofte ikke-gjenbrukbare og tillater ikke miljømessige manipulering (narkotika behandlinger, såret osv.) etter at fisken har blitt montert.
Derfor ble zWEDGI enheten (figur 1) utviklet for å overvinne noen av ulempene med agar montering for langsiktig live bildebehandling av sebrafisk Larvene mens tillater manipulering av prøven. ZWEDGI består av tre halvåpne compartmentalized kamre (figur 1A) å tillate lasting, tilbakeholdenhet, såret og bildebehandling for 2 til 4 dager etter befruktning sebrafisk larver. Enheten er fabrikkert fra Polydimethylsiloxane (PDMS) og plassert på cover slip av en 60 mm glass bunn tenkelig rett. Design presenteres her var ment for sår helbredelse studier, men bruk av et modulært design og standard fabrikasjon teknologier gjør zWEDGI design kan endres og mottagelig for en rekke eksperimentelle prosedyrer, spesielt for prosedyrer som kreve minimalt tilbakeholdenhet med eksperimentelle manipulasjon og langsiktig bildebehandling.

<table border="0" cellpadding="0" cellspacing="0" width="920">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Fabricate molds</td>
			<td style="width:127px;"></td>
			<td style="width:145px;"></td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Solidworks Professional Accedemic Research 3D modeling software</td>
			<td>Dassault Systemes</td>
			<td>SPX0117-01</td>
			<td>Fisher Unitech</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Viper Si2 SLA 3D printer</td>
			<td>3D Systems Inc.</td>
			<td>23200-902</td>
			<td>3D Systems Inc.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accura 60 photopolymer resin</td>
			<td>3D Systems Inc.</td>
			<td>24075-902</td>
			<td>3D Systems Inc.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">denatured alcohol</td>
			<td>Sunnyside</td>
			<td>5613735</td>
			<td>Menards</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV post cure apparatus</td>
			<td>3D Systems Inc.</td>
			<td>23363-101-00</td>
			<td>3D Systems Inc.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TouchNTuff nitrile gloves</td>
			<td>Ansell</td>
			<td>92-600</td>
			<td>McMaster Carr</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">220B, 400B, 600 grit T414 blue-bak sandpaper&#160;</td>
			<td>Norton</td>
			<td>66261139359, 54, 52</td>
			<td>MSC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">borosilicate glass disc, 2&#34; diameter</td>
			<td>McMaster-Carr</td>
			<td>MIL-G-47033</td>
			<td>McMaster-Carr</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ultrasonicator cleaner</td>
			<td>Branson</td>
			<td>1510R-MTH</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">isopropyl rubbing alcohol 70%</td>
			<td>Hydrox</td>
			<td>54845T43</td>
			<td>McMaster-Carr</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10oz clear plastic cup</td>
			<td>WNA Masterpiece</td>
			<td>557405</td>
			<td>Amazon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6&#34;craft stick</td>
			<td>Perfect Stix</td>
			<td>Craft WTD-500</td>
			<td>Amazon</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fabricate zWEDGI PDMS device</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sylgard 184 silicon elastomeric kit&#160;</td>
			<td>Dow-Corning</td>
			<td>4019862</td>
			<td>Ellworth Adhesives&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10mL syringe</td>
			<td>Becton Dickinson</td>
			<td>305219</td>
			<td>Vitality Medical Inc</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">desiccator</td>
			<td>Bel-Art Scienceware</td>
			<td>F42027-0000</td>
			<td>Amazon</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4 in ratcheting bar clamp</td>
			<td>Pittsburgh</td>
			<td>68974</td>
			<td>Harbor Freight</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">lab oven</td>
			<td>Quincy Lab Inc.</td>
			<td>20GC</td>
			<td>Global Industrial</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tweezer set</td>
			<td>Aven</td>
			<td>549825</td>
			<td>McMaster-Carr</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">compressed air filtered nozzle</td>
			<td>Innotech</td>
			<td>TA-N2-2000FT</td>
			<td>Cleanroom Supply</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">vacuum bench vise</td>
			<td>Wilton Tool Group</td>
			<td>63500</td>
			<td>MSC Industrial</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">55mm glass bottom dish; 30mm micro-well #1.5 cover glass</td>
			<td>Cellvis</td>
			<td>D60-30-1.5-N</td>
			<td>Cellvis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td>Harrick Plasma</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Loading Larvae</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipetteman, P200</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100% ethanol (diluted to 70% with water prior to use)</td>
			<td>Pharmco-aaper</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Fisherbrand</td>
			<td>13-711-5A</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">powdered skim milk</td>
			<td></td>
			<td>2902887</td>
			<td>MP Biomedicals</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">double distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-phenylthiorurea</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tricaine (ethyl 3-aminobenzoate)</td>
			<td></td>
			<td>C-FINQ-UE</td>
			<td>Western Chemical</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">low melting point agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A0701</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">heat block (dry bath incubator)</td>
			<td>Fisher Scientific</td>
			<td>11-718-2</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E3 buffer&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">large orifice pipette tip, 200 uL</td>
			<td>Fisherbrand</td>
			<td>02-707-134</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">General purpose pipette tip, 200 uL</td>
			<td>Fisherbrand</td>
			<td>21-197-8E</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">#15 scalpel blade&#160;</td>
			<td>Feather</td>
			<td>2976</td>
			<td>Amazon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25G syringe needle</td>
			<td>BD&#160;</td>
			<td>BD305122</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">inverted microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris imaging software</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

long-term live imaging device for improved experimental manipulation of zebrafish larvae

Sebrafisk Larven er en viktig modell organisme for både utviklingsbiologi og sårtilheling. Videre, sebrafisk Larven er en verdifull system for live høyoppløselig mikroskopiske bildebehandling av dynamisk biologiske fenomener i rom og tid med cellular oppløsning. Den tradisjonelle metoden for agarose innkapsling for live bildebehandling kan imidlertid hindre larver utvikling og vev regrowth. Derfor dette manuskriptet beskriver zWEDGI (sebrafisk Wounding og Entrapment enhet for vekst og bildebehandling), som ble designet og fabrikkert som funksjonelt compartmentalized apparat å orientere Larvene for høy oppløsning mikroskopi mens tillater halefinnen transection innen enheten og påfølgende hemningsløs hale utvikling og re-vekst. Denne enheten gir såret og langsiktig imaging samtidig levedyktighet. Gitt at zWEDGI mold er 3D trykt, customizability sin geometrityper gjør det enkelt endres for mangfoldig sebrafisk tenkelig søknadene. Videre tilbyr zWEDGI mange fordeler, for eksempel tilgang til Larvene under eksperimentering såret eller anvendelsen av reagenser, paralleller retning av flere Larvene for strømlinjeformet bildebehandling og gjenbruk av enheten.

ZWEDGI PDMS microfluidic enheten er et funksjonelt compartmentalized enhet designet for å huse fire hovedfunksjoner (nedenfor) forbundet med live bildebehandling av halefinne såret healing og gjenvekst i sebrafisk larvene. PDMS ble valgt zWEDGI fabrikasjon fordi det ikke er bare lett tilgjengelig og en industristandard for biocompatibility, men også fungerer godt i formene. I tillegg gjør PDMS enheten gjenbrukbare og blottet for hardt eller skarpe kanter når enheten er dannet. ZWEDGI...

Watch this Scientific Journal Video about Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae at JoVE.com

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

Dette manuskriptet beskriver zWEDGI (sebrafisk Wounding og Entrapment enhet for vekst og bildebehandling), som er en compartmentalized enhet konstruert for å orientere og holde sebrafisk larver. Design tillater hale transection og langsiktig samling av høyoppløselige fluorescerende mikroskopi bilder av sårheling og gjenfødelse.

Langsiktig Live bildeenheten forbedret eksperimentelle manipulering av sebrafisk larver

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The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Bruke Microfluidics Chips for Live Imaging and Study of Injury Responses i<em&gt; Drosophila</em&gt; Larver

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Langsiktig Intravital Immunfluorescens Imaging av Tissue Matrix Komponenter med epifluorescence og To-foton mikroskopi

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Høyoppløselig Spatiotemporal Analyse av Receptor Dynamics av ​​Single-molekyl fluorescens mikroskopi

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

Multi-orgel Chip - En mikrofluid plattform for langsiktig Multi-vev coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Live Cell Imaging during Mechanical Stretch

Live-Cell Imaging under Mekanisk Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Hybrid &#181;CT-FMT imaging and image analysis

Hybrid Ãƒâ€šÃ‚ÂµCT-FMT imaging and image analysis

Hybrid μCT-FMT bildebehandling og bildeanalyse

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Høyoppløselig bilde av Nuclear Dynamics i levende celler under Uniaxial strekk belastning

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the ...

Targeting the Rat's Small Bowel: Long-Term Infusion into the Superior Mesenteric Artery

Målretting av rottens lille tarm: Langsiktig infusjon i den overlegne mesenteriske arterien

The superior mesenteric artery can be cannulated in humans through minimally invasive radiological catheterization of the femoral or axillary artery. SMA ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Avanserte 3D levermodeller for in vitro gentoksisitetstesting etter langvarig nanomaterialeeksponering

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...