Name: a microfluidic platform for longitudinal imaging in caenorhabditis elegans
Uploaded: 2018-05-02T13:30:00
Description: Watch this Scientific Journal Video about A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans at JoVE.com

Denne forskning blev støttet af National Science Foundation gennem tilskud PHY-1205494 og MCB-1413134 (EL) og National Research Foundation i Korea grant 2017R1D1A1B03035671 (bekymret).

Protokollen nedenfor bruger WormSpa5, en tidligere beskrevet mikrofluid enhed for langsgående billeddannelse af orme. Fabrikation af WormSpa (begyndende med CAD-filer, der kan opnås fra forfatterne på anmodning) er ligetil, men kræver nogle ekspertise. I de fleste tilfælde gøres der let fabrikation af en core facilitet eller en kommerciel virksomhed, der leverer sådanne tjenester. Når opdigte enheden, Sørg for at angive, at højden af funktionerne er 50 µm.
1. e...

<ol>
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Mikrofluid værktøjer giver flere fordele i at studere orme. Imaging i en PDMS tilbyder indretning højere billeddiagnostiske kvalitet sammenlignet med en standard NGM agar plade. Flere billeder kan tages fra et enkelt worm, i modsætning til traditionelle metoder, hvor dyr er plukket fra pladen og monteret på et objektglas til billedbehandling. Derudover kan mikromiljø, orme bor holdes konstant eller moduleret som ønsket, tillader præcis kortlægning mellem sammensætningen af miljøet og svar af dyrene.
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Ændringer i de miljømæssige forhold kan føre til aktivering af genetiske programmer ledsaget af induktion og repression af udtryk af specifikke gener1,2. Disse kinetic ændringer kan være variabel blandt væv i de samme dyr og mellem forskellige dyr. Undersøgelser af sådanne genetiske programmer kræver derfor metoder, der tillader langsgående billeddannelse af enkelte dyr og opnå præcis dynamisk styring af miljøforhold.
I de seneste år, har microfabricated fluidic enheder været brugt til at studere mange aspekter af reaktion og opførsel i små dyr, herunder orme, fluer, vand bjørne og flere3,4,5,6, 7. Programmer omfatter, for eksempel dybe fænotyper, optogenetic optagelse af neuronal aktivitet som svar på kemiske stimuli, og sporing af motor adfærd som bevægelse og pumpe8,9,10 , 11.
Mikrofluid-baserede tilgange holde mange egenskaber, der kunne gavne langsigtede langsgående billeddannelse af reaktion på miljømæssige stikord, herunder præcise dynamisk kontrol af de lokale mikromiljø, fleksibelt design, der giver mulighed for vedligeholdelsen af enkelte dyr i separate kvartaler og gunstige attributter for billeddannelse. Opretholde dyr i en mikrofluid afdeling i lang tid med minimal negativ indvirkning på deres godt væsener er dog en udfordring, som kræver særlig opmærksomhed i udformningen af mikrofluid enheden og i udførelsen af forsøget.
Her demonstrere vi brugen af WormSpa, en mikrofluid enhed for langsgående billeddannelse af Caenorhabditis elegans. 5 individuelle orme er begrænset i kamre. En konstant lav flow af flydende og bakteriel suspension sikrer orme er velnærede og tilstrækkelig aktiv til at bevare et godt helbred og lindre stress, og strukturen i afdelingerne tillader orme at lægge æg. Enkelheden i konstruktionen og driften af WormSpa bør tillade forskere uden tidligere erfaring i mikrofluidik at integrere denne enhed i deres egen forskningsplaner.

<table border="0" cellpadding="0" cellspacing="0" style="width:1012px;" width="1011">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">WormSpa</td>
			<td style="width:123px;">N/A</td>
			<td style="width:344px;">N/A</td>
			<td style="width:329px;">The CAD file for WormSpa is available from the Levine lab.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Compound Microscope</td>
			<td>Zeiss</td>
			<td>AxioObserver Z1</td>
			<td>An inverted fluorescence microscope with a motorized stage</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Syringe Pump</td>
			<td>New Era Pump Systems</td>
			<td>NE-501</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tubing</td>
			<td>SCI Scientific Commodities Inc.</td>
			<td>BB31695-PE/5</td>
			<td>0.034&#8221; (0.86 mm) I.D. x 0.052&#8221; (1.32 mm) O.D</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Syringe Tip</td>
			<td>CMLsupply</td>
			<td>901-20-050</td>
			<td>20 Gauge x 1/2&#8221; blunt tip stainless steel canula</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Syringe Filter</td>
			<td>PALL</td>
			<td>4650</td>
			<td>Acrodisc 32 mm Syringe Filter with 5 um Supor Membrane</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Syringe</td>
			<td>Qosina</td>
			<td>C3307</td>
			<td>10 mL Male Luer Lock Syringe</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">3 Way Valve</td>
			<td>ColeParmer</td>
			<td>FF-30600-23</td>
			<td>Large-bore 3-way, male-lock, stopcocks, 10/pack, Non-sterile</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dowel Pin</td>
			<td>McMaster-Carr</td>
			<td>90145A317</td>
			<td>18-8 Stainless Steel Dowel Pins (1/32&#34; Dia. x 1/2&#34; Lg.)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Low Binding Microcentrifuge Tube</td>
			<td style="width:123px;">Corning</td>
			<td>CL S3206</td>
			<td>0.65 mL low binding snap cap microcentrifuge tube</td>
		</tr>
	</tbody>
</table>

a microfluidic platform for longitudinal imaging in caenorhabditis elegans

I det sidste årti, mikrofluid teknikker har været anvendt til at studere små dyr, herunder nematode Caenorhabditis elegans, og har vist sig nyttige som en bekvem live imaging platform giver præcis kontrol af kapaciteter eksperimentelle betingelser i realtid. I denne artikel vil vise vi live billeddannelse af enkelte orme ansætte WormSpa, en tidligere udgivet brugerdefineret mikrofluid enhed. I enheden, er flere orme individuelt begrænset til separate kamre, så multipleksede langsgående overvågning af forskellige biologiske processer. For at illustrere kapacitet, udførte vi proof-of-principle eksperimenter hvor orme var smittet i enhed med patogene bakterier, og dynamikken i udtryk af immunrespons gener og æglægning blev overvåget løbende i de enkelte dyr. Den enkle design og drift af denne enhed gør det velegnet til brugere uden tidligere erfaring med mikrofluid-baserede eksperimenter. Vi foreslår, at denne tilgang vil være nyttig for mange forskere interesseret i længderetningen observationer af biologiske processer på veldefinerede betingelser.

Alder-synkroniseret unge voksne orme (46 timer post L1 larver anholdelse ved 25 ° C)12 blev indlæst på enheden, som beskrevet i protokollen. Ormene var individuelt placeret i separate kanaler, aktivering af langsgående måling af dyrenes svar til patogenet. Når eksperimentet er vellykket, forbliver de fleste orme i deres kanaler for varigheden af forsøget. I dette tilfælde tages billeder af individuelle orme simpelthen ved at placere deres kanal i imaging st...

Watch this Scientific Journal Video about A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans at JoVE.com

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

En mikrofluid Platform for langsgående Imaging i Caenorhabditis elegans

I denne artikel vil vise vi live billeddannelse af enkelte orme beskæftiger en brugerdefineret mikrofluid enhed. I enheden, er flere orme individuelt begrænset til separate kamre, så multipleksede langsgående overvågning af forskellige biologiske processer.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

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Bioengineering

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

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Simple mikrofluidenheder for<em&gt; In vivo</em&gt; Scanning af<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; Og zebrafisk

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Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

Mikrofluid-baserede Electrotaxis for On-demand kvantitativ analyse af<em&gt; Caenorhabditis elegans</em&gt; &#39;Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

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A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

En Mikrofluid platform for High-throughput Single-celle Isolering og Kultur

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

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Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

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Imaging rod hår morfologi af Arabidopsis stiklinger i en to-lags mikrofluid Platform

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An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

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Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

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Immobilisering af levende Caenorhabditis elegans enkeltpersoner ved hjælp af en ultra-tynd Polydimethylsiloxan mikrofluid Chip med væskeophobning

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...