Name: using a microfluidics device for mechanical stimulation and high resolution imaging of c. elegans
Uploaded: 2018-02-19T13:00:00
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Vi takker Sandra N. Manosalvas-Kjono, Purim Ladpli, Farah Memon, Divya Gopisetty og Veronica Sanchez for støtte i enhed design og produktion af mutant dyr. Denne forskning blev støttet af NIH tilskud R01EB006745 (til BLP), R01NS092099 (til MBG), K99NS089942 (til MK), F31NS100318 (til ALN) og modtaget støtte fra Det Europæiske Forskningsråd (ERC) under EUs Horisont 2020 forskning og innovation program () tilskudsaftale No. 715243 til MK).

1. enhed fabrikation
<ol><li>Hent filen vedhæftet maske (supplerende fil 1) og generere en chrome maske ved hjælp af en kommerciel tjeneste eller interne facility. Som den mindste dimension på enheden er 10 µm (aktuator membran tykkelse), sikre, at masken har tilstrækkelig høj opløsning inden for ± 0,25 µm, pålideligt producere funktionerne.</li>
	<li>
Følg SU-8 fotolitografi standardmetoder (f.eks.referencer24,<sup class="...

<ol>
	<li>Eastwood, A. L., 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=Tissue+mechanics+govern+the+rapidly+adapting+and+symmetrical+response+to+touch.">Tissue mechanics govern the rapidly adapting and symmetrical response to touch.</a> Proc. Natl. Acad. Sci. 15 (50), E6955-E6963 (2015).</li><li>Katta, S., Krieg, M., Goodman, 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=Feeling+Force:+Physical+and+Physiological+Principles+Enabling+Sensory+Mechanotransduction.">Feeling Force: Physical and Physiological Principles Enabling Sensory Mechanotransduction.</a> Annu. Rev. Cell Dev. Biol. 31, 347-371 (2015).</li><li>Krieg, M., Dunn, A. R., Goodman, M. 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M., M&#252;ller, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stimulation+of+Piezo1+receptors+depends+on+extracellular+matrix+proteins+and+directionality+of+force.">Mechanical stimulation of Piezo1 receptors depends on extracellular matrix proteins and directionality of force.</a> Nano Lett. 17 (3), 2064-2072 (2017).</li><li>Geffeney, S. L., 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=DEG/ENaC+but+not+TRP+channels+are+the+major+mechanoelectrical+transduction+channels+in+a+c.+Elegans+nociceptor.">DEG/ENaC but not TRP channels are the major mechanoelectrical transduction channels in a c. Elegans nociceptor.</a> Neuron. 71 (5), 845-857 (2011).</li><li>O'Hagan, R., Chalfie, M., Goodman, 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=The+MEC-4+DEG/ENaC+channel+of+Caenorhabditis+elegans+touch+receptor+neurons+transduces+mechanical+signals.">The MEC-4 DEG/ENaC channel of Caenorhabditis elegans touch receptor neurons transduces mechanical signals.</a> Nat. Neurosci. 8 (1), 43-50 (2005).</li><li>Suzuki, H., 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=In+Vivo+Imaging+of+C.+elegans+Mechanosensory+Neurons+Demonstrates+a+Specific+Role+for+the+MEC-4+Channel+in+the+Process+of+Gentle+Touch+Sensation.">In Vivo Imaging of C. elegans Mechanosensory Neurons Demonstrates a Specific Role for the MEC-4 Channel in the Process of Gentle Touch Sensation.</a> Neuron. 39 (6), 1005-1017 (2003).</li><li>Kopito, R. 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B., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DEG/ENaC+protein+MEC-10+regulates+the+transduction+channel+complex+in+Caenorhabditis+elegans+touch+receptor+neurons.">The DEG/ENaC protein MEC-10 regulates the transduction channel complex in Caenorhabditis elegans touch receptor neurons.</a> J. Neurosci. 31 (35), 12695-12704 (2011).</li><li>Lockhead, D., 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=The+tubulin+repertoire+of+Caenorhabditis+elegans+sensory+neurons+and+its+context-dependent+role+in+process+outgrowth.">The tubulin repertoire of Caenorhabditis elegans sensory neurons and its context-dependent role in process outgrowth.</a> Mol. Biol. Cell. 27 (23), 3717-3728 (2016).</li><li>Goodman, M. B., Ernstrom, G. G., Chelur, D. S., O'hagan, R., Yao, C. A., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MEC-2+regulates+C.+elegans+DEG/ENaC+channels+needed+for+mechanosensation.">MEC-2 regulates C. elegans DEG/ENaC channels needed for mechanosensation.</a> Nature. 415 (6875), 1039-1042 (2002).</li><li>Nekimken, A., Fehlauer, H., Kim, A., Goodman, M., Pruitt, B. L., Krieg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumatic+stimulation+of+C.+elegans+mechanoreceptor+neurons+in+a+microfluidic+trap.">Pneumatic stimulation of C. elegans mechanoreceptor neurons in a microfluidic trap.</a> Lab Chip. , (2017).</li><li>Brower, K., White, A. K., Fordyce, P. 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Denne protokol viser en metode til at levere præcise mekaniske stimulering til huden af en rundorm, der er fanget i en mikrofluid chip. Det er beregnet til at lette integrationen af fysiske stimuli for at besvare biologiske spørgsmål og sigter mod at strømline mechanobiology forskning i biologiske laboratorier. Denne metode udvider tidligere assays for at vurdere funktionen af mechanosensory neuroner i C. elegans. Tidligere kvantitative og semi-kvantitative teknikker målt styrker1<su...

Følelse af touch giver dyr med vigtige oplysninger om deres miljø. Afhængigt af den anvendte kraft opfatter touch som harmløst, lystbetonet eller smertefuld. Væv deformation under touch er opdaget af specialiserede mechanoreceptor celler indlejret i huden, der udtrykker receptorproteiner, oftest Ionkanaler. Skridt forbinder kraft opfattelse at ion-kanal aktivering under touch og smerte er ikke fuldt forstået. Endnu mindre er kendt om hvordan hudvæv filtre mekanisk deformation og om mechanoreceptors registrerer ændringer i stamme eller stress1,2,3. Dette hul i forståelse opstår delvis fra en mangel på egnede værktøjer til at anvende præcis mekaniske stimulering til overfladen af huden af en levende dyr under iagttagelse af svarene på cellulært niveau. Mens atomic force mikroskopi er blevet brugt flittigt til at anvende og måle kræfter i isolerede celler4,5 og også aktivere Piezo1 receptorer i levende celler6, lignende forsøg med levende dyr, især C. elegans, har været notorisk udfordrende på grund af den iboende mobilitet af emnet. Denne udfordring er traditionelt omgået ved hjælp af veterinær - eller kirurgisk-grade ren lim til at immobilisere enkelte dyr på agar puder1,7,8,9. Denne tilgang har været produktive, men har begrænsninger vedrører færdigheder kræves for immobilisering af limning og bløde agar overfladen på mekaniske overholdelse. En mikrofluidik strategi er et gratis alternativ, der undgår nogle af de komplikationer knyttet til limning.
Nematode C. elegans er en organisme, genetiske model med en helt kortlagt nervesystem, der på grund af dyrets størrelse, er en god pasform for mikrofluidik teknologi. Mikrofluidik-baserede enheder tilbud den fordel, at de ellers ekstremt mobile dyr kan være tilbageholdende mens de udfører høj opløsning imaging og levering af relevante neuro-modulerende stimuli. Ved hjælp af mikrofluid kan teknologier, levende dyr være immobiliseret uden skade10,11, at aktivere overvågning af adfærdsmæssige aktivitet over hele levetid12,13 og høj opløsning Imaging af neuronal aktivitet14,15,16,17. Yderligere, mange mechanoreceptor neuroner behov for følelsen af touch og smerte kan karakteriseres på deres fysiologiske1,8, mekanisk4,18,19, og molekylære niveau20,21,22.
C. elegans sanser blid mekaniske stimuli til sin krop væg ved hjælp af seks TRNs, hvoraf tre innerverer dyrets forreste (ALML/R og AVM) og tre af innerverer dyrets posterior (PLML/R og PVM). Ion-kanal molekyler til transducing en kraft til en biokemiske signal er blevet grundigt undersøgt i sin TRNs8. Denne artikel præsenterer en mikrofluid platform23 , der gør det muligt for forskere at anvende præcis mekaniske kræfter på huden af en immobiliseret C. elegans spolorm, mens du læser ud af deformation af dens indre væv af optiske billeddannelse. Ud over fremlæggelsen veldefinerede mekaniske stimuli, kan calcium transienter registreres i mechanoreceptor neuroner med subcellulært opløsning og korreleret med anatomiske og morfologiske funktioner. Enheden består af en central fældefangst kanal, der holder et enkelt dyr og præsenterer sin hud ved siden af seks pneumatisk aktivering kanaler (figur 1 og figur 2). Seks kanaler er placeret langs diffusering kanalen til at levere mekaniske stimuli til hver af worm's seks TRNs. Disse kanaler er adskilt fra fældefangst kammer med tynde PDMS membraner, der kan være drevet af en ydre luft tryk source (figur 1). Vi kalibreret fordrejning med hensyn til presset og giver målinger i denne artikel. Hvert aktuator kan behandles individuelt og bruges til at stimulere en mechanoreceptor valg. Trykket leveres ved hjælp af et piezo-drevet tryk pumpe, men eventuelle alternative enhed kan bruges. Vi viser, at pres-protokollen kan bruges til at aktivere TRNs i vivo og demonstrere Transporterende enheder velegnet til levere mekaniske stimuli til voksen C. elegans, indlæse voksne dyr i enheder, udfører calcium imaging eksperimenter, og analysere resultaterne. Enheden fabrikation består af to hovedtrin: 1) fotolitografi at gøre en Skimmelsvamp fra SU-8; og 2) molding PDMS for at gøre en enhed. Kortfattethed og klarhed henvises læsere til tidligere publicerede artikler og protokoller24,25 for instruktioner om, hvordan til at fremstille forme og enheder.

<table>
	<tbody>
		<tr>
			<td>Chrome mask</td>
			<td>Compugraphics (http://www.compugraphics-photomasks.com/)</td>
			<td></td>
			<td>5&#39;&#39;, designed in AutoCAD (Autodesk, Inc.)</td>
		</tr>
		<tr>
			<td>Chrome mask</td>
			<td>Mitani-Micronics (http://www.mitani-micro.co.jp/en/)</td>
			<td></td>
			<td>5&#39;&#39;, designed in AutoCAD (Autodesk, Inc.)</td>
		</tr>
		<tr>
			<td>Chrome mask</td>
			<td>Kuroda-Electric (http://www.kuroda-electric.eu/</td>
			<td></td>
			<td>5&#39;&#39;, designed in AutoCAD (Autodesk, Inc.)</td>
		</tr>
		<tr>
			<td>4&#39;&#39; Silicon wafer (B-test)</td>
			<td>Stanford Nanofabrication Facility</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2002</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2050</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spin-coater</td>
			<td>Laurell Technologies</td>
			<td>WS-400BZ-6NPP/LITE</td>
			<td></td>
		</tr>
		<tr>
			<td>Exposure timer</td>
			<td>Optical Associates, Inc</td>
			<td>OAI 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumination controller</td>
			<td>Optical Associates, Inc</td>
			<td>2105C2</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 developer</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Fisher Scientific</td>
			<td>A426F-1GAL</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>A18-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloromethylsilane (TCMS)</td>
			<td>Sigma-Aldrich</td>
			<td>92361-500ML</td>
			<td>Caution: TCMS is toxic and water-reactive</td>
		</tr>
		<tr>
			<td>Sylgard 184 Elastomer Kit</td>
			<td>Dow Corning</td>
			<td></td>
			<td>PDMS prepolymer</td>
		</tr>
		<tr>
			<td>Biopsy punch, 1 mm</td>
			<td>VWR</td>
			<td>95039-090</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen Plasma Asher</td>
			<td>Branson/IPC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small metal tubing (0.635 mm OD, 0.4318 mm ID, 12.7 mm long); gage size 23TW</td>
			<td>New England Small Tube Corporation</td>
			<td>NE-1300-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene syringe filter, 0.22 &#956;m</td>
			<td>Thermo Scientific</td>
			<td>725-2520</td>
			<td>to filter all solution, small particles would clog the chip</td>
		</tr>
		<tr>
			<td>Polyethylene tubing; 0.9652 mm OD, 0.5842 mm ID</td>
			<td>Solomon Scientific</td>
			<td>BPE-T50</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 1 ml</td>
			<td>BD Scientific</td>
			<td>309628</td>
			<td>for worm trapping and release</td>
		</tr>
		<tr>
			<td>Syringe, 20 ml</td>
			<td>BD Scientific</td>
			<td>309661</td>
			<td>for gravity-based flow</td>
		</tr>
		<tr>
			<td>Gilson Minipuls 3, Peristaltic pump</td>
			<td>Gilson</td>
			<td></td>
			<td>to suck solutions and worms out of the chip</td>
		</tr>
		<tr>
			<td>Microfluidic flow controller, equipped with 0&#8211;800 kPa pressure channel</td>
			<td>Elveflow</td>
			<td>OB1 MK3</td>
			<td>pressure delivery</td>
		</tr>
		<tr>
			<td>Water-Resistant Clear Poly- urethane Tubing, 4 mm ID and 6 mm OD</td>
			<td>McMaster-Carr</td>
			<td>5195 T52</td>
			<td>connection from house air to pressure pump</td>
		</tr>
		<tr>
			<td>Water-Resistant Clear Polyurethane Tubing, 2.6mm ID and 4mm OD</td>
			<td>McMaster-Carr</td>
			<td>5195 T51</td>
			<td>connect pressure pump to small tubng</td>
		</tr>
		<tr>
			<td>Push-to-Connect Tube Fitting for Air</td>
			<td>McMaster-Carr</td>
			<td>5111K468</td>
			<td>metric - imperial converter</td>
		</tr>
		<tr>
			<td>Straight Connector for 6 mm &#215; 1/4&#8243; Tube OD</td>
			<td>McMaster-Carr</td>
			<td>5779 K258</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMI 4000 B microscopy system</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>63&#215;/1.32 NA HCX PL APO oil objective</td>
			<td>Leica</td>
			<td>506081</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamamatsu Orca-Flash 4.0LT digital CMOS camera</td>
			<td>Hamamatsu</td>
			<td>C11440-42U</td>
			<td></td>
		</tr>
		<tr>
			<td>Lumencor Spectra X light engine</td>
			<td>Lumencor</td>
			<td></td>
			<td>With cyan and green/yellow light source</td>
		</tr>
		<tr>
			<td>Excitation beam splitter</td>
			<td>Chroma</td>
			<td>59022bs</td>
			<td>in the microscope</td>
		</tr>
		<tr>
			<td>Hamamatsu W-view Gemini Image splitting optics</td>
			<td>Hamamatsu</td>
			<td>A12801-01</td>
			<td>to split green and red emission and project them on different areas on the camera chip</td>
		</tr>
		<tr>
			<td>Emission beam splitter</td>
			<td>Chroma</td>
			<td>T570lpxr</td>
			<td>in the image splitter</td>
		</tr>
		<tr>
			<td>Emission filters GCamp6s</td>
			<td>Chroma</td>
			<td>ET525/50m</td>
			<td>in the image splitter</td>
		</tr>
		<tr>
			<td>Emission filters mCherry</td>
			<td>Chroma</td>
			<td>ET632/60m</td>
			<td>in the image splitter</td>
		</tr>
	</tbody>
</table>

using a microfluidics device for mechanical stimulation and high resolution imaging of c. elegans

Et centralt mål for mechanobiology er at forstå den gensidige effekt af mekanisk stress på proteiner og celler. Trods dens betydning, er mekanisk stress indflydelse på cellulær funktion stadig dårligt forstået. Delvis eksisterer denne videnskløft fordi par værktøjer aktiverer samtidige deformation af væv og celler, billeddannelse af cellulære aktivitet i levende dyr og effektiv begrænsning af motilitet i ellers meget mobile modelorganismer, såsom nematode Caenorhabditis elegans. Den lille størrelse af C. elegans gør dem en fremragende match til enheder, mikrofluidik-baseret forskning og løsninger for immobilisering har været præsenteret ved hjælp af mikrofluid enheder. Selv om disse enheder giver mulighed for høj opløsning imaging, er dyret helt indkapslet i Polydimethylsiloxan (PDMS) og glas, begrænse fysisk adgang til levering af mekanisk kraft eller elektrofysiologiske optagelser. For nylig har skabt vi en enhed, der integrerer pneumatisk aktuatorer med en diffusering design, der er kompatibel med høj opløsning Fluorescens mikroskopi. Aktivering-kanal er adskilt fra den orm-fældefangst kanal af en tynd PDMS membran. Denne membran er afbøjet i siden af en orm ved at anvende pres fra en ekstern kilde. Enheden kan målrette individuelle mechanosensitive neuroner. Aktivering af disse neuroner er afbildet på høj opløsning med genetisk kodet calcium indikatorer. Denne artikel præsenterer den generelle metode ved hjælp af C. elegans stammer udtryk for calcium-følsomme aktivitet indikator (GCaMP6s) i deres touch receptor neuroner (TRNs). Metoden, men er ikke begrænset til TRNs eller calcium sensorer som en sonde, men kan udvides til andre mekanisk-følsomme celler eller sensorer.

SU-8 litografi og Chip limning Litografi protokol og PDMS støbning følge standard procedurer. Detaljer kan findes andetsteds23,24,25,26. PDMS skulle skrælle wafer uden problemer efter hærdning. Hvis SU-8 funktioner rip off under PDMS peeling, var enten SU-8 friktion lag eller silanisering utilstrækkelig. Hvis plasma-aktivering af glas co...

Watch this Scientific Journal Video about Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans at JoVE.com

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

Nye værktøjer til mechanobiology forskning er nødvendig for at forstå hvordan mekanisk stress aktiveres biokemiske veje og fremkalder biologiske svar. Her, fremvise vi en ny metode til selektiv mekaniske stimulation af immobiliserede dyr med en mikrofluid fælde så høj opløsning billeddannelse af cellulære svar.

Ved hjælp af en mikrofluidik anordning for mekanisk Stimulation og høj opløsning Imaging af C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

Research

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Neuroscience

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Assaying ÃƒÅ½Ã‚Â²-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers

Aktivering Høj gråtoner højopløste displays og præcise svar tidsmålinger på konventionelle computere

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Høj opløsning funktionel magnetisk resonans Metoder til human midthjernen

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

Høj opløsning Live-Imaging af celle adfærd i udviklingslandene neuroepitel

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Automated Quantification of Synaptic Fluorescence in C. elegans

Automatiseret Kvantificering af Synaptic Fluorescens in<em&gt; C. elegans</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Calcium Imaging i C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...