Name: a microfluidic platform for longitudinal imaging in caenorhabditis elegans
Uploaded: 2018-05-02T13:30:00.000+00:00
Duration: 9 min
Description: Watch this Scientific Journal Video about A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans at JoVE.com

Questa ricerca è stata sostenuta dal National Science Foundation attraverso sovvenzioni PHY-1205494 e MCB-1413134 (EL) e dalla 2017R1D1A1B03035671 di grant National Research Foundation of Korea (KSL).

Il protocollo sottostante utilizza WormSpa5, un dispositivo microfluidico precedentemente descritto per l'imaging longitudinale di vermi. Fabbricazione di WormSpa (a partire con i file CAD che possono essere ottenuti dagli autori su richiesta) è semplice, ma richiede alcune competenze. Nella maggior parte dei casi, fabbricazione può essere fatto facilmente da una struttura o da una società commerciale che fornisce tali servizi. Quando fabbricando il dispositivo, assicurarsi di specificare l'altezza delle caratteristiche è di 50 µm.
1. organizzazione sperimentale
<ol><li>Montare il dispositivo microfluidico su un microscopio a fluorescenza invertito con un tavolino motorizzato.</li>
	<li>Posto un agitatore orbitale vicino al microscopio, ma assicurarsi che la vibrazione da shaker non influisce direttamente al microscopio. Ad esempio, inserire nello shaker su un ripiano fissato su una parete che non rende nessun contatto con il tavolo ottico.</li>
	<li>Posto una pompa a siringa su agitatore orbitale. Nastro della pompa a shaker affinché esso non jiggle mentre si stringono.</li>
</ol>2. preparazione dell'ambiente microfluidica
Nota: Durante l'esperimento, quattro siringhe sono collegati al dispositivo microfluidico tramite tubi di siringa, come indicato nella Figura 1. Questo passaggio descrive la preparazione di queste siringhe. A meno che non diversamente specificato, è possibile mantenere le provette di siringa più breve possibile senza che li rende teso.
<ol><li>Preparare una siringa di presa e un tubo siringa. Questa siringa (S1 nella Figura 1) successivamente sarà collegata alla presa dell'apparecchio attraverso il tubo siringa e utilizzato per l'archiviazione temporanea di fluido proveniente dal dispositivo.<ol>
<li>Rendono un adattatore ago rimuovendo parti in plastica di una punta della siringa 20 x 1/2 pollice, mantenendo solo l'ago (parte metallica). Farlo tenendo le parti in plastica e metallo con una pinza e li separano. Residui di plastica dell'ago possono essere bruciato con un becco Bunsen.</li>
		<li>Piegare l'ago secondo la geometria del setup sperimentale tenendo le due estremità con le pinze. Nel disegno qui descritto, piegare l'ago nella sua metà ad un angolo di ~ 110° è più conveniente.</li>
		<li>Inserire l'ago di adattatore a metà strada nel tubo. L'altra metà sarà successivamente collegata al dispositivo. Il tubo siringa dovrebbe essere compatibile con una punta della siringa 20 G senza una perdita (ad esempio, tubi con diametro interno di 0,86 mm e diametro esterno di 1,32 mm). La lunghezza del tubo consigliato è ~ 50 cm.</li>
		<li>Collegare una siringa da 10 mL a altra estremità del tubo siringa tramite una punta della siringa 20 x 1/2 pollice (aperta).</li>
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</li>
	<li>Preparare buffer-ingresso siringhe e tubi a siringa. Uno di queste siringhe (S2 nella Figura 1) è usato come un serbatoio per il liquido che va al dispositivo e per controllare il flusso di iniezione. Altre la siringa (S3 nella Figura 1) è necessario per scambio di buffer, come spiegato nel punto 3.2.<ol>
<li>Collegare una siringa luer lock 10 mL (S2) direttamente ad una valvola a 3 vie e un altro siringa (S3) per la stessa valvola tramite un tubo siringa (Figura 1): collegare S3 al tubo tramite una punta della siringa e collegare il tubo alla valvola tramite un'altra punta della siringa. Nota: Non importa l'ordine in cui questi materiali sono collegati.</li>
		<li>Collegare un tubo siringa alla porta restante della valvola 3 vie. Preparare un altro ago adattatore come descritto al punto 2.1.2 e collegare l'altra estremità (aperta) del tubo siringa l'ago a metà strada.</li>
		<li>Posizionare la valvola in modo tale che il tubo siringa al punto 2.2.2 e S2 sono collegati mentre S3 è chiuso (Figura 1).</li>
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</li>
	<li>Preparare una siringa di vite senza fine-aspirazione e un tubo siringa. Questa siringa (S4 nella Figura 1) è utilizzato per il caricamento di vermi nel dispositivo.<ol>
<li>Collegare un tubo siringa lunga a una siringa luer lock 10 mL (S4). Determinare la lunghezza di questo tubo dal volume del fluido che verrà utilizzato per il caricamento; ad esempio, 100 cm di tubo supporta oltre 2 mL di liquido (vedere anche punto 4.2).</li>
		<li>Preparare un altro ago adattatore come descritto al punto 2.1.2 e collegare una metà dell'ago all'estremità (aperta) del tubo siringa.</li>
	</ol>
</li>
	<li>Sciacquare tutte le siringhe e tubi con S-medio12 due volte. Riempire le siringhe e i tubi con S-medio, avendo cura di eliminare tutte le bolle d'aria.</li>
	<li>Collegare il tubo di uscita siringa all'uscita del dispositivo.<ol>
<li>Inserire l'ago di adattatore all'estremità del tubo siringa alla porta di uscita.</li>
		<li>Iniettare un piccolo volume di S-medio attraverso l'uscita per riempire il dispositivo, fino a quando un po ' S-medio esce sia l'ingresso di buffer e l'ingresso del verme.</li>
	</ol>
</li>
	<li>Collegare il tubo di siringa buffer-ingresso alla porta tampone-ingresso del dispositivo, mentre collegando la bocca di vite senza fine con un pin (spina di centraggio in acciaio inox con un diametro di 1/32 di pollice).</li>
	<li>Iniettare un flusso costante di buffer nel dispositivo. Caricare la siringa di buffer-ingresso S2 su una siringa pompa13e impostare la pompa in modo che il mezzo in S2 continuamente viene iniettato il dispositivo ad una portata di 3 µ l/min.</li>
</ol>3. caricamento di batteri nel dispositivo microfluidico
<ol><li>Preparare Escherichia coli OP50 sospesa in medio-S12.<ol>
<li>A seguito di un protocollo standard, inoculare un pallone da 250 mL con 50 mL di LB e una singola Colonia e incubare per una notte a 37 ° C.</li>
		<li>Centrifugare la coltura durante la notte a 4000 giri/min per 10 minuti e risospendere il pellet in 30 mL di medio-S.</li>
		<li>Filtrare la sospensione attraverso un filtro per siringa 5 µm. Misurare la concentrazione batterica dalla sua densità ottica a 600 nm (OD600) e regolare la concentrazione di un OD600 di 3. Questa ad alta densità è necessaria per mantenere i vermi ben nutriti.</li>
	</ol>
</li>
	<li>Scambiare il buffer nel dispositivo con la sospensione di OP50.<ol>
<li>Preparare una siringa pulita (S5) riempita con la sospensione di OP50. Tenere la siringa in posizione verticale e toccare più volte per raccogliere tutte le bolle di aria nella parte superiore.</li>
		<li>Girare la valvola 3 vie delle siringhe buffer-ingresso per chiudere la connessione al dispositivo e collegare le due siringhe, S2 e S3. Disinnestare S2 da pompa a siringa.</li>
		<li>Scollegare la valvola S2 e collegare S5 alla valvola. Spingere fuori tutta l'aria raccolto nella parte superiore della S5 a S3 attraverso la valvola fino a quando un po ' di buffer OP50 entra in S3. In tal modo, assicurarsi che nessuna bolla di aria rimane in S5 o la valvola.</li>
		<li>Girare la valvola a 3 vie torna alla posizione originale per chiudere la connessione tra S3 e S5 e connettersi al dispositivo di S5. Carico S5 sulla pompa a siringa. Accendere l'agitatore orbitale che tiene la pompa. Impostare la velocità di agitazione per circa 200 giri/min.</li>
		<li>Impostare la portata da 100 µ l/min. Il tempo che necessario per il nuovo buffer di arrivare ai vermi nel dispositivo dipende dalla lunghezza del tubo siringa buffer-ingresso (circa 5 minuti con il tubo sopra descritto). Un più alto tasso di flusso può essere impiegato se è necessario un più rapido scambio di buffer.</li>
		<li>Una volta che il buffer OP50 si diffonde in tutto il dispositivo, è possibile impostare la portata indietro a 3 µ l/min.</li>
	</ol>
</li>
</ol>4. caricamento Worms nel dispositivo microfluidico
<ol><li>Preparare un tubo del microcentrifuge piccola associazione basso (650 µ l) e riempirlo con 100 µ l di sospensione OP50. Trasferimento intorno 20-30 sincronizzato di età giovani vermi adulti (46 ore dopo l'arresto larvale L1 a 25 ° C) da una piastra di agar NGM (crescita del nematode medio) al tubo di raccoglierli uno per uno con un verme pick. Età-sincronizzazione può essere fatta seguendo un protocollo standard12.</li>
	<li>Riempire la vite senza fine-aspirazione siringa e siringa tube (S4 nella Figura 1) con la sospensione di OP50. Iniettare ~ 500 µ l del fluido nel tubo del microcentrifuge con i vermi. Disegnare la sospensione con i vermi nel tubo siringa vite senza fine-aspirazione. Assicurarsi che il tubo siringa è abbastanza a lungo (> 1 m) e che il worm disegnato soggiornare nel tubo siringa e non fare tutto il senso alla siringa S4.</li>
	<li>Collegare S4 all'entrata del verme. Iniettare tutti i vermi nel tubo siringa verme-ingresso nel dispositivo microfluidico. Scollegare il tubo siringa e S4. Collegare la bocca di vite senza fine con un pin.</li>
	<li>Disinnestare la siringa di buffer-ingresso S2 dalla pompa meccanica ( Figura 1) e controllare manualmente la S1 e S2 per spingere tutti i vermi in canali separati. Premendo S2 si muovono vermi nei canali, mentre premendo S1 li sposterà nella direzione inversa. Una volta caricato un canale, il worm in ciascun canale bloccherà l'ingresso di altri worm.</li>
	<li>Lasciare che il worm al nuovo ambiente per 2-3 ore.</li>
</ol>5. creazione di un protocollo di Imaging
<ol><li>Trovare tutti i vermi che si trovano in modo sicuro nel dispositivo e impostare una posizione di imaging per ciascuno di essi nel software di acquisizione di immagine che controlla il microscopio. Si noti che in alcuni casi (ad esempio quando si desidera ottenere maggiore ingrandimento, o quando si utilizzano fotocamere con un più piccolo sensore CCD) posizioni multiple di formazione immagine sono necessari per ogni canale. Nota: Mentre questo può essere fatto manualmente, alcuni pacchetti di software di acquisizione possono caricare un file di testo che elenca le posizioni dove le immagini dovrebbero essere prese. Poiché queste posizioni vengono ordinate regolarmente in WormSpa, un utente può scrivere un piccolo script (ad esempio, in Python o al software commerciale) che crea automaticamente tale elenco. Il formato preciso di questo script sarebbe dipendono dal formato file previsto dal software di acquisizione (Vedi un esempio in 1 materiale supplementare).</li>
	<li>Impostare la frequenza richiesta di time-lapse imaging. Ad esempio, formazione immagine della risposta di gene immune all'infezione avviene ad una frequenza di 10 minuti per ogni frame. Assicurarsi che tutti i canali necessari (ad es. bright-field e la fluorescenza di GFP) vengono acquisiti in ogni momento.</li>
</ol>6. risposta ospite all'infezione di Pseudomonas
Nota: Questo passaggio è specifico per lo studio delle interazioni ospite-patogeno. In alternativa, si può preparare un buffer che contiene altri stimoli ambientali di interesse (fattori di stress biotici e abiotici, droghe, segnalazione di molecole, ecc.).
<ol><li>Preparare Pseudomonas aeruginosa PA14 sospesa in SK-medio14.<ol>
<li>Inoculare un pallone da 250 mL con 50 mL di LB e una singola Colonia e incubare per una notte a 37 ° C.</li>
		<li>Inoculare un altro pallone da 250 mL con 50 mL di LB e un'aliquota della cultura e incubare per una notte a 37 ° C.</li>
		<li>Centrifugare la coltura durante la notte a 4000 giri/min per 10 minuti e risospendere il pellet in 10 mL di SK-medio. Misurare la concentrazione batterica e regolare la concentrazione di OD600 = 4.</li>
		<li>Trasferire la sospensione in un pallone pulito e incubare a 37 ° C per 24 ore e a 25 ° C per altre 24 ore mentre si stringono. Questo passaggio imita il passo piatto-incubazione nello standard uccidendo saggio14.</li>
		<li>Filtrare la sospensione attraverso un filtro per siringa 5 µm. Questo è per impedire l'intasamento del dispositivo di aggregazioni batteriche.</li>
	</ol>
</li>
	<li>Sostituire la sospensione OP50 nel dispositivo con la sospensione di PA14, seguendo la procedura descritta al punto 3.2. Tenere imaging per 10 ore.</li>
</ol>

<ol>
	<li>Lopez-Maury, L., Marguerat, S., Bahler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+gene+expression+to+changing+environments:+from+rapid+responses+to+evolutionary+adaptation.">Tuning gene expression to changing environments: from rapid responses to evolutionary adaptation.</a> Nat Rev Genet. 9 (8), 583-593 (2008).</li><li>de Nadal, E., Ammerer, G., Posas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+gene+expression+in+response+to+stress.">Controlling gene expression in response to stress.</a> Nat Rev Genet. 12 (12), 833-845 (2011).</li><li>Hulme, S. E., Shevkoplyas, S. S., Samuel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics:+Streamlining+discovery+in+worm+biology.">Microfluidics: Streamlining discovery in worm biology.</a> Nat Methods. 5 (7), 589-590 (2008).</li><li>San-Miguel, A., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+as+a+tool+for+C.+elegans+research.">Microfluidics as a tool for C. elegans research.</a> WormBook. , (2013).</li><li>Kopito, R. B., Levine, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Durable+spatiotemporal+surveillance+of+Caenorhabditis+elegans+response+to+environmental+cues.">Durable spatiotemporal surveillance of Caenorhabditis elegans response to environmental cues.</a> Lab Chip. 14 (4), 764-770 (2014).</li><li>Mishra, B., 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=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.</a> J Vis Exp. (84), e50998 (2014).</li><li>Grisi, 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=NMR+spectroscopy+of+single+sub-nL+ova+with+inductive+ultra-compact+single-chip+probes.">NMR spectroscopy of single sub-nL ova with inductive ultra-compact single-chip probes.</a> Sci Rep. 7, 44670 (2017).</li><li>Crane, M. M., Chung, K., Stirman, J., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics-enabled+phenotyping,+imaging,+and+screening+of+multicellular+organisms.">Microfluidics-enabled phenotyping, imaging, and screening of multicellular organisms.</a> Lab Chip. 10 (12), 1509-1517 (2010).</li><li>Leifer, A. M., Fang-Yen, C., Gershow, M., Alkema, M. J., Samuel, A. D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+manipulation+of+neural+activity+in+freely+moving+Caenorhabditis+elegans.">Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans.</a> Nat Meth. 8 (2), 147-152 (2011).</li><li>Lee, K. S., Lee, L. E., Levine, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HandKAchip+-+Hands-free+killing+assay+on+a+chip.">HandKAchip - Hands-free killing assay on a chip.</a> Sci Rep. 6, 35862 (2016).</li><li>Lee, K. S., 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=Serotonin-dependent+kinetics+of+feeding+bursts+underlie+a+graded+response+to+food+availability+in+C.+elegans.">Serotonin-dependent kinetics of feeding bursts underlie a graded response to food availability in C. elegans.</a> Nat Commun. 8, 14221 (2017).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> NE-1000 Series of Programmable Syringe Pumps. , (2017).</li><li>Tan, M. W., Mahajan-Miklos, S., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killing+of+Caenorhabditis+elegans+by+Pseudomonas+aeruginosa+used+to+model+mammalian+bacterial+pathogenesis.">Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis.</a> Proc Natl Acad Sci U S A. 96 (2), 715-720 (1999).</li><li>Kim, D. 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=A+conserved+p38+MAP+kinase+pathway+in+Caenorhabditis+elegans+innate+immunity.">A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity.</a> Science. 297 (5581), 623-626 (2002).</li><li>Troemel, E. R., 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=p38+MAPK+regulates+expression+of+immune+response+genes+and+contributes+to+longevity+in+C.+elegans.">p38 MAPK regulates expression of immune response genes and contributes to longevity in C. elegans.</a> PLoS Genet. 2 (11), 183 (2006).</li><li>Estes, K. A., Dunbar, T. L., Powell, J. R., Ausubel, F. M., Troemel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bZIP+transcription+factor+zip-2+mediates+an+early+response+to+Pseudomonas+aeruginosa+infection+in+Caenorhabditis+elegans.">bZIP transcription factor zip-2 mediates an early response to Pseudomonas aeruginosa infection in Caenorhabditis elegans.</a> Proc Natl Acad Sci U S A. 107 (5), 2153-2158 (2010).</li><li>Byerly, L., Cassada, R. C., Russell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life+cycle+of+the+nematode+Caenorhabditis+elegans:+I.+Wild-type+growth+and+reproduction.">The life cycle of the nematode Caenorhabditis elegans: I. Wild-type growth and reproduction.</a> Dev Biol. 51 (1), 23-33 (1976).</li><li>Chalancon, G., 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=Interplay+between+gene+expression+noise+and+regulatory+network+architecture.">Interplay between gene expression noise and regulatory network architecture.</a> Trends Genet. 28 (5), 221-232 (2012).</li><li>Sanchez, A., Choubey, S., Kondev, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+noise+in+gene+expression.">Regulation of noise in gene expression.</a> Annu Rev Biophys. 42, 469-491 (2013).</li><li>Norman, T. M., Lord, N. D., Paulsson, J., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+Switching+of+Cell+Fate+in+Microbes.">Stochastic Switching of Cell Fate in Microbes.</a> Annu Rev Microbiol. 69, 381-403 (2015).</li><li>Gardner, T. S., di Bernardo, D., Lorenz, D., Collins, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inferring+genetic+networks+and+identifying+compound+mode+of+action+via+expression+profiling.">Inferring genetic networks and identifying compound mode of action via expression profiling.</a> Science. 301 (5629), 102-105 (2003).</li><li>Samuelson, A. V., Carr, C. E., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+activities+that+mediate+increased+life+span+of+C.+elegans+insulin-like+signaling+mutants.">Gene activities that mediate increased life span of C. elegans insulin-like signaling mutants.</a> Genes Dev. 21 (22), 2976-2994 (2007).</li><li>Edwards, C. B., Copes, N., Brito, A. G., Canfield, J., Bradshaw, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malate+and+Fumarate+Extend+Lifespan+in+Caenorhabditis+elegans.">Malate and Fumarate Extend Lifespan in Caenorhabditis elegans.</a> PLoS ONE. 8 (3), 58345 (2013).</li><li>Riddle, D. L., Blumenthal, T., Meyer, B. J., Priess, J. R., Riddle, D. L., Blumenthal, T., Meyer, B. J., Priess, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans II. , (1997).</li><li>Churgin, M. A., 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=Longitudinal+imaging+of+Caenorhabditis+elegans+in+a+microfabricated+device+reveals+variation+in+behavioral+decline+during+aging.">Longitudinal imaging of Caenorhabditis elegans in a microfabricated device reveals variation in behavioral decline during aging.</a> eLife. 6, 26652 (2017).</li><li>Scholz, M., Lynch, D. J., Lee, K. S., Levine, E., Biron, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+scalable+method+for+automatically+measuring+pharyngeal+pumping+in+C.+elegans.">A scalable method for automatically measuring pharyngeal pumping in C. elegans.</a> J Neurosci Methods. 274, 172-178 (2016).</li><li>Scholz, M., Dinner, A. R., Levine, E., Biron, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+feeding+dynamics+arise+from+the+need+for+information+and+energy.">Stochastic feeding dynamics arise from the need for information and energy.</a> Proc Natl Acad Sci U S A. 114 (35), 9261-9266 (2017).</li></ol>

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Strumenti di microfluidica forniscono molteplici vantaggi nello studio di vermi. Imaging in un PDMS dispositivo offre qualità delle immagini superiore rispetto ad una piastra di agar NGM standard. Immagini multiple possono essere assunto da una vite senza fine, in contrasto con i metodi tradizionali in cui gli animali sono raccolte dalla piastra e montati su un vetrino da microscopio per l'imaging. Inoltre, il microambiente in cui si trovano vermi può essere mantenuto costante o modulata come desiderato, permettendo pr...

Cambiamenti nelle condizioni ambientali possono portare all'attivazione di programmi genetici accompagnato da induzione e repressione dell'espressione di specifici geni1,2. Questi cambiamenti cinetici possono essere variabile tra tessuti negli stessi animali e tra animali diversi. Studi di tali programmi genetici pertanto chiamano per metodi che consentono di imaging longitudinale dei singoli animali e forniscono un controllo dinamico preciso delle condizioni ambientali.
Negli ultimi anni, sono stati utilizzati fluidico dispositivi microfabbricati per studiare molti aspetti della risposta e comportamento nei piccoli animali, tra cui vermi, mosche, acqua orsi e altre3,4,5,6, 7. Le applicazioni includono, ad esempio, profonda phenotyping, optogenetica registrazione dell'attività neuronale in risposta a stimoli chimici e il monitoraggio dei comportamenti motori come locomozione e pompaggio8,9,10 , 11.
Gli approcci basati su microfluidici tenere molte proprietà che potrebbero beneficiare di formazione immagine longitudinale a lungo termine della risposta a stimoli ambientali, tra cui controllo dinamico preciso del microambiente locale, design flessibile che permette la manutenzione dei singoli animali in quartieri separati e attributi favorevoli per l'imaging. Tuttavia, mantenere gli animali in una camera di microfluidica per lungo tempo con minimo impatto negativo sulla loro esseri ben è una sfida, che richiede particolare cura nella progettazione del dispositivo microfluidico nonché nell'esecuzione dell'esperimento.
Qui dimostriamo l'uso di WormSpa, un dispositivo microfluidico per formazione immagine longitudinale del Caenorhabditis elegans. 5 singoli vermi sono confinati negli alloggiamenti. Un basso flusso costante di liquido e batterica sospensione garantisce che i vermi sono ben nutriti e sufficientemente attivo per mantenere una buona salute e alleviare lo stress, e la struttura delle camere permette di vermi per deporre le uova. La semplicità del design e il funzionamento del WormSpa dovrebbe permettere ai ricercatori con nessuna esperienza precedente in microfluidica per incorporare i propri piani di ricerca di questo dispositivo.

<table><tbody><tr><td>WormSpa</td><td>N/A</td><td>N/A</td><td>The CAD file for WormSpa is available from the Levine lab.</td></tr><tr><td>Compound Microscope</td><td>Zeiss</td><td>AxioObserver Z1</td><td>An inverted fluorescence microscope with a motorized stage</td></tr><tr><td>Syringe Pump</td><td>New Era Pump Systems</td><td>NE-501</td><td></td></tr><tr><td>Tubing</td><td>SCI Scientific Commodities Inc.</td><td>BB31695-PE/5</td><td>0.034&amp;rdquo; (0.86 mm) I.D. x 0.052&amp;rdquo; (1.32 mm) O.D</td></tr><tr><td>Syringe Tip</td><td>CMLsupply</td><td>901-20-050</td><td>20 Gauge x 1/2&amp;rdquo; blunt tip stainless steel canula</td></tr><tr><td>Syringe Filter</td><td>PALL</td><td>4650</td><td>Acrodisc 32 mm Syringe Filter with 5 um Supor Membrane</td></tr><tr><td>Syringe</td><td>Qosina</td><td>C3307</td><td>10 mL Male Luer Lock Syringe</td></tr><tr><td>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><td>Dowel Pin</td><td>McMaster-Carr</td><td>90145A317</td><td>18-8 Stainless Steel Dowel Pins (1/32&quot; Dia. x 1/2&quot; Lg.)</td></tr><tr><td>Low Binding Microcentrifuge Tube</td><td>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

Nell'ultimo decennio, microfluidica tecniche sono state applicate per lo studio di piccoli animali, tra cui il nematode Caenorhabditis eleganse si sono rivelati utili come una comoda piattaforma di imaging live fornendo funzionalità per un controllo preciso di condizioni sperimentali in tempo reale. In questo articolo, dimostriamo live imaging dei singoli vermi che impiegano WormSpa, un dispositivo microfluidico personalizzato precedentemente pubblicato. Nel dispositivo, più vermi individualmente sono confinati per separare chambers, permettendo multiplex sorveglianza longitudinale di vari processi biologici. Per illustrare le funzionalità, abbiamo effettuato esperimenti di prova-de-principio in cui vermi sono stati infettati nel dispositivo con i batteri patogeni, e la dinamica dell'espressione di geni di risposta immunitaria e la deposizione delle uova sono stata monitorata in continuo in individuo animali. Il design semplice e il funzionamento di questo dispositivo lo rendono adatto per utenti con nessuna esperienza precedente con esperimenti basati su microfluidica. Proponiamo che questo approccio sia utile per molti ricercatori interessati nelle osservazioni longitudinali dei processi biologici in condizioni ben definite.

Vermi adulti giovani età-sincronizzati (46 ore post arresto larvale L1 a 25 ° C)12 sono stati caricati nel dispositivo, come descritto nel protocollo. I vermi si trovavano individualmente in canali separati, Abilitazione misura longitudinale della risposta degli animali all'agente patogeno. Quando l'esperimento è riuscito, la maggior parte dei worm rimangono nei loro canali per tutta la durata dell'esperimento. In questo caso, immagini di worms individuali sono ...

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

Una piattaforma microfluidica per Imaging longitudinale in Caenorhabditis elegans

In questo articolo, dimostriamo live imaging dei singoli vermi che impiegano un dispositivo microfluidico personalizzato. Nel dispositivo, più vermi individualmente sono confinati per separare chambers, permettendo multiplex sorveglianza longitudinale di vari processi biologici.

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

a-microfluidic-platform-for-longitudinal-imaging-caenorhabditis

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

6.4K Views.  Kongju National University. In questo articolo, dimostriamo live imaging dei singoli vermi che impiegano un dispositivo microfluidico personalizzato. Nel dispositivo, più vermi individualmente sono confinati per separare chambers, permettendo multiplex sorveglianza longitudinale di vari processi biologici.

Video: A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

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.

Semplici dispositivi microfluidici per<em&gt; In vivo</em&gt; Imaging di<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; E Zebrafish

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

Ricerca

Bioingegneria

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

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

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

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

Electrotaxis microfluidica-based per On-demand analisi quantitativa della<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 ...

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.

Microscopia isotropica leggera e analisi di lignaggio cellulare automatizzate al catalogo Caenorhabditis elegans embriogenesi con risoluzione subcellulare

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

Biologia dello sviluppo

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.

Un metodo di imaging semi-alto e un toolkit di visualizzazione dei dati per analizzare lo sviluppo embrionale C. elegans

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

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Microchambers Agarose per lungo termine Imaging di calcio<em&gt; Caenorhabditis elegans</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Neuroscienze

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 Gli embrioni utilizzando un microscopio epifluorescente e Software Open Source

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

Biologia

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

Tramite un Chip olfattiva di microfluidica adattato per l'Imaging dell'attività neuronale in risposta ai feromoni in neuroni di testa maschio C. Elegans

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

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

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

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

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

Utilizzando un dispositivo di microfluidica per stimolazione meccanica e alta risoluzione Imaging di c. elegans

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

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.

Un semplice chip microfluidico per la crescita a lungo termine e l'imaging di Caenorhabditis elegans

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

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

Coltura a lungo termine di singoli Caenorhabditis elegans su terreni solidi per il monitoraggio longitudinale della fluorescenza e interventi avversivi

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

Una piattaforma microfluidica per Imaging longitudinale in Caenorhabditis elegans

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Electrotaxis microfluidica-based per On-demand analisi quantitativa della