Name: simple microfluidic devices for in vivo imaging of c. elegans, drosophila and zebrafish
Uploaded: 2012-09-30T14:00:00
Description: Watch this Scientific Journal Video about Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish at JoVE.com

Vi takker Dr. Krishanu Ray til Drosophila aktier, Tarjani Agarwal for at opretholde en Drosophila bur, Peter Juo for nuIs25 og CGC for C. elegans stammer. SPK lavet jsIs609 i Michael Nonet laboratorium. Vi takker Arpan Agnihotri (BITS Pilani) for hans hjælp i time-lapse imaging af mitokondrier transport af jsIs609 dyr i mikrofluidenheder. Vi er taknemmelige for Dr. Vatsala Thirumalai og Surya Prakash for at forsyne os med zebrafisk embryoner. Vi takker Dr. Krishna og CIFF på NCB&#39;erne til brug af spinning disc konfokal mikroskop støttet af Ministeriet for Videnskab og Teknologi-Centre for nanoteknologi (nr. SR/55/NM-36-2005). Vi takker også Kaustubh Rau, V. Venkatraman og Chetana Sachidanand for drøftelserne. Dette arbejde blev finansieret af DBT postdoktoralt stipendium (SM), DST fast-track ordning (SM) og en DBT tilskud til (SPK). SA blev støttet af DST og CSIR tilskud til SPK.

1. SU8 Master Fabrication <ol><li> Designe de mikrofluide strukturer ved hjælp Clewin software og udskrive det ved hjælp af 65.024 DPI laser kortplotter med minimal træk størrelse på 8 um på printkortet film. </li><li> Ren 2 cm x 2 cm silicium wafers med nativt oxid i 20% KOH i 1 min og skylles i demineraliseret vand, en wafer hver for strømmen lag og dets tilsvarende kontrollag. </li><li> Føntørre stykkerne med nitrogengas og dehydrere på en varm plade ved 120 ° C i 4 timer. Lad brikkerne til at køle ned ...

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PDMS mikrofluidenheder er optisk transparent derfor kan anvendes til høj opløsning in vivo-afbildning af en transparent / translucent model organisme. Vores design er egnet til stor forstørrelse spatio-temporale billeddannelse af cellulære og subcellulære begivenheder i intakte levende dyr. Microfabrication med bløde litografiteknikker tillader nem manipulation af enhedens dimensioner for forskellige størrelser af modelorganismer. Indretninger af forskellige størrelser, fremstilles til forskellige C...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> Siliciumskiver </td><td> University wafer </td><td> 150 mm (100) Mech Grade SSP Si </td><td></td></tr><tr><td> Clewin Software </td><td> WieWeb software </td><td> Version 2,90 </td><td></td></tr><tr><td> Laser plotter </td><td> Fine Line Imaging </td><td> 65.024 DPI </td><td></td></tr><tr><td> HMDS </td><td> Sigma-Aldrich </td><td> 440.191-100ML </td><td></td></tr><tr><td> SU8 </td><td> Microchem </td><td> SU8-2025, SU8-2050 </td><td></td></tr><tr><td> Udvikler </td><td> Microchem </td><td> SU8 Udvikler </td><td></td></tr><tr><td> Silan </td><td> Sigma-Aldrich </td><td> 448931-10G </td><td></td></tr><tr><td> PDMS </td><td> DOW CORNING </td><td> Sylgard 184 </td><td></td></tr><tr><td> UV-lampe </td><td> Oriel </td><td> 66.943 </td><td> 200W Hg Oriel Light </td></tr><tr><td> Varmluftsovn </td><td> Ultra Instruments </td><td> Skræddersyet </td><td> Indstillet på 50 ° C </td></tr><tr><td> Varmeplade </td><td> IKA Laboratory Equipment </td><td> 3810000 </td><td> <a href="http://www.ika.com" target="_blank">http://www.ika.com</a> </td></tr><tr><td> Plasma renere </td><td> Harrick Plasma </td><td> PDC-32G </td><td></td></tr><tr><td> Spinner </td><td> Halvleder Produktionssystemer </td><td> SPIN150-NPP </td><td> <a href="http://www.SPS-Europe.com" target="_blank">www.SPS-Europe.com</a> </td></tr><tr><td> Dækglas </td><td> Gold Seal </td><td> 22 X 22 mm, nr. 1 tykkelse </td><td></td></tr><tr><td>C. elegans </td><td> Caenorhabditis Genetics Center (CGC) </td><td> e1265, ayIs4 </td><td></td></tr><tr><td> Drosophila </td><td> Bloomington </td><td> P {chaGAL4} / cyo, UAS-syt.eGFP </td><td></td></tr><tr><td> Zebrafisk </td><td> Indian vildtype </td><td> Vildtype </td><td></td></tr><tr><td> Tygon slange </td><td> Sigma </td><td> Z279803 </td><td></td></tr><tr><td> Micro nål </td><td> Sigma </td><td> Z118044 </td><td> Skæres i 1 cm stykker </td></tr><tr><td> 3-vejs stophane </td><td> Sigma </td><td> S7521 </td><td></td></tr><tr><td> Harris puncher </td><td> Sigma </td><td> Z708631 </td><td></td></tr><tr><td> Komprimeret nitrogengas </td><td> Lokale gasleverandør </td><td></td><td> Brug en regulator til at styre trykket </td></tr><tr><td> Stereomikroskop &lt;/ Td&gt; <td> Nikon </td><td> SMZ645 </td><td></td></tr><tr><td> Konfokalt mikroskop </td><td> Andor &amp; Olympus </td><td> Yokogawa rotationsskiveapparatet konfokalt mikroskop </td><td></td></tr><tr><td> ImageJ </td><td> National Institutes of Health </td><td> <a href="http://www.rsbweb.nih.gov/ij" target="_blank">www.rsbweb.nih.gov / ij</a> </td><td> Java-baseret billedbehandling program </td></tr></tbody></table>

simple microfluidic devices for in vivo imaging of c. elegans, drosophila and zebrafish

Micro fabrikerede strømningstekniske giver et tilgængeligt mikro-miljø for in vivo undersøgelser af små organismer. Simple fremstillingsprocesser er tilgængelige for mikrofluide enheder ved hjælp af bløde litografiteknikker 1-3. Mikrofluide anordninger er blevet anvendt til subcellulære billeddannelse 4,5, in vivo laser mikrokirurgi 2,6 og cellulær billeddannelse 4,7. In vivo-billeddannelse kræver immobilisering af organismer. Dette er opnået ved hjælp af sugning 5,8, tilspidsede kanaler 6,7,9, deformerbare membraner 2-4,10, suge med yderligere afkøling 5, bedøvende gas 11, temperaturfølsomme geler 12, cyanoacrylat lim 13 og anæstetika, såsom levamisol 14 , 15. Almindeligt anvendte anæstetika indflydelse synaptisk transmission 16,17 og er kendt for at have skadelige virkninger for sub-cellulær neuronal transport 4. I denne study vi påvise en membran baseret poly-dimethyl-siloxan (PDMS) enhed, der tillader anæstetisk fri immobilisering af intakte genetiske modelorganismer såsom Caenorhabditis elegans (C. elegans), Drosophila-larver og zebrafisk larver. Disse modelorganismer er egnet til in vivo-undersøgelser i mikrofluidenheder på grund af deres små diametre og optisk transparente eller translucente organer. Organ diametre i området fra ~ 10 um til ~ 800 um for tidlige larvestadier af C. elegans og zebrafisk larver og kræver mikrofluidenheder af forskellig størrelse for at opnå fuldstændig immobilisering for høj opløsning time-lapse billeddannelse. Disse organismer er immobiliseret ved hjælp af tryk fra komprimeret nitrogengas gennem en væskesøjle og afbildet under anvendelse af et omvendt mikroskop. Dyr frigives fra fælden vende tilbage til normal locomotion indenfor 10 min. Vi viser fire ansøgninger om time-lapse imaging i C. elegansnemlig imaging mitokondriel transport i neuroner, præ-synaptisk vesikel transport i en transport-defekt mutant, glutamat receptor transport og Q neuroblast celledeling. Data opnået fra sådanne film viser, at mikrofluid immobilisering er et nyttigt og præcise middel til at opnå in vivo data af cellulære og subcellulære begivenheder sammenlignet med bedøvede dyr (fig. 1J og 3C-F 4). Enhedens dimensioner blev ændret for at give tid-lapse imaging af forskellige stadier af C. elegans, første stadium Drosophila larver og zebrafisk larver. Transport af vesikler markeret med synaptotagmin mærket med GFP (syt.eGFP) i sensoriske neuroner viser rettet bevægelse af synaptiske vesikel markører udtrykt i cholinerge sensoriske neuroner i intakt 1:a stadie Drosophila larver. En lignende indretning er blevet anvendt til at udføre time-lapse billeddannelse af hjerteslag i ~ 30 timer efter befrugtning (HPF)zebrafisk larver. Disse data viser, at de simple indretninger vi har udviklet, kan anvendes til en række forskellige modelsystemer til at studere adskillige cellebiologiske og udviklingsmæssige fænomener in vivo.

Immobiliseringen indretning er et dobbeltlag PDMS block fremstilles ved binding to lag: a flow layer (Layer 1) og et kontrol-lag (lag 2) som vist i figur 1.. Den vigtigste fælde er forbundet til en nitrogengas cylinderen gennem en regulator og en 3-vejs stophane til at anvende nødvendigt (3-14 psi) på membranen gennem en væskesøjle (figur 1A). Den afbøjede membran immobiliserer C. elegans, Drosophila og zebrafisk larver i strømningskanalen udformet med forskellige dimens...

Watch this Scientific Journal Video about Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish at JoVE.com

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

En simpel mikrofluid enhed er udviklet til at udføre bedøvelse free<em&gt; In vivo</em&gt; Billeddannelse af<em&gt; C. elegans</em&gt;, Intakt<em&gt; Drosophila</em&gt; Larver og zebrafisk larver. Enheden benytter en deformerbar PDMS membran til at immobilisere disse modelorganismer for at udføre tid bortfalder billeddannelse af mange forskellige processer, såsom puls, celledeling og subcellulært neuronal transport. Vi demonstrerer brugen af ​​denne enhed og viser eksempler på forskellige typer af data indsamlet fra forskellige modelsystemer.

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

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A Microfluidic Technique to Probe Cell Deformability

En Mikrofluid Teknik til sonde Cell deformerbarhed

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Layer-by-lags kollagenaflejring i mikrofluidapparater for Microtissue Stabilisering

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

Electrotaxis Studier af lungecancerceller ved hjælp af en Multichannel Dual-elektriske-felt Mikrofluid Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Multi-trins Variabel Højde Fotolitografi for med ventiler Multilayer mikrofluidenheder

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Fremstilling af Tre-dimensionel papir-baserede mikrofluidenheder for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

Polydimethylsiloxan-polykarbonat mikrofluidenheder for Cell Migrationsstudier Under Perpendicular Kemiske og Oxygen farveforløb

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

En simpel og skalerbar Fabrication Metode til Økologisk Elektroniske enheder om tekstiler

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

En mikrofluid Platform for langsgående Imaging i Caenorhabditis elegans

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