We thank Dr. Krishanu Ray for Drosophila stocks, Tarjani Agarwal for maintaining a Drosophila cage, Peter Juo for nuIs25 and CGC for C. elegans strains. SPK made jsIs609 in Michael Nonet's laboratory. We thank Arpan Agnihotri (BITS Pilani) for his help in time-lapse imaging of mitochondria transport of jsIs609 animals in microfluidic devices. We are grateful to Dr. Vatsala Thirumalai and Surya Prakash for providing us with zebrafish embryos. We thank Dr. Krishna and CIFF at NCBS for use of the spinning disc confocal microscope supported by the Department of Science and Technology- Centre for Nanotechnology (No. SR/55/NM-36-2005). We also thank Kaustubh Rau, V. Venkatraman and Chetana Sachidanand for discussions. This work was funded by the DBT post-doctoral fellowship (S.M.), DST fast-track scheme (S.M.) and a DBT grant to (S.P.K.). S.A. was supported by DST and CSIR grants to SPK.

1. SU8 Master Fabrication
<ol>
 <li>Design the microfluidic structures using Clewin software and print it using 65,024 DPI laser plotter with minimum feature size of 8 &mu;m on circuit board film. </li>
 <li>Clean 2 cm X 2 cm silicon wafers with native oxide in 20% KOH for 1 min and rinse in deionized water; one wafer each for the flow layer and its corresponding control layer. </li>
 <li>Blow dry the pieces with nitrogen gas and dehydrate on a hot plate at 120 &deg;C for 4 hr. Allow the pieces to cool down to ro...

<ol>
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No conflicts of interest declared.

PDMS microfluidic devices are optically transparent therefore can be used for high resolution in vivo imaging of any transparent/translucent model organism. Our design is suitable for high magnification spatio-temporal imaging of cellular and sub-cellular events in intact live animals. Microfabrication using soft lithography techniques allows easy manipulation of device dimensions for various sizes of model organisms. Devices of various sizes are fabricated for different stages of C. elegans, Drosop...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Silicon wafers</td>
 <td>University wafer</td>
 <td>150 mm (100) Mech Grade SSP Si</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Clewin Software</td>
 <td>WieWeb software</td>
 <td>Version 2.90</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Laser plotter</td>
 <td>Fine Line Imaging</td>
 <td>65,024 DPI</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HMDS</td>
 <td>Sigma-Aldrich</td>
 <td>440191-100ML</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>SU8</td>
 <td>Microchem</td>
 <td>SU8-2025, SU8-2050</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Developer</td>
 <td>Microchem</td>
 <td>SU8 Developer</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Silane</td>
 <td>Sigma-Aldrich</td>
 <td>448931-10G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PDMS</td>
 <td>Dow corning</td>
 <td>Sylgard 184</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>UV lamp</td>
 <td>Oriel</td>
 <td>66943</td>
 <td>200W Hg Oriel Light</td>
 </tr>
 <tr>
 <td>Hot air oven</td>
 <td>Ultra Instruments</td>
 <td>Custom made</td>
 <td>Set at 50 &deg;C</td>
 </tr>
 <tr>
 <td>Hot plate</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 cleaner</td>
 <td>Harrick Plasma</td>
 <td>PDC-32G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Spinner</td>
 <td>Semiconductor Production Systems</td>
 <td>SPIN150-NPP</td>
 <td><a href="http://www.SPS-Europe.com" target="_blank">www.SPS-Europe.com</a></td>
 </tr>
 <tr>
 <td>Glass cover slip</td>
 <td>Gold Seal</td>
 <td>22 X 22 mm, No. 1 thickness</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>C. elegans</td>
 <td>Caenorhabditis Genetics Center (CGC)</td>
 <td>e1265, ayIs4</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Drosophila</td>
 <td>Bloomington</td>
 <td>P{chaGAL4}/cyo, UAS-syt.eGFP</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Zebrafish</td>
 <td>Indian wild type</td>
 <td>Wild type</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tygon tube</td>
 <td>Sigma</td>
 <td>Z279803</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micro needle</td>
 <td>Sigma</td>
 <td>Z118044</td>
 <td>Cut into 1 cm pieces</td>
 </tr>
 <tr>
 <td>3-way stopcock</td>
 <td>Sigma</td>
 <td>S7521</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Harris puncher</td>
 <td>Sigma</td>
 <td>Z708631</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Compressed nitrogen gas</td>
 <td>Local Gas supplier</td>
 <td>&nbsp;</td>
 <td>Use a regulator to control the pressure</td>
 </tr>
 <tr>
 <td>Stereo microscope</td>
 <td>Nikon</td>
 <td>SMZ645</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Confocal microscope</td>
 <td>Andor &amp; Olympus</td>
 <td>Yokogawa spinning disc confocal microscope</td>
 <td>&nbsp;</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 based image processing program</td>
 </tr>
 </tbody>
</table>

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.

The immobilization device is a bilayer PDMS block fabricated by bonding two layers: a flow layer (Layer 1) and a control layer (Layer 2) as shown in Figure 1. The main trap is connected to a nitrogen gas cylinder through a regulator and a 3-way stop cock to apply necessary (3-14 psi) pressure onto the membrane through a liquid column (Figure 1A). The deflected membrane immobilizes C. elegans, Drosophila or zebrafish larvae in the flow channel designed with different dim...

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

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.

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 ...

simple-microfluidic-devices-for-vivo-imaging-c-elegans-drosophila

Research

JoVE Journal

Bioengineering

17.0K Görüntüleme.  NCBS-TIFR. Basit bir mikroakışkan cihaz anestezik ücretsiz gerçekleştirmek için geliştirilmiştir In vivo görüntüleme C. elegans, Intakt Drosophila Larva ve zebrafish larva. Cihaz, kalp ritmi, hücre bölünmesi ve hücre altı nöronal ulaşım gibi birçok süreçlerin zaman atlamalı görüntüleme gerçekleştirmek için bu model organizmalar hareketsiz bir deforme PDMS membran kullanır. Biz bu cihazın kullanımını gö...