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

Summary

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.

Abstract

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 μm to ~800 μm 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.

Protocol

1. SU8 Master Fabrication

1. Design the microfluidic structures using Clewin software and print it using 65,024 DPI laser plotter with minimum feature size of 8 μm on circuit board film.
2. 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.
3. Blow dry the pieces with nitrogen gas and dehydrate on a hot plate at 120 °C for 4 hr. Allow the pieces to cool down to room temperature before proceeding to the next step.
4. Place silicon pieces one at a time on the spinner chuck and turn on the vacuum. Cover th....

Results

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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Name of the reagent	Company	Catalogue number	Comments (optional)
Silicon wafers	University wafer	150 mm (100) Mech Grade SSP Si
Clewin Software	WieWeb software	Version 2.90
Laser plotter	Fine Line Imaging	65,024 DPI
HMDS	Sigma-Aldrich	440191-100ML
SU8	Microchem	SU8-2025, SU8-2050
Developer	Microchem	SU8 Developer
Silane	Sigma-Aldrich	448931-10G
PDMS	Dow corning	Sylgard 184
UV lamp	Oriel	66943	200W Hg Oriel Light
Hot air oven	Ultra Instruments	Custom made	Set at 50 °C
Hot plate	IKA Laboratory Equipment	3810000	http://www.ika.com
Plasma cleaner	Harrick Plasma	PDC-32G
Spinner	Semiconductor Production Systems	SPIN150-NPP	www.SPS-Europe.com
Glass cover slip	Gold Seal	22 X 22 mm, No. 1 thickness
C. elegans	Caenorhabditis Genetics Center (CGC)	e1265, ayIs4
Drosophila	Bloomington	P{chaGAL4}/cyo, UAS-syt.eGFP
Zebrafish	Indian wild type	Wild type
Tygon tube	Sigma	Z279803
Micro needle	Sigma	Z118044	Cut into 1 cm pieces
3-way stopcock	Sigma	S7521
Harris puncher	Sigma	Z708631
Compressed nitrogen gas	Local Gas supplier		Use a regulator to control the pressure
Stereo microscope	Nikon	SMZ645
Confocal microscope	Andor & Olympus	Yokogawa spinning disc confocal microscope
ImageJ	National Institutes of Health	www.rsbweb.nih.gov/ij	Java based image processing program