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 the silicon surfaces with ~20 μl hexamethyl-disilazane (HMDS) and coat them using a SPIN150 spinner at 500 rpm for 5 sec followed by 3,000 rpm for 30 sec.
5. Cover silicon wafers completely with ~1.5 ml of SU8-2025 (http://www.microchem.com/Prod-SU82000.htm) and coat wafers using SPIN150 spinner at 500 rpm for 5 sec followed by 2,000 rpm for 30 sec. This spinning protocol produces a photoresist thickness of ~40 μm that is suitable for the control and flow layers for early larval stages of C. elegans.
6. Spin ~1.5 ml SU8-2050 using SPIN150 spinner at 500 rpm for 5 sec followed by 2,000 rpm for 30 sec to obtain a photoresist thickness of ~80 μm that is suitable for flow layer fabrication for late larval stages of C. elegans, basal flow layer for Drosophila/zebrafish larvae and their corresponding control layers.
7. Place the silicon pieces on hot plate with the SU8 coated layer on top. Soft bake the coated silicon pieces at 65 °C for 1 min followed by 95 °C for 10 min. Allow the pieces to cool down to room temperature before proceeding to the next step.
8. Place the soft-baked silicon pieces on the exposure stage with SU8-2025 coated surface on the top facing the UV lamp. Use the photo mask with design I (L1, Figure 1B) and design II (L2, Figure 1B) for the flow layer and control layer pattern respectively for early larval stages of C. elegans. Place the photo mask on top of the SU8 layer and ensure the mask is flat against the coated layer. Open the shutter of the UV lamp and expose the soft-baked SU8 wafers to 200 Watt UV lamp through the photo mask for 15 sec.
9. Use the photo mask with design I (L1, Figure 1B) and design II (L2, Figure 1B) on SU8-2050 coated pieces (prepared in step 1.6) to fabricate the flow layer and control layer for late larval stages of C. elegans. Use the photo mask with the design I (L1, Figure 1C) and design II (L2, Figure 1C) for the basal flow layer and control layer respectively for Drosophila/zebrafish larvae on wafers coated with SU8-2050 (prepared in step 1.6). Expose the SU8 surfaces through the photo mask to the 200 Watt UV lamp for 15 sec.
10. Place the exposed silicon pieces on a hot plate with the coated layer on top. Post bake the wafers at 65 °C for 1 min followed by 95 °C for 10 min. Allow the pieces to cool down before proceeding to the next step.
11. Develop the pieces using the SU8 developer solution for 20 min. Rinse the pieces in Iso-Propyl Alcohol (IPA) and blow dry using nitrogen gas.
12. Place the silicon pieces in a desiccator with the SU8 pattern on top. Coat the pieces with 50 μl of tricholoro (1H,1H,2H,2H-perfluorooctyl) silane vapor in a desiccator for 2 hr.

Precautions: Take safety precautions for chemical handling during KOH treatment, photoresist coating and developing. Silane vapor coating should be performed inside a desiccator in a ventilated area. Protect SU8 resist from over exposure to light. Use protective eyewear with an UV light source.

2. PDMS Mold Fabrication

1. Prepare 10:1 PDMS by combining 50 g of Sylgard 184 base with 5 g of curing agent in a plastic cup. Mix the content manually for ~3 min. Degas the mixture inside a desiccator to remove all air bubbles formed during mixing.
2. Pour a 5 mm thick PDMS mixture over the silicon wafers with SU8 pattern of design II, for control layer, gently to avoid formation of air bubbles. In case bubbles are formed during pouring, degas the PDMS on the SU8 pattern in low vacuum to remove all the bubbles.
3. Place the silicon wafers with SU8-2025 pattern of design I, for the flow layer, on spinner chuck and turn on the vacuum to hold them. Cover the wafers with ~1 ml of PDMS mix and coat the wafers using SPIN150 spinner at 500 rpm for 5 sec followed by 1,500 rpm for 30 sec.
4. Coat ~1 ml PDMS mix on silicon wafers with the 80 μm SU8-2050 pattern of design I (L1, Figure 1B), for the flow layer for late larval stages of C. elegans using the SPIN150 spinner at 500 rpm for 5 sec followed by 1,000 rpm for 30 sec. Coat a thick layer of PDMS mix on the silicon pieces with SU8-2050 pattern of flow layer design I (L1, Figure 1C) for Drosophila/zebrafish larvae, using SPIN150 spinner at 500 rpm for 35 sec.
5. Bake PDMS coated silicon pieces of design I and wafers with corresponding design II for control layer containing PDMS for C. elegans and/or Drosophila/zebrafish larvae in a hot air convection oven at 50 °C for 6 hr.
6. Cut out the PDMS piece from the silicon substrate containing the SU8 pattern II for C. elegans and/or Drosophila/zebrafish larvae using a sharp blade. Punch a small hole of ~1 mm diameter on top of the reservoir connecting the main trap in the PDMS mold using a Harris puncher.
7. Place the punched PDMS block (containing the 40 μm thick mold for control layer L2) on a plastic tray, with the molded side of the control layer facing up. Place the PDMS coated silicon wafer containing the 40 μm thick SU8 design I on the same tray, with the PDMS coated surface facing up. Insert the tray inside the chamber of plasma cleaner and turn on the vacuum for 2 min. Subsequently, turn on the plasma power and lower the chamber pressure until the chamber turns light pink in color. Expose both surfaces to 18 Watt air plasma under low vacuum for 2 min.
8. Place the punched PDMS block removed from the 80 μm thick SU8 master containing pattern II for late larval stages of C. elegans or Drosophila/zebrafish larvae on a plastic tray, with the molded side of the control layer facing up. Place the corresponding baked PDMS layer spin coated on the silicon wafer containing 80 μm thick design I for later C. elegans stages or Drosophila/zebrafish larvae simultaneously on the same tray with the flat PDMS coated surface facing up. Use the same protocol mentioned above to expose them to air plasma.
9. Place the two plasma treated surfaces for C. elegans and/or Drosophila/zebrafish larvae together with gentle pressure and bake them in hot air convection oven at 50 °C for 2 hr.
10. Cut out the bonded devices from the silicon substrate with SU8 design I for C. elegans and/or Drosophila/zebrafish larvae. Punch access holes at the inlet and outlet reservoirs of the flow channel.
11. Place the bonded PDMS mold on a plastic tray for C. elegans, with the molded side of the flow layer design facing up. Clean glass cover slip (22 X 22 mm, No. 1 thickness) and place it on the same plastic tray. Insert the tray containing PDMS mold and glass cover slip inside the plasma chamber. Expose the bottom PDMS surface of the device and glass cover slip to 18 Watt air plasma at low pressure for 2 min. Place the two plasma treated surfaces with gentle pressure and bake them in hot air convection oven at 50 °C for 2 hr.
12. Store the device in a clean environment for future use.

3. Additional Steps for Drosophila/Zebrafish Device

1. Expose clean glass surface of size 2 cm X 2 cm with 50 μl of tricholoro(1H,1H,2H,2H-perfluorooctyl) silane vapor in a desiccator for 2 hr.
2. Spin ~1 ml of 10:1 PDMS mix prepared in step 2.1 on the silanized glass surface using SPIN150 spinner at 500 rpm for 35 sec. Bake the PDMS coated glass substrates in a hot air oven at 50 °C for 6 hr with the coated surface facing up. Allow the PDMS layer to cool down to room temperature before proceeding to the next step.
3. Punch the coated layer using a custom built sharp metal puncher at the middle of the PDMS, to form the PDMS spacer layer. The shape of the metal puncher is designed similar to the flow design for Drosophila/zebrafish (L1, Figure 1C).
4. Expose the PDMS spacer layer and the single-bonded block (flow layer and control layer, made in step 2.9 for Drosophila/zebrafish larvae) to 18 Watt air plasma using plasma cleaner at low vacuum. Place the two plasma treated surfaces together and bake them in a hot air oven at 50 °C for 2 hr.
5. Cut the bonded device from the glass, punch access holes at the inlet and outlet reservoirs and bond it to a glass cover slip (22 X 22 mm, No. 1 thickness) using a plasma cleaner as mentioned in step 2.11. This would produce an immobilization device for first instar Drosophila larvae with a channel height of ~500 μm. For zebrafish larvae, duplicate the PDMS spacer layer fabrication process with an additional bonding step. The dual layer of PDMS-S produces a channel height of 900 μm for 30 hfp zebrafish larvae.

Precautions: Avoid dust particles during device fabrication. Two plasma cleaned surfaces need to be completely dust free for proper bonding. Store the devices in a desiccator in situations where a special clean room is not available in order to minimize accumulation of dust particles in devices.

4. Using PDMS Membrane

1. Connect one end of a micro-flex tube (inner diameter ~1.6 mm, outer diameter ~4.8 mm) to a pressurized nitrogen gas supply regulator and the other end to the main trap reservoir through an 18 gauge needle (outer diameter ~1.25 mm) glued to the tube. A 3-way stopcock used in the middle of the tube connection allows application or release of pressure on the membrane.
2. Fill the tube connected to the PDMS device with a 10 cm column of distilled water before connecting it to the reservoir of the main trap of a C. elegans and/or Drosophila/zebrafish larvae device.
3. Turn the valve of the nitrogen supply to read 14 psi and monitor the membrane of the main trap deflecting towards the flow channel at low magnification of an inverted microscope. Length of the water column in the micro-flex tube is compressed whenever the 3-way stopcock is open. Edges of the deflected membrane are visible in transmitted light (Figure 1I and 1J). Membrane deflection causes displacement of dust particles/bubbles under the PDMS membrane and pushes them to the channel boundaries.
4. Wait till the liquid front fills the microchannel completely without any trapped air.
5. Release the pressure by using the 3-way stopcock to relax the membrane to its resting position.

5. Inserting C. elegans, Drosophila and Zebrafish Larvae into the Device and Immobilizing Them Under the Flexible PDMS Membrane

1. Fill the flow channel with M9 buffer [3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl, 1 ml 1 M MgSO₄, H₂O to 1 L, sterilize by autoclaving] for C. elegans 18 or 1X PBS [8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, 0.24 g KH₂PO₄, H₂O to 1 L, sterilize by autoclaving] for Drosophila/zebrafish larvae 19 for 10 min before an experiment.
2. Locate a single C. elegans of required stage on a NGM plate ¹⁸ (or first instar Drosophila larvae from agar egg plate or manually dechorionated 30 hpf zebrafish larvae in liquid medium ²⁰) using a low magnification stereo microscope. Pick a single animal using a small volume of buffer solution into a micro tip. Use a micro tip with its end cut to accommodate the larger Drosophila or zebrafish larvae in buffer.
3. Push the single organism through the flow channel inlet. Adjust the pipetting pressure to position the individual animal under the main trap.
4. Increase the pressure of the PDMS membrane slowly to position the animal against the channel boundary and immobilize it with 14 psi pressure for C. elegans, 7 psi for Drosophila and 3 psi for zebrafish larvae.
5. Position the immobilized animal at the center of the microscopic field of view. Use an inverted microscope at desired settings for high resolution bright field or fluorescence imaging. Acquire single or time-lapse fluorescence images at predefined frame rate.
6. Release the trap pressure and monitor the locomotion of the animal for 5-10 min at low magnification.
7. Flush the animal and insert a fresh animal to repeat the above steps.
8. Wash all the animals from the waste reservoir using distilled water applied using a syringe. Dry the channel by pushing air using a syringe for future reuse.

Precautions: Animals requiring higher pipetting pressure to flow inside the main channel tend to show poor locomotion/health after release from the trap and should be avoided for imaging. Clean the flow channel for any trace of buffer to prevent clogging of channel by crystals. If oil is used for high resolution imaging, clean the glass cover slip to be able to maintain good signal to noise ratio for subsequent experiments.

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