Authors: Kwanghun Chung, Matthew Crane & Hang Lu
Microscopy, phenotyping, and visual screens are frequently applied to model organisms in combination with genetics. Although widely used, these techniques for multicellular organisms have mostly remained manual and low-throughput. We recently reported a fully automated microsystem for sorting and imaging of C. elegans (Fig. 1a) [ref 1]. This protocol describes how to fabricate microfluidic devices and how to operate the system. The microfluidic device is fabricated using conventional soft-lithography by molding a silicone material (polydimethylsiloxane or PDMS) to the masters created using microfabrication methods. To create the negative masters, photoresists are deposited on a silicon wafer, and patterned to create micron-scale structures using optical masks; a separate master is created for each of the three layers of the final device. The silicone components are aligned and bonded to one another to create the final device. One of the significant advantages of these devices is that they can be used with any microscope, camera and stage of choice. Additionally, the device can be easily scaled for C. elegans of different sizes as well as for other small model organisms. The devices are inexpensive to create, and once the masters have been fabricated will costly only pennies for the materials per device.
The general setup to operate the microchip is illustrated in Fig. 1b.
The following details the procedure for creating microfluidic masters, devices and running the experiments. Devices can be prepared ahead of time.
I. Microfluidic device fabrication:
A. Mold fabrication – the mold wafers should be fabricated in a Cleanroom environment if possible for a high yield in device fabrication. Follow Cleanroom protocols and safety precautions.
B. PDMS device fabrication
II. C. elegans culture and sample preparation
III. System operation
Fabricating the masters will take several hours in a cleanroom by a trained user, and fabricating the PDMS devices typically requires a few hours per batch. Preparing the synchronized animals will take less than half an hour, and should be done sufficiently in advance of using the system so that the animals can grow to the correct age. Setting up the system to run a batch of animals will take ~30 minutes and operation time will depend on the application and the number of animals to be screened and sorted.
Reflow: The reflow process for the AZ photoresist is critical. This process allows the rectangular channels originally fabricated on the master to transform into rounded channels and allow the valves to completely close. The rounded profile of the channel can be confirmed by measuring height profile of the channel using a profilometer.
Silane: Depositing a thin layer of silane on the silicon master prevents the PDMS from adhering to the photoresist structures. Without this, the photoresist will be removed from the wafer when the PDMS device is peeled off. Silane allows the master to be used many times and minimizes the total cost.
Bonding: Bonding the different PDMS layers must be done carefully and with great precision. By exposing the PDMS to oxygen plasma, it is possible to activate the surface so that it will bond irreversibly to glass or another PDMS layer. If the PDMS is exposed to plasma for too short or too long, however, it will fail to create a strong bond. Once activated, the layers must be bonded within two minutes.
Alignment: Accurate alignment is critical for robust device operation. Typically we perform alignment by hand using a stereomicroscope. With practice, it is possible to get reliable alignment with 5-micron precision.
Pressure: The pressure for injecting the animal suspension and actuating on-chip valves will likely have to be fine-tuned at the beginning of every experiment due to individual variations in the devices as they are manufactured.
Leakage: If the needles used to punch holes in the device are not sufficiently sharp, they can tear the PDMS. When the device is subsequently used, the tears may prevent a good seal from forming, and cause leakage. Make sure that the holes in each device are punched cleanly. Inspect the devices under a microscope once assembled.
Silane treatment: If the PDMS is hard to be peeled off from the wafer and/or removes the photoresist, this is likely a result of ineffective silane treatment. This is possily a result of either silane that has been left open and exposed to the ambient for an extended period of time, or an insufficiently strong vacuum. Test the vacuum desiccator to insure that it is pulling a strong vacuum, and if the silane is believed to be significantly oxidized, purchase new silane.
Bonding issue: If the bonds between layers in the microfluidic device do not appear sufficiently strong, either causing leakage or is non-conforming, there could be several culprits. If the layers thermo-bonded are delaminating, it is possible that one or all of the layers are overcured. Repeat the fabrication process but bake the thin, spin-coated layer for a few minutes less. If the bond between layers that are plasma activated is weak, there could be several causes. If the plasma chamber is not as clean, the plasma activation process may not work as properly. Clean according to manufacturer’s recommendations and test by bonding blank pieces of PDMS to each other or to glass. Properly bonded PDMS should be very difficult to remove. Additionally, evaluate the protocol and be sure that the bonding surfaces are clean and are not touched by gloves or tweezers. You may need to vary the plasma treatment time to find an optimal condition.
Alignment: Proper alignment is critical. If the device is offset slightly the partial closure valves will operate slightly differently and change the hydrodynamic resistance. To facilitate good alignment, we use a ring-LED light source connected to a stereomicroscope, and perform bonding at high magnification. Your Cleanroom may have equipment (e.g. aligners) that may facilitate these processes.
Leakage: Leakage typically results from one of two sources: bonding issues, or a poor seal between the device and connected tubing/pins. If the cause seems to be bonding, see the bonding troubleshooting entry. If the problem appears to be a result of a bad seal between the connected pins and the device, it could be as a result of either incorrectly sized pins or a tearing around the holes in the device. The silicone material of the device is used to form a strong seal around pins and tubing connected to it. When the holes in the device are punched, however, it is possible to create small tears around the hole that prevent a good seal from forming. If this is the case, make sure to take care when punching holes, and possibly sharpen the pins used for punching holes in the device.
Cooling: If animals do not appear to be immobilized, the temperature on chip may not be sufficiently low. To find the optimal temperature for the coolant, measure the coolant temperature before arriving on chip and optimize the power input to the Peltier. An optimal starting point is ~3 °C, but the exact condition also depends on the room temperature and the flow rates of the coolant and the animal suspension.
Device clogging: When operating the device, occasionally multiple animals or a piece of debris may clog the channels. Increase the pressure that drives the flow to push the animals and debris out of the channel. Some animals may be lost in that process, so depending on whether more false positives or false negatives can be tolerated, open either the sorted sample outlet or the waste outlet.
We acknowledge Jeffrey Stirman for assistance in designing the injection manifold and troubleshooting.
Figure 1: Schematics of the system.
Figure 2a,b: AutoCAD design of the worm imaging device.
Figure 2c: AutoCAD design of the worm imaging device.
Figure 3: AutoCAD design of the filtering device.
Figure 4: PDMS device fabrication process for the imaging/sorting device.
Figure 5: Schematic diagrams summarizing the valve control sequence in the worm-sorting process.
Automated on-Chip Rapid Microscopy, Phenotyping, and Sorting of C. elegans, Kwanghun Chung, Matthew M Crane, and Hang Lu, Nature Methods 5 (7) 637 - 643 22/06/2008 doi:10.1038/nmeth.1227
Kwanghun Chung, School of Chemical and Biomolecular Engineering, Georgia Institute of Technology
Matthew Crane, Interdisciplinary Program in Bioengineering, Georgia Institute of Technology
Hang Lu, School of Chemical and Biomolecular Engineering, Interdisciplinary Program in Bioengineering, Georgia Institute of Technology
Source: Protocol Exchange (2009) doi:10.1038/nprot.2009.105. Originally published online 20 May 2009.