Since oscillations are a phenomena that require observation at a small scale (level of very few cells or even single cells), we designed microfluidic chambers in order to load our cells and observe oscillations.
We used standard soft lithography techniques to generate microfluidic channels. In brief, SU8 photoresist was spin coated on a silicon wafer to the height of 50μm. The desired pattern was generated using maskless lithography. A silicone elastomer was added with its curing agent in 10:1 volume ratio and poured over the micro-mold. After 4 hours of incubation at 65oC, PDMS was peeled off the silicon wafer and inlet and outlet holes were punched. The surface of PDMS and a cover slip were modified using a plasma cleaner and microfluidic channels were created by bonding the two together.
Cell culture grown overnight containing LB media was loaded directly into the channels, or was diluted 1:50 and flowed through the main channel. Air bubbles were introduced at the T-junction. Hence droplets of water surrounded by air on either side were created in the channel. Cells trapped in these droplets were studied under a fluorescence microscope using a 40x objective.
Two types of fluorescence microscopes were used for our studies. The first had a mercury lamp as the light source, with a black and white camera. We were able to observe fluorescence in cells that were constitutively expressing GFP, containing the reporter under the PhlF repressible promoter. The system was on a high copy, and PhlF was not being produced, thereby rendering the promoter to be constitutively ON.
However, for the purpose of our further work, we used the Etaluma Lumascope S40 fluorescence microscope, which had an LED light source, with the appropriate excitation, emission and dichroic filters for observing our GFP levels. Loaded in the channel, our cells showed fluorescence, and a sample image is shown below –
Maintaining Flow Rate
In order to visualize the oscillations, we needed to maintain a flow rate in the system. This is because as cells would grow, they would produce toxic compounds inside the chamber, and also reach stationary phase, which would cause a slowdown in protein production, which could kill the oscillations. Flow rate maintenance is something that commonly needs to be done in microfluidic chambers where oscillations are required to be seen, but such sophisticated systems for flow rate maintenance are extremely costly and require specialized equipment to handle.
We designed our own cost effective solution to the problem, by using commonly available material. From the required velocity of the fresh medium that would be needed, the flow rate was calculated. This was in turn used to calculate the pressure difference using the Hagen Poiseuille law. This pressure difference that would be required for the system was achieved by filling media inside a rubber pipe of small diameter (0.4 mm), and placing it up to a height h such that the hydrostatic pressure would be equal to the pressure difference required.
The height could be modulated to generate different flow rates and tune them specifically for our requirements. The following videos show the different flow rates that could be achieved –
Low Flow Rate –
Intermediate Flow Rate –
High Flow Rate –
Further, the droplets that were generated could also be modified to allow cells to pass through or not, depending on the initial pressure and volume of air that was pumped into the system. The following video shows how when low amounts of air is pushed through, the cells are able to pass through from droplet to droplet, from the edges of the air bubbles.
