Difference between revisions of "Team:IIT Delhi/Microfluidics"
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<a href="/Team:IIT_Delhi/Circuit_Design">Circuit design and construction</a> | <a href="/Team:IIT_Delhi/Circuit_Design">Circuit design and construction</a> | ||
| − | <a href="/Team:IIT_Delhi/Microfluidics">Microfluidics and | + | <a href="/Team:IIT_Delhi/Microfluidics">Microfluidics and Fluorescence</a> |
<a href="/Team:IIT_Delhi/Photobleaching">Photobleaching</a> | <a href="/Team:IIT_Delhi/Photobleaching">Photobleaching</a> | ||
<a href="/Team:IIT_Delhi/Promoter">Promoter strength</a> | <a href="/Team:IIT_Delhi/Promoter">Promoter strength</a> | ||
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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 – <br><br> | 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 – <br><br> | ||
Low Flow Rate –<br> | Low Flow Rate –<br> | ||
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Intermediate Flow Rate –<br> | Intermediate Flow Rate –<br> | ||
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High Flow Rate –<br> | High Flow Rate –<br> | ||
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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. | 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. | ||
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Latest revision as of 23:08, 1 November 2017
Microfluidic
Chamber Design
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
