This procedure shows how to assemble and use the microfluidic probe or MFP. The microfluidic components of the MFP are gas tight syringes, glass capillary tubes, micro tight connectors from Upchurch and the MFP head. This video will present the critical steps of assembly and alignment.
Once assembled, the MFP can be used with a large variety of substrates and reagents for surface patterning and processing. Hi, I am Cecil Perot. I'm a postdoctoral fellow in the laboratory of David Yaker in the Department of Biomedical Engineering at McGill University in Montreal, Canada.
Hi, I'm Mohammad Kasime. I am a PhD candidate on David Y's lab on the biomedical engineering department, McGill University. Hi, I'm David Yoker.
I'm an assistant professor in the Department of Biomedical Engineering, and I would like to welcome you today to our demonstration on the microfluidic probe. Today we'll show you a procedure of the operation and use of the microfluidic probe. We use this procedure in our laboratory for surface processing and patterning.
So let's get started To start this protocol. Plastic syringes and needles are used to fill gas type glass syringes with reagents, ensuring that no air bubbles are present. Typically, we use a one to 10 microliter syringe for injection and a syringe with five to 10 times larger volume for aspiration.
Next, using nano tight fittings with low dead volume, the syringes are connected to capillary tubing. The capillaries are then filled and checked for bubbles under the microscope. The MFP chip is prefilled with buffer solution to prevent the trapping of bubbles when connecting the capillaries.
Finally, the capillaries are plugged into the PDMS connection piece in the micro fabricated prop head. With the MFP now assembled, we are ready to begin setting it up. The substrate is inserted into a homemade holder.
There's affixed to the microscope stage. The gap between the MFP and the substrate is critical for surface patterning processes. Using a three point support formed by micrometer screws, the horizontal alignment is adjusted with micrometer precision.
This is necessary because the substrate is processed by being scanned below the MFP. To begin setting up the MFP, the probe head is clamped into the probe holder and mounted on the probe station on top of an inverted microscope. The syringes are then placed into high precision syringe pumps.
Next, using a pair of goniometers, the alignment of the meza of the MFP with the substrate is adjusted by observing the Newton's rings, also known as interference fringes. These appear when the MFP is brought into contact with the substrate, the point of contact and the frequency of the rings serve as indication of the tilt. When the MFP is aligned with the surface, a single interference ring extends over the entire surface.
This measure also serves to calibrate the separation between the MFP and substrate. With the MFP set up, we are now ready to move on to operation of the MFP. During operation of the MFP dispensing is controlled by lab view software.
Device operation is visualized by eye and with a CCD camera. Before starting proper aspiration, inject some liquid to make sure the aspiration syringe is oriented properly and that there are no bubbles. The MFP is then immersed in the buffer.
We can now begin the injection of liquid while monitoring the flown confinement of beads. The injection aspiration ratio varies from one to three to one to 10, depending on the diffusivity of the reagent with a surrounding buffer and the desired geometrical flow pattern. Finally, the probe can now be used for a variety of applications.
The surface patterns shown here were achieved by the MFP with fluorescein labeled biotin on strep divide, ENC coated hydrogel glass slides. The injection flow rate was one nanoliter per second, and the aspiration flow rate was 10 nanoliters per second using 10 microliter and 50 microliter syringes respectively. The gap between the surface and the probe was five micrometers.
The MFP can also be used for local staining and processing of cells and tissues. The flow confinement prohibits diffusion of the reagent and allows precise chemical staining of cells. As shown here, a monolayer of fibroblast cells was stained with 100 micrograms per milliliter of dai.
So we've shown you how to operate the microfluidic probe. When doing this procedure, you have to make sure to avoid any bubbles on the system and to make sure that the microfluidic prop surface is parallel to the substrate. So that's it.
Thanks for watching and good luck with your experiments.
