To begin, fill a one milliliter syringe with 500 microliters of continuous phase. Attach the PTFE microtubing to a 27 gauge 0.75 inch needle. Mount the assembly onto the syringe and subsequently onto the syringe pump.
Then punch a hole using a 0.75 millimeter biopsy punch in the middle of a PDMS cutout. Pull approximately three centimeters of PTFE tubing through the hole in the cutout, and push the assembly into the top of a 200 microliter pipette tip. Seal the connector with UV curable glue on top of the pipette, and attach the other side of the tube to a 23 gauge 1.25 inch needle.
Next, fill two one milliliter syringes with 500 microliters of light mineral oil. Attach two 23 gauge needles with the custom-made attachments and mount them onto the syringe pump. Using the syringe pump control software, aspirate functionalized nanoparticle and cell solution into the pipette tip of the aqueous phases.
After cleaning the prepared observation chamber, clamp the chamber into the printed chamber holder equipped with two neodymium magnets at 30 degrees. Now, open both ports and plug a paper towel in the upper port to absorb the excess outer phase during filling. Connect the continuous phase to the top inlet of the microfluidic chip.
Then, flush the chip with the continuous phase for approximately 30 seconds at a flow rate of 1800 microliters per hour. Attach the pipette tips of the aqueous solutions to the two middle inlets of the chip. Start the flow of the aqueous solution at 200 microliters per hour for each solution.
Once the liquid appears at the outlet, begin the fluorinated oil phase flow at 800 microliters per hour. Wait until stable droplet production is established, indicated by a homogeneous gray, shiny solution at the outlet. Then connect the PTFE microtubing to the outlet port to collect the droplets.
Using the ferrule module of a finger-tight one piece fitting, direct them into an observation chamber. Once the chamber is filled, stop the flow and close off the ports with port plugs using finger-tight pressure. After droplet production, flush the chip with fluorinated oil, followed by nitrogen, to blow out any remaining liquid.
Mount the chamber holder on the microscope with a well plate format stage. Then, using the 10x objective, switch the microscope to the brightfield channel. Focus on the immobilized droplets in the brightfield, and move to the center of the chamber.
Then, activate the automated focusing system. Adjust the measurement plane on the brightfield channel so that droplet edges appear as sharp black circles easily distinguishable from the oil phase and background. Go through each of the fluorescence channels, setting the optimal measurement plane for them.
For relocation measurements, focus on the nanoparticle aggregate in the FITC, TRITC, and Cyanine5 channels.
