This protocol allows for a simple and quick synthesis of optically vaporized perfluorocarbon nanodroplets and provides a method to enhance the performance of these particles. Person version allows enhancement of contrast by just manipulating the phase of the ultrasound imaging first. It requires no additional equipment and can be performed on any programmable ultrasound system.
Begin by rinsing a 10 milliliter round bottom flask with chloroform and wash out a 10 microliter and one milliliter gas tight glass syringe with chloroform by repeatedly aspirating the full syringe volume and expelling it a total of three times. Using the syringes, add 200 microliters of 25 milligrams per milliliter DSPE-mPEG-2000, eight microliters of 10 milligrams per milliliter DSPC, and one milliliter of one milligram per milliliter IR-1048 into the round bottom flask. Remember to clean out the syringes between lipids or dye to prevent contamination in the stock.
Remove the solvent utilizing a rotary evaporator. Ensure that the vacuum is slowly adjusted to 332 millibars to prevent bumping. After five minutes, reduce the pressure to 42 millibars to remove any water that may have entered the solution.
Suspend the lipid cake in one milliliter of PBS and sonicate or vortex at room temperature for five minutes or until all lipid cake has been suspended and dissolved in the solution. Transfer the solution to a seven milliliter glass vial and place the vial in a glass dish filled with ice to allow the solution to cool down for five minutes. Rinse out a gas-tight glass syringe with perfluorohexane.
Then add 50 microliters of perfluorohexane to the vial using the syringe. Probe sonicate the mixture with the amplitude set to one, the process time set to 20 seconds, pulse on set to one second, and pulse off set to five seconds. Then change the settings as mentioned in the text manuscript and sonicate the mixture once more.
Transfer the nano droplet solution to a 1.5 milliliter centrifuge tube and centrifuge at 300g for three minutes to separate the larger droplets which are more than one micrometer from the smaller droplets. Discard the pellet and transfer the supernatant to another 1.5 milliliter centrifuge tube. Wash the supernatant by centrifuging at 3, 000g for five minutes to pellet all the droplets in the solution.
Re-suspend the PFCnDs in one milliliter of PBS by pipetting the pellet up and down and then sonicate in a bath sonicater for one minute. Dilute the stock PFCnDs 100 fold by adding 10 microliters of PFCnD stock to 990 microliters of PBS and bath sonicate to disperse the PFCnDs before measuring their size. Measure the size of the droplets using dynamic light scattering.
Degas water by filling a 500 milliliter vacuum flask with 400 milliliters of deionized water, seal with a rubber cork, and connect the flask to the vacuum line. Open the vacuum line and submerge the bottom of the flask in the bath sonicater. Sonicate for five minutes or until no gas bubble formation is visible.
Prepare 10%ammonium per sulfate solution by dissolving 500 milligrams in five milliliters of degassed water. Gently swirl the solution if the ammonium persulfate does not fully dissolve. In a 400 milliliter beaker with a stir bar on a stir plate, add 150 milliliters of degassed water and 50 milliliters of 40 acrylamide bisacrylamide solution to form 200 milliliters of 10%acrylamide bisacrylamide solution.
Stir the mixture at 200 RPM to allow for proper mixing without introducing bubbles. Weigh out 400 milligrams of silica and add it to the 10%acrylamide bisacrylamide solution to form a 0.2%of silica and acrylamide solution. Prepare a square mold with a cylindrical inclusion by cutting off the tips from a plastic transfer pipette and supporting it in the mold with lab tape.
Add two milliliters of 10%ammonium persulfate solution to the beaker to make a final concentration of 0.1%and add 250 microliters of TEMED to the phantom solution. Allow the solution to stir for less than a minute. Quickly pour the solution into the mold while being careful not to introduce air bubbles into the solution.
The solution should polymerize within 10 minutes. Remove the phantom by running the flat end of a lab spatula around the edge of the mold and inverting the mold. Turn on and warm up the pulsed laser system for approximately 20 minutes following manufacturer instructions.
Ensure that the fiber optical bundle is properly connected to the laser output and the two legs are properly placed within the fiber bundle holder. After turning on the ultrasound imaging system, connect the array imaging transducer to the system and fix the transducer within the holder to align its imaging plane with laser cross section. Set the pulse repetition frequency of the laser system to 10 hertz and place a power meter at the end of the fiber bundle to measure energy.
Tune the Q switch delay until the estimated fluence is 70 milijoule per square centimeter. Backfill one of the channels in the poly acrylamide phantom with the ultrasound gel and PFCnD mixture using a one milliliter plastic slip tip syringe. Liberally cover the top of the channel with ultrasound gel and remove any bubbles with a one milliliter plastic slip tip syringe.
Finally, place the poly acrylamide phantom underneath the transducer and fiber bundle. Successful formulation and centrifugal separation of the PFCnDs yield droplets around 200 to 300 nanometers in diameter. The size of the droplets increases over time due to coalescing and diffusion in a process known as Ostwald ripening.
The contrast from the inclusion for the end pulse was found to be approximately 3.2 times greater, that is a 220%improvement than the P-pulse. The hyperechoic area was calculated for each frame and normalized by the hyperechoic area of the first frame and then fitted to an exponential decay model. The characteristic decay time of normalized hyperechoic area was up to 3.5 times longer in N-pulse imaging compared to P-pulse.
The B-mode differential image frames were recorded in time for each N-pulse and P-pulse imaging. One of the most important things is to expel the lipids at the bottom of the flask to ensure that they get properly incorporated into the lipid cake.
