This method can help answer key questions in the high intensity focused ultrasound field, such as can laser ultrasound nucleated cavitation be used to guide, and also enhance HIFU cancer treatments. The main advantage of this technique is that through the combination of nano-particles, HIFU, and laser illumination, it can overcome the limitations of each of these modalities by themselves. Manufacture phantoms to demonstrate the nucleation method.
Start with de-ionized, de-gassed, room temperature water in a glass beaker. In addition, prepare 40%weight by volume acrylamide/bis-acrylamide solution. Add the solution to the water, followed by a buffer, and ammonium persulfate.
Place the beaker inside a vacuum chamber situated on a magnetic stirrer plate. Add a 40-millimeter long PTFE magnetic stirring bar to the beaker and stir at medium speed. Slowly add bovine serum albumin powder.
When done, close the vacuum chamber and start the vacuum pump. Maintain the target vacuum and continue stirring for 60 minutes. Next, release the vacuum and continue working with the solution.
Add nanoparticles for phantoms that require them. For all phantoms, add the catalyst. After five minutes of mixing, pour the solution into individual molds and wait 20 minutes.
This is an example of a phantom that is set and been removed from the mold. It is ready to use in the experiment. Once the phantoms are set, store the demolded phantoms in an airtight container.
To produce an alignment phantom, begin with the phantom solution. Place the beaker and stir bar in a vacuum chamber on a magnetic stirrer. Begin stirring at medium speed, and pump the chamber to the target vacuum.
After retrieving the solution, pour 25 milliliters into a mold and add the catalyst. Wait 20 minutes before placing a one-millimeter spherical metal target in the phantom's center. Then, pour another 25 milliliters of the phantom solution into the mold.
Add the catalyst and wait for another 20 minutes. When set, the alignment phantom is ready for use or storage in an airtight container. Prepare the setup for the experiment.
For this, have an acrylic water tank with 4.5 liters of de-ionized, de-gassed water. At one end of the tank, place an acoustic absorber. Next, turn attention to the high intensity focused ultrasound transducer.
Mount it and it's co-aligned broadband hydrophone on a three-axis micrometer stage. Fully submerge the transducer and hydrophone in the tank to face the absorber. Connect the transducer to an impedance matching circuit that will allow it to be driven at its third harmonic.
This circuit is connected directly to the output of an RF power amplifier, which has a digital function generator as its input. The function generator is programmed remotely. After calibration, get an alignment phantom to continue setting up.
The phantom should be in a 3D printed holder and mounted on an automated 3D stage. Position the phantom so the magnetic target is at the approximate focal peak of the transducer. Now, connect the hydrophone directly to the data acquisition card.
Use the transducer and hydrophone to pulse echolocate the alignment target. Send a three microsecond, 10-cycle burst, and view the detected signal in real time on the computer. Adjust the transducer's micrometer stage to change the time of flight and signal amplitude.
The system is aligned once the time of flight is 85 microseconds and the signal amplitude is maximized. Next, connect the broadband hydrophone directly to a five megahertz high-pass filter. Send the signal through a 40-decibel pre-amplifier and then to a data acquisition card.
Now, set up laser illumination for the sample. Synchronize a 532 nanometer pulse laser with the function generator by a TTL digital delay pulse generator. Use the laser to pump an optical parametric oscillator.
Couple its output in the phantom with a two-millimeter fiber bundle. At the tank, mount this fiber on a micrometer stage. Position the fiber in front of the phantom at an angle of 45 degrees from the acoustic axis.
For alignment, use visible light. Position the beam to have the alignment target at the center of a 15-millimeter laser spot. Finally, position a digital microscope and a white light source on opposite sides of the water tank.
Mount the microscope on a micrometer stage. Position it to have the alignment target in focus in its field of view. Ensure the correct phantom is in place.
In this case, the appropriate tissue phantom replaces the alignment phantom. Tune the laser wavelength to the nanoparticle's surface plasmon resonance. At the control computer, set the transducer to produce a 10-cycle burst, and to set the laser fluence.
Target the focal peak of the high intensity focused ultrasound burst 10 millimeters deep and at 13 unique locations in the vertical direction spaced by five millimeters. Ensure that a tissue phantom is in place in the tank. Then, with the software, set the fluence and continuous wave exposure parameters.
Use the microscope to record thermal lesion formation as the high intensity focused ultrasound with a chosen peak negative pressure is directed at one location. These data are detector voltage versus time for short, high intensity focused ultrasound exposure for different phantoms under different conditions. In these data sets, the phantoms were also exposed to laser illumination.
However, one phantom did not have nanorods and one phantom did. In these data sets, both phantoms had nanorods, but one was exposed to laser illumination and the other phantom was not. This demonstrates that broadband emissions are detected only when nanoparticles, ultrasound exposure, and laser illumination are all present.
This microscope video provides an example of the formation of thermal and cavitation lesions in a gel phantom with nanorods exposed to high intensity focused ultrasound and laser illumination. While attempting this procedure, it is important to remember to wear suitable personal protective equipment when handling the chemicals and ensure correct eye protection is used when using the laser. After its development, this technique could pave the way for researchers in the field of high intensity focused ultrasound therapy to explore using molecular targeted nanoparticles to enhance cancer treatments through targeted and fast thermal ablations.
