Ultrasound contrast-mediated medical imaging and therapy both rely on the dynamics of micron- and nanometer-sized ultrasound cavitation nuclei, such as phospholipid-coated microbubbles and phase-change droplets. Ultrasound cavitation nuclei respond non-linearly to ultrasound on a nanosecond time scale that necessitates the use of ultra-high-speed imaging to fully visualize these dynamics in detail. In this study, we developed an ultra-high-speed optical imaging system that can record up to 20 million frames per second (Mfps) by coupling two small-sized, commercially available, 10-Mfps cameras. The timing and reliability of the interleaved cameras needed to achieve 20 Mfps was validated using two synchronized light-emitting diode strobe lights. Once verified, ultrasound-activated microbubble responses were recorded and analyzed. A unique characteristic of this coupled system is its ability to be reconfigured to provide orthogonal observations at 10 Mfps. Acoustic droplet vaporization was imaged from two orthogonal views, by which the 3-D dynamics of the phase transition could be visualized. This optical imaging system provides the temporal resolution and experimental flexibility needed to further elucidate the dynamics of ultrasound cavitation nuclei to potentiate the clinical translation of ultrasound-mediated imaging and therapy developments.

An in vivo range verification technology for proton beam cancer therapy, preferably in real-time and with submillimeter resolution, is desired to reduce the present uncertainty in dose localization. Acoustical imaging technologies exploiting possible local interactions between protons and microbubbles or nanodroplets might be an interesting option. Unfortunately, a theoretical model capable of characterising the acoustical field generated by an individual proton on nanometer and micrometer scales is still missing. In this work, such a model is presented. The proton acoustic field is generated by the adiabatic expansion of a region that is locally heated by a passing proton. To model the proton heat deposition, secondary electron production due to protons has been quantified using a semi-empirical model based on Rutherford's scattering theory, which reproduces experimentally obtained electronic stopping power values for protons in water within 10% over the full energy range. The electrons transfer energy into heat via electron-phonon coupling to atoms along the proton track. The resulting temperature increase is calculated using an inelastic thermal spike model. Heat deposition can be regarded as instantaneous, thus, stress confinement is ensured and acoustical initial conditions are set. The resulting thermoacoustic field in the nanometer and micrometer range from the single proton track is computed by solving the thermoacoustic wave equation using k-space Green's functions, yielding the characteristic amplitudes and frequencies present in the acoustic signal generated by a single proton in an aqueous medium. Wavefield expansion and asymptotic approximations are used to extend the spatial and temporal ranges of the proton acoustic field.

Superheated nanodroplet (ND) vaporization by proton radiation was recently demonstrated, opening the door to ultrasound-based in vivo proton range verification. However, at body temperature and physiological pressures, perfluorobutane nanodroplets (PFB-NDs), which offer a good compromise between stability and radiation sensitivity, are not directly sensitive to primary protons. Instead, they are vaporized by infrequent secondary particles, which limits the precision for range verification. The radiation-induced vaporization threshold (i.e., sensitization threshold) can be reduced by lowering the pressure in the droplet such that ND vaporization by primary protons can occur. Here, we propose to use an acoustic field to modulate the pressure, intermittently lowering the proton sensitization threshold of PFB-NDs during the rarefactional phase of the ultrasound wave. Simultaneous proton irradiation and sonication with a 1.1 MHz focused transducer, using increasing peak negative pressures (PNPs), were applied on a dilution of PFB-NDs flowing in a tube, while vaporization was acoustically monitored with a linear array. Sensitization to primary protons was achieved at temperatures between 29 °C and 40 °C using acoustic PNPs of relatively low amplitude (from 800 to 200 kPa, respectively), while sonication alone did not lead to ND vaporization at those PNPs. Sensitization was also measured at the clinically relevant body temperature (i.e., 37 °C) using a PNP of 400 kPa. These findings confirm that acoustic modulation lowers the sensitization threshold of superheated NDs, enabling a direct proton response at body temperature.

The potential of proton therapy to improve the conformity of the delivered dose to the tumor volume is currently limited by range uncertainties. Injectable superheated nanodroplets have recently been proposed for ultrasound-based in vivo range verification, as these vaporize into echogenic microbubbles on proton irradiation. In previous studies, offline ultrasound images of phantoms with dispersed nanodroplets were acquired after irradiation, relating the induced vaporization profiles to the proton range. However, the aforementioned method did not enable the counting of individual vaporization events, and offline imaging cannot provide real-time feedback. In this study, we overcame these limitations using high-frame-rate ultrasound imaging with a linear array during proton irradiation of phantoms with dispersed perfluorobutane nanodroplets at 37°C and 50°C. Differential image analysis of subsequent frames allowed us to count individual vaporization events and to localize them with a resolution beyond the ultrasound diffraction limit, enabling spatial and temporal quantification of the interaction between ionizing radiation and nanodroplets. Vaporization maps were found to accurately correlate with the stopping distribution of protons (at 50°C) or secondary particles (at both temperatures). Furthermore, a linear relationship between the vaporization count and the number of incoming protons was observed. These results indicate the potential of real-time high-frame-rate contrast-enhanced ultrasound imaging for proton range verification and dosimetry.

Phospholipid-coated microbubbles are ultrasound contrast agents that can be employed for ultrasound molecular imaging and drug delivery. For safe and effective implementation, microbubbles must respond uniformly and predictably to ultrasound. Therefore, we investigated how lipid handling and phase distribution affected the variability in the acoustic behavior of microbubbles. Cholesterol was used to modify the lateral molecular packing of 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC)-based microbubbles. To assess the effect of lipid handling, microbubbles were produced by a direct method, i.e., lipids directly dispersed in an aqueous medium or indirect method, i.e., lipids first dissolved in an organic solvent. The lipid phase and ligand distribution in the microbubble coating were investigated using confocal microscopy, and the acoustic response was recorded with the Brandaris 128 ultra-high-speed camera. In microbubbles with 12 mol% cholesterol, the lipids were miscible and all in the same phase, which resulted in more buckle formation, lower shell elasticity and higher shell viscosity. Indirect DSPC microbubbles had a more uniform response to ultrasound than direct DSPC and indirect DSPC-cholesterol microbubbles. The difference in lipid handling between direct and indirect DSPC microbubbles significantly affected the acoustic behavior. Indirect DSPC microbubbles are the most promising candidate for ultrasound molecular imaging and drug delivery applications.