A new light and sound sensitive nanoemulsion contrast agent is presented. The agents feature a low boiling point liquid perfluorocarbon core and a broad light spectrum absorbing polypyrrole (PPy) polymer shell. The PPy coated nanoemulsions can reversibly convert from liquid to gas phase upon cavitation of the liquid perfluorocarbon core. Cavitation can be initiated using a sufficiently high intensity acoustic pulse or from heat generation due to light absorption from a laser pulse. The emulsions can be made between 150 and 350 nm in diameter and PPy has a broad optical absorption covering both the visible spectrum and extending into the near-infrared spectrum (peak absorption ~1053 nm).
The size, structure, and optical absorption properties of the PPy coated nanoemulsions were characterized and compared to PPy nanoparticles (no liquid core) using dynamic light scattering, ultraviolet-visible spectrophotometry, transmission electron microscopy, and small angle X-ray scattering. The cavitation threshold and signal intensity were measured as a function of both acoustic pressure and laser fluence. Overlapping simultaneous transmission of an acoustic and laser pulse can significantly reduce the activation energy of the contrast agents to levels lower than optical or acoustic activation alone. We also demonstrate that simultaneous light and sound cavitation of the agents can be used in a new sono-photoacoustic imaging method, which enables greater sensitivity than traditional photoacoustic imaging.
Ultrasound-induced inertial cavitation is a mechanical process used for site-localized therapies such as non-invasive surgery. Initiating cavitation in tissue requires very high intensity focused ultrasound (HIFU) and low-frequencies. Hence, some applications like thrombolysis require targeted contrast agents to reduce peak intensities and the potential for secondary effects. A new type of theranostic nanoemulsion has been developed as a combined ultrasound (US)/photoacoustic(PA) agent for molecular imaging and therapy. It includes a nanoscale emulsion core encapsulated with a layer of gold nanospheres at the water/ oil interface. Its optical absorption exhibits a spectrum broadened up to 1100 nm, opening the possibility that 1064 nm light can excite cavitation nuclei. If optically-excited nuclei are produced at the same time that a low-frequency US wave is at peak negative pressure, then highly localized therapies based on acoustic cavitation may be enabled at very low US pressures. We have demonstrated this concept using a low-cost, low energy, portable 1064 nm fiber laser in conjunction with a 1.24 MHz US transducer for simultaneous laser/US excitation of nanoemulsions. Active cavitation detection from backscattered signals indicated that cavitation can be initiated at very low acoustic pressures (less than 1 MPa) when laser excitation coincides with the rarefaction phase of the acoustic wave, and that no cavitation is produced when light is delivered during the compressive phase. US can sustain cavitation activity during long acoustic bursts and stimulate diffusion of the emulsion, thus increasing treatment speed. An in vitro clot model has been used to demonstrate combined US and laser excitation of the nanoemulsion for efficient thrombolysis.
A nanoemulsion contrast agent with a perfluorohexane core and optically absorptive gold nanospheres (GNSs) assembled on the surface, is presented to improve the specificity of photoacoustic (PA) molecular imaging in differentiating targeted cells or aberrant regions from heterogeneous background signals. Compared to distributed GNSs, clustered GNSs at the emulsion oil-water interface produce a red-shifted and broadened absorption spectrum, exhibiting fairly high absorption in the near-infrared region commonly used for deep tissue imaging. Above a certain laser irradiation fluence threshold, a phase transition creating a microbubble in the emulsion core leads to more than 10 times stronger PA signals compared with conventional thermal-expansion-induced PA signals. These signals are also strongly non-linear, as verified by a differential scheme using recorded PA images at different laser fluences. Assuming a linear relation between laser fluence and the PA signal amplitude, differential processing results in nearly perfect suppression of linear sources, but retains a significant residue for the non-linear nanoemulsion with more than 35 dB enhancement. This result demonstrates that contrast specificity can be improved using the nanoemulsion as a targeting agent in PA molecular imaging by suppressing all background signals related to a linear PA response. Furthermore, combined with a system providing simultaneous laser/ultrasound excitation, cavitation-generated bubbles have the potential to be a highly specific contrast agent for ultrasound molecular imaging and harmonic imaging, as well as a targeted means for noninvasive ultrasound-based therapies.
A new technique using pulsed laser heating of a nanocomposite contrast agent resulting in local bubble formation and
concomitant harmonic generation in a scattered probe ultrasound (US) beam is proposed to increase specific contrast in
both US imaging and laser-induced photoacoustic (PA) imaging. The composite combines an emulsion bead core with
amphiphilic gold nanospheres (GNSs) assembled at the interface. Clustered GNSs result in a broadened absorption
spectrum in the near infrared range (700-1000 nm) compared to the typical 520 nm peak of distributed GNSs, enabling
their use at depth in tissue. Illuminating the composite with a pulsed laser with appropriately chosen parameters heats the
composite through optical absorption by the GNSs and results in a phase transition of the emulsion bead to form a
transient bubble. By delivering a probe US pulse simultaneously, or immediately after the laser pulse is delivered,
harmonic signals are produced in the scattered US beam. The results show that a residual signal created by subtracting a
US signal from the simultaneous US/laser probing signal of the emulsion bead sample is 1.7 dB higher than the laser
alone generated PA signal and 20 dB higher than the PA signal of a control homogeneous GNSs dispersion with the
same optical absorption, indicating the nonlinear contrast enhancement from bubble dynamics. The proposed technique
of local activation of this designed contrast agent can be used to dramatically enhance both the specificity and sensitivity
of integrated US/PA molecular imaging.
It is important to understand the interaction of pulsed ultrasound with ultrasound contrast agents so that the agents can be utilized to their fullest. Towards this goal, we have optimized light-scattering to provide quantitative information about microbubble oscillations when subjected to diagnostic ultrasound pulses. Initial experiments were performed with individual microbubbles. Pulses from actual diagnostic imaging systems were used to 'activate' the microbubbles. Bubble oscillations were measured by focusing the scattered light onto a photodetector. Data was collected with a fast oscilloscope set up to capture instantaneous bubble oscillation data for many consecutive ultrasound pulses. Results for individual bubbles show that at low pressures, the bubbles can be stable for many pulses. Sometimes, the bubbles evolve, presumably due to shell permeabilization and shell fatigue. Bubble dynamics models compare favorably with the observed oscillations. Light scattering can be an important tool for understanding and optimizing newer bubble imaging modes such as pulse inversion.
Vision-based measurement methods were used to measure bubble sizes in this sonoluminescence experiment. Bubble imaging was accomplished by placing the bubble between a bright light source and a microscope-CCD
camera system. A collimated light-emitting diode was operated in a pulsed mode with an adjustable time delay with respect to the piezo-electric transducer drive signal. The light-emitting diode produced a bubble shadowgraph consisting of a multiple exposure made by numerous light pulses imaged onto a charge-couple device camera. Each image was transferred from the camera to a computer-controlled machine vision system via a frame grabber. The frame grabber was equipped with on-board memory to accommodate sequential image buffering while images were transferred to the host processor and analyzed. This configuration allowed the host computer to perform diameter
measurements, centroid position measurements and shape estimation in "real-time" as the next image was being acquired. Bubble size measurement accuracy with an uncertainty of 3 microns was achieved using standard lenses and machine vision algorithms. Bubble centroid position accuracy was also within the 3 micron tolerance of the
vision system. This uncertainty estimation accounted for the optical spatial resolution, digitization errors and the edge detection algorithm accuracy. The vision algorithms include camera calibration, thresholding, edge detection, edge position determination, distance between two edges computations and centroid position computations.
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