Ultrasound-guided photoacoustic imaging has shown great potential for many clinical applications including vascular visualization, detection of nanoprobes sensing molecular profiles, and guidance of interventional procedures. However, bulky and costly lasers are usually required to provide sufficient pulse energies for deep imaging. The low pulse repetition rate also limits potential real-time applications of integrated photoacoustic/ultrasound (PAUS) imaging. With a compact and low-cost laser operating at a kHz repetition rate, we aim to integrate photoacoustics (PA) into a commercial ultrasound (US) machine utilizing an interleaved scanning approach for clinical translation, with imaging depth up to a few centimeters and frame rates > 30 Hz. Multiple PA sub-frames are formed by scanning laser firings covering a large scan region with a rotating galvo mirror, and then combined into a final frame. Ultrasound pulse-echo beams are interleaved between laser firings/PA receives. The approach was implemented with a diode-pumped laser, a commercial US scanner, and a linear array transducer. Insertion of an 18-gauge needle into a piece of chicken tissue, with subsequent injection of an absorptive agent into the tissue, was imaged with an integrated PAUS frame rate of 30 Hz, covering a 2.8 cm × 2.8 cm imaging plane. Given this real-time image rate and high contrast (> 40 dB at more than 1-cm depth in the PA image), we have demonstrated that this approach is potentially attractive for clinical procedure guidance.
Due to the high scattering coefficient of tissue over the wavelength range used for photoacoustic (PA) imaging, most studies employ bulky, low repetition rate lasers to provide sufficient pulse energies at depth to image within the body. The size and cost of these lasers has impeded integration of photoacoustics into conventional, routinely-used ultrasound (US) scanners. Here, we present an approach leveraging the capabilities of modern, high repetition rate fiber lasers to produce a clinically translatable system providing integrated US/PA images at frame rates > 30 Hz. The system uses a portable, low-cost, low pulse-energy (1 mJ/pulse), high repetition rate (1 kHz), 1064 nm laser and is designed for integrated US/PA imaging of the peripheral vasculature or any relevant diseased region, such as a tumor. Using a rotating galvo-mirror system, the incident laser beam is quickly scanned over the imaging area. Multiple PA images covering the scan area are integrated to form a single PA image. Additionally, ultrasound firings are integrated into the scan sequence to provide an US image reconstructed over the same frame period. We acquired PA images of a 1.5-mmdiameter cylindrical absorber (absorption coefficient 5 cm-1) embedded in a tissue-mimicking gelatin phantom at 6-cm depth. A 2 cm × 1 cm (depth × lateral) area was reconstructed. We obtained a signal-to-noise ratio of more than 30 dB, comparable to conventional PA methods using high energy, low repetition rate lasers. The current system produces an integrated US/PA frame at a 32 Hz rate, and 100 Hz frame rates are possible with our present approach.
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.
Photoacoustic (PA) imaging has been widely used in molecular imaging to detect diseased cells by targeting them with
nanoparticle-based contrast agents. However, the sensitivity and specificity are easily degraded because contrast agent
signals can be masked by the background. Magnetomotive photoacoustic imaging uses a new type of multifunctional
composite particle combining an optically absorptive gold nanorod core and magnetic nanospheres, which can
potentially accumulate and concentrate targeted cells while simultaneously enhancing their specific contrast compared to
background signals. In this study, HeLa cells molecularly targeted using nanocomposites with folic acid mimicking
targeted rare circulating tumor cells (CTCs) were circulated at a 6 ml/min flow rate for trapping and imaging studies.
Preliminary results show that the cells accumulate rapidly in the presence of an externally applied magnetic field
produced by a dual magnet system. The sensitivity of the current system can reach up to 1 cell/ml in clear water. By
manipulating the trapped cells magnetically, the specificity of detecting cells in highly absorptive ink solution can be
enhanced with 16.98 dB background suppression by applying motion filtering on PA signals to remove unwanted
background signals insensitive to the magnetic field. The results appear promising for future preclinical studies on a
small animal model and ultimate clinical detection of rare CTCs in the vasculature.
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.
Results on magnetically trapping and manipulating micro-scale beads circulating in a flow field mimicking metastatic cancer cells in human peripheral vessels are presented. Composite contrast agents combining magneto-sensitive nanospheres and highly optical absorptive gold nanorods were conjugated to micro-scale polystyrene beads. To efficiently trap the targeted objects in a fast stream, a dual magnet system consisting of two flat magnets to magnetize (polarize) the contrast agent and an array of cone magnets producing a sharp gradient field to trap the magnetized contrast agent was designed and constructed. A water-ink solution with an optical absorption coefficient of 10 cm−1 was used to mimic the optical absorption of blood. Magnetomotive photoacoustic imaging helped visualize bead trapping, dynamic manipulation of trapped beads in a flow field, and the subtraction of stationary background signals insensitive to the magnetic field. The results show that trafficking micro-scale objects can be effectively trapped in a stream with a flow rate up to 12 ml/min and the background can be significantly (greater than 15 dB) suppressed. It makes the proposed method very promising for sensitive detection of rare circulating tumor cells within high flow vessels with a highly absorptive optical background.
Contrast-enhanced photoacoustic (PA) imaging has been proposed to identify circulating metastatic cancer cells magnetically trapped in the vasculature. However, its sensitivity is limited by the presence of a strong blood-background signal. This technique can be further improved by the significant suppression of blood background. In the phantom study presented here, significant background suppression is demonstrated with magnetomotive photoacoustic imaging. Magnetic particles with a mean diameter of 10 μm were integrated (concentration of 0.05 mg / ml) into an ink-water solution with an optical absorption coefficient of 5 cm − 1 to mimic cells targeted with magnetic nanoparticles and magnetically trapped in the human vasculature. Two mechanically moveable permanent magnets were used to accumulate microparticles in the investigated solution and manipulate them within a thin, 1.6-mm-diameter Teflon tube mimicking a blood vessel. Our results clearly indicate that the undesirable background can be effectively suppressed using the difference of PA images corresponding to different locations of accumulated particles.
Trapping and manipulation of micro-scale objects mimicking metastatic cancer cells in a flow field have been
demonstrated with magnetomotive photoacoustic (mmPA) imaging. Coupled contrast agents combining gold nanorods
(15 nm × 50 nm; absorption peak around 730 nm) with 15 nm diameter magnetic nanospheres were targeted to 10 μm
polystyrene beads recirculating in a 1.6 mm diameter tube mimicking a human peripheral vessel. Targeted objects were
then trapped by an external magnetic field produced by a dual magnet system consisting of two disc magnets separated
by 6 cm to form a polarizing field (0.04 Tesla in the tube region) to magnetize the magnetic contrast agents, and a
custom designed cone magnet array with a high magnetic field gradient (about 0.044 Tesla/mm in the tube region)
producing a strong trapping force to magnetized contrast agents. Results show that polystyrene beads linked to
nanocomposites can be trapped at flow rates up to 12 ml/min. It is shown that unwanted background in a photoacoustic
image can be significantly suppressed by changing the position of the cone magnet array with respect to the tube, thus
creating coherent movement of the trapped objects. This study makes mmPA imaging very promising for differential
visualization of metastatic cells trafficking in the vasculature.
Magnetic nanoparticles (MNPs) have been used extensively ex vivo for cellular and molecular separations. We recently
showed that a coupled nanoparticle combining a superparamagnetic core with a thin, isolated gold shell providing strong
absorption in the near infrared can be used for magnetomotive photoacoustic imaging (mmPA), a new technique in
which magnetic manipulation of the particle during PA imaging greatly enhances molecular contrast specificity. This
particle can also be biologically targeted for in vivo applications, where mmPA imaging provides a spatially localized
readout of magnetic manipulations. As an initial test of potential in vivo molecular assays and integrated molecular
therapeutics using magnetic manipulation of nanoparticles, we present experiments demonstrating PA readout of trapped
magnetic particles in a flow field. An aqueous solution containing a concentration of 0.05-mg/ml 10-μM
superparamagnetic iron oxide particles flowed in a 1.65-mm diameter Zeus PTFE (Teflon) sublite wall tubing at three
velocities of 0.8, 1.5 and 3.0-mm/s. Opposed permanent magnets separated by 40-mm were positioned on both sides of
the tube. As expected, the targeted objects can be magnetically captured and accumulated locally. By translating the
magnets, a dynamic magnetic field (0.1-0.3-T) was alternately generated on the side of the tube closest to one of the
magnets and created a synchronous PA motion from accumulated targeted objects. This synchronized motion can be
used to differentiate the stationary background or other PA sources moving asynchronously with magnetic manipulations
(e.g., moving blood) from targeted cells moving synchronously with the magnetic field. This technology can potentially
provide sensitive molecular assays of cellular targets travelling in the vasculature (e.g., metastatic tumor cells).
Current systems designed for deep photoacoustic (PA) imaging typically use a low repetition rate, high power pulsed
laser to provide a ns-scale pulse illuminating a large tissue volume. Acoustic signals recorded on each laser firing can be
used to reconstruct a complete 2-D (3-D) image of sources of heat release within that region. Using broad-beam
excitation, the maximum frame rate of the imaging system is restricted by the pulse repetition rate of the laser.
An alternate illumination approach is proposed based on fast scanning by a low energy (~ 1 mJ) high repetition rate (up
to a few kHz) narrow laser beam (~1 mm) along the tissue surface over a region of interest. A final PA image is
produced from the summation of individual PA images reconstructed at each laser beam position. This concept can take
advantage of high repetition rate fiber lasers to create PA images with much higher frame rates than current systems,
enabling true real-time integration of photoacoustics with ultrasound imaging. As an initial proof of concept, we compare
conventional broad beam illumination to a scanned beam approach in a simple model system.
Two transparent teflon tubes with diameters of 1.6 mm and 0.8 mm were filled with ink having an absorption coefficient
of 5 cm-1. These tubes were buried inside chicken breast tissue acting as an optical scattering medium. They were
separated by 3 mm or 10 mm to test spatial and contrast resolution for the two scan formats. The excitation wavelength
was 700 nm. The excitation source is a traditional OPO pumped by a
Q-switched Nd:YAG laser with doubler.
Photoacoustic images were reconstructed using signals from a small, scanned PVDF transducer acting as an acoustic
array. Two different illumination schemes were compared: one was 15 mm x 10 mm in cross section and acted as the
broad beam; the other was 5 mm x 2 mm in cross section (15 times smaller than the broad beam case) and was scanned
over an area equivalent to broad beam illumination. Multiple images obtained during narrow beam scanning were added
together to form one PA image equivalent to the single-shot broad beam one.
Results of the phantom study indicate that PA images formed by narrow beam scanning excitation can be equivalent to
one shot broad beam illumination in signal to noise ratio and spatial resolution. Future studies will focus on high
repetition-rate laser sources and scan formats appropriate for real-time, integrated deep photoacoustic/ultrasonic imaging.
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