This PDF file contains the front matter associated with SPIE Proceedings Volume 9323, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and Conference Committee listing.

Premature, very-low-birth-weight (VLBW; ≤1500 g) and low-birth-weight (LBW; 1500-2499 g) infants are at increased risk for severe neurological disability. 25-50% of the 63,000 VLBW infants born annually in the USA have major longterm cognitive or neurobehavioral deficits in which cerebral hypoxia plays an important role. At present, no technology is capable of noninvasive, accurate monitoring of cerebral oxygenation in newborns. We proposed to use an optoacoustic technique for noninvasive cerebral hypoxia monitoring by probing the superior sagittal sinus (SSS). Recently, we developed and built a medical grade, multi-wavelength, near-infrared optoacoustic system suitable for neonatal applications. We designed and built an adjustable patient interface for neonates with VLBW, LBW, and normal weight (NBW) that provides single or continuous measurements of the SSS blood oxygenation in both the reflection and transmission modes. We performed pilot clinical tests of the system in VLBW, LBW, and NBW hemodynamically stable infants admitted to the neonatal intensive care unit. The system was capable of detecting SSS signals through the open anterior and posterior fontanelles and through the skull and allowed for monitoring of the SSS blood oxygenation in both modes. To the best of our knowledge this paper reports for the first time detection of optoacoustic signals from human cerebral blood vessels in both the transmission mode and the reflection mode. Analysis of the signals allows for noninvasive measurement of cerebral venous blood oxygenation in newborns in both modes. The transmission mode can be used for accurate measurement of the total hemoglobin concentration as well. The method and system proposed in this study can be used for optoacoustic human brain imaging, tomography, and mapping.

In this paper, wavelength selection for multispectral photoacoustic/ultrasound tomography was optimized to obtain accurate images of hemoglobin oxygen saturation (sO₂) in vivo. Although wavelengths can be selected by theoretical methods, in practice the accuracy of reconstructed images will be affected by wavelength-specific and system-specific factors such as laser source power and ultrasound transducer sensitivity. By performing photoacoustic spectroscopy of mouse tumor models using 14 different wavelengths between 710 and 840 nm, we were able to identify a wavelength set which most accurately reproduced the results obtained using all 14 wavelengths via selection criteria. In clinical studies, the optimal wavelength set was successfully used to image human ovaries in vivo and noninvasively. Although these results are specific to our co-registered photoacoustic/ultrasound imaging system, the approach we developed can be applied to other functional photoacoustic and optical imaging systems.

We developed a handheld photoacoustic microscope (PAM) to detect melanoma and determine tumor depth in nude mice in vivo. Compared to our previous PAM system for melanoma imaging, a new light delivery mechanism is introduced to improve light penetration. We show that melanomas with 4.1 mm and 3.3 mm thicknesses can be successfully detected in phantom and in vivo experiments, respectively. With its deep melanoma imaging ability and novel handheld design, this system is promising for clinical melanoma diagnosis, prognosis, and surgical planning for patients at the bedside.

Real-time ultrasound/photoacoustic in-vivo imaging will be demonstrated using a portable, dual-modality photoacoustic and ultrasound imager that features a pulsed diode laser integrated into an hand-held ultrasound probe. The 800-nm-wavelength diode provides 0.6 mJ pulses after the probe aperture in a 20 mm × 4 mm rectangular beam and can be fired at 210 Hz, limited by the Maximum Permissible Exposure. In-vivo imaging that will be demonstrated will include finger-joint imaging for future early detection of inflammation in rheumatoid diseases. New photoacoustic techniques that might increase the sensitivity of the detection of inflammation, such as PA Doppler, will also be discussed.

Accurate endoscopic detection and dysplasia in patients with Barrett’s esophagus (BE) remains a major unmet clinical need. Current diagnosis use multiple biopsies under endoscopic image guidance, where up to 99% of the tissue remains unsampled, leading to significant risk of missing dysplasia. We conducted an ex vivo clinical trial using photoacoustic imaging (PAI) in patients undergoing endoscopic mucosal resection (EMR) with known high-grade dysplasia for the purpose of characterizing the esophageal microvascular pattern, with the long-term goal of performing in vivo endoscopic PAI for dysplasia detection and therapeutic guidance. EMR tissues were mounted immediately on an agar layer and covered with ultrasound gel. Digital photography guided the placement of the PAI transducer (40 MHz center frequency). The luminal side of the specimen was scanned over a field of view of 14 mm (width) by 15 mm (depth) at 680, 750, 824, 850 and 970 nm. Acoustic images were simultaneously acquired. Tissues were then sliced and fixed in formalin for histopathology with H and E staining. Analysis consisted of co-registration and correlation between the intrinsic PAI features and the histological images. The initial PAI + ultrasound images from 8 BE patients have demonstrated the technical feasibility of this approach and point to the potential of PAI to reveal the microvascular pattern within EMR specimens. There are several technical factors to be considered in rigorous interpretation of the PAI characteristics, including the loss of blood from the ex vivo specimens and the limited depth penetration of the photoacoustic signal.

Current radiofrequency cardiac ablation procedures lack real-time lesion monitoring guidance, limiting the reliability and efficacy of the treatment. The objective of this work is to demonstrate that optoacoustic imaging can be applied to develop a diagnostic technique applicable to radiofrequency ablation for cardiac arrhythmia treatment with the capabilities of real-time monitoring of ablated lesion size and geometry. We demonstrate an optoacoustic imaging method using a 256-detector optoacoustic imaging probe and pulsed-laser illumination in the infrared wavelength range that is applied during radiofrequency ablation in excised porcine myocardial tissue samples. This technique results in images with high contrast between the lesion volume and unablated tissue, and is also capable of capturing time-resolved image sequences that provide information on the lesion development process. The size and geometry of the imaged lesion were shown to be in excellent agreement with the histological examinations. This study demonstrates the first deep-lesion real-time monitoring for radiofrequency ablation generated lesions, and the technique presented here has the potential for providing critical feedback that can significantly impact the outcome of clinical radiofrequency ablation procedures.

Melanoma, one of the most common types of skin cancer, has a high mortality rate, mainly due to a high propensity for tumor metastasis. The presence of circulating tumor cells (CTCs) is a potential predictor for metastasis. Label-free imaging of single circulating melanoma cells in vivo provides rich information on tumor progress. Here we present photoacoustic microscopy of single melanoma cells in living animals. We used a fast-scanning optical-resolution photoacoustic microscope to image the microvasculature in mouse ears. The imaging system has sub-cellular spatial resolution and works in reflection mode. A fast-scanning mirror allows the system to acquire fast volumetric images over a large field of view. A 500-kHz pulsed laser was used to image blood and CTCs. Single circulating melanoma cells were imaged in both capillaries and trunk vessels in living animals. These high-resolution images may be used in early detection of CTCs with potentially high sensitivity. In addition, this technique enables in vivo study of tumor cell extravasation from a primary tumor, which addresses an urgent pre-clinical need.

With the capability of assessing high resolution optical information in soft tissues at imaging depth up to several centimeters, innovative biomedical photoacoustic imaging (PAI) offers benefits to diagnosis and treatment monitoring of inflammatory arthritis, particularly in combination with more established ultrasonography (US). In this work, a PAI and US dual-modality system facilitating both imaging functions in a real-time fashion was developed and initially tested for its clinical performance on patients with active inflammatory arthritis. Photoacoustic (PA) images of metacarpophalangeal (MCP) joints were acquired at 580-nm wavelength that provides a desired balance between optical absorption of blood and attenuation in background tissue. The results from six patients and six normal volunteers used as a control demonstrated the satisfactory sensitivity of PAI in assessing the physiological changes in the joints, specifically enhanced blood flow as a result of active synovitis. This preliminary study suggests that PAI, by revealing vascular features suggestive of joint inflammation, could be a valuable supplement to musculoskeletal US for rheumatology clinic.

We previously introduced photoacoustic imaging to detect blood vessels surrounded by bone and thereby eliminate the deadly risk of carotid artery injury during endonasal, transsphenoidal surgeries. Light would be transmitted through an optical fiber attached to the surgical drill, while a transcranial probe placed on the temporal region of the skull receives photoacoustic signals. This work quantifies changes in photoacoustic image contrast as the sphenoid bone is drilled. Frontal bone from a human adult cadaver skull was cut into seven 3 cm x 3 cm chips and sanded to thicknesses ranging 1-4 mm. For 700-940 nm wavelengths, the average optical transmission through these specimens increased from 19% to 44% as bone thickness decreased, with measurements agreeing with Monte Carlo simulations within 5%. These skull specimens were individually placed in the optical pathway of a 3.5 mm diameter, cylindrical, vessel-mimicking photoacoustic target, as the laser wavelength was varied between 700-940 nm. The mean optical insertion loss and photoacoustic image contrast loss due to the bone specimens were 56-80% and 46-79%, respectively, with the majority of change observed when the bone was ≤2 mm thick. The decrease in contrast is directly proportional to insertion loss over this thickness range by factors of 0.8-1.1 when multiple wavelengths are considered. Results suggest that this proportional relationship may be used to determine the amount of bone that remains to be drilled when the thickness is 2 mm or less.

We demonstrate, by means of internal light delivery, photoacoustic imaging of the deep brain of rats in vivo. With fiber illumination via the oral cavity, we delivered light directly into the bottom of the brain, much more than can be delivered by external illumination. The study was performed using a photoacoustic computed tomography (PACT) system equipped with a 512-element full-ring transducer array, providing a full two-dimensional view aperture. Using internal illumination, the PACT system provided clear cross sectional photoacoustic images from the palate to the middle brain of live rats, revealing deep brain structures such as the hypothalamus, brain stem, and cerebral medulla.

Linear transducer arrays are readily available for ultrasonic detection in photoacoustic computed tomography. They offer low cost, hand-held convenience, and conventional ultrasonic imaging. However, the elevational resolution of linear transducer arrays, which is usually determined by the weak focus of the cylindrical acoustic lens, is about one order of magnitude worse than the in-plane axial and lateral spatial resolutions. Therefore, conventional linear scanning along the elevational direction cannot provide high-quality three-dimensional photoacoustic images due to the anisotropic spatial resolutions. Here we propose an innovative method to achieve isotropic resolutions for three-dimensional photoacoustic images through combined linear and rotational scanning. In each scan step, we first elevationally scan the linear transducer array, and then rotate the linear transducer array along its center in small steps, and scan again until 180 degrees have been covered. To reconstruct isotropic three-dimensional images from the multiple-directional scanning dataset, we use the standard inverse Radon transform originating from X-ray CT. We acquired a three-dimensional microsphere phantom image through the inverse Radon transform method and compared it with a single-elevational-scan three-dimensional image. The comparison shows that our method improves the elevational resolution by up to one order of magnitude, approaching the in-plane lateral-direction resolution. In vivo rat images were also acquired.

MultiSpectral optoacoustic tomography (MSOT) is an emerging modality that combines the high contrast of optical imaging with the spatial resolution and penetration depth of ultrasound, to provide detailed images of hemoglobin concentration and oxygenation. To facilitate accurate determination of changes in the vascularity and oxygenation of a biological tissue over time, a tumor in response to cancer therapy for example, an extensive study of stability and reproducibility of a small animal MSOT system has been performed. Investigations were first made with a stable phantom imaged repeatedly over time scales of hours, days and months to evaluate the reproducibility of the system over time. We found that the small animal MSOT system exhibited excellent reproducibility with a coefficient of variation (COV) in the measured MSOT signals of less than 8% over the course of 30 days and within 1.5% over a single day. Experiments performed in vivo demonstrated the potential for measurement of oxyhemoglobin over time in a realistic experimental setting. The effect of breathing medical air or oxygen under conditions of fixed respiration rate and body temperature within normal organs, including the spleen and kidneys, were investigated. The COV for oxyhemoglobin signals retrieved from spectral unmixing was assessed within both biological (different mouse) and imaging (different scan) replicates. As expected, biological replicates produced a large COV (up to 40% within the spleen) compared to imaging replicates within a single mouse (up to 10% within the spleen). Furthermore, no significant difference was found between data acquired by different operators. The data presented here suggest that MSOT is highly reproducible for both phantom and in vivo imaging, hence could reliably detect changes in oxygenation occurring in living subjects.

Capitalizing on endogenous hemoglobin contrast, photoacoustic computed tomography (PACT), a deep-tissue highresolution imaging modality, has drawn increasing interest in neuro-imaging. However, most existing studies are limited to functional imaging on the cortical surface, and the deep-brain structural imaging capability of PACT has never been demonstrated. Here, we explicitly studied the limiting factors of deep-brain PACT imaging. We found that the skull distorted the acoustic signal and blood suppressed the structural contrast from other chromophores. When the two effects are mitigated, PACT can provide high-resolution label-free structural imaging through the entire mouse brain. With 100 μm in-plane resolution, we can clearly identify major structures of the brain, and the image quality is comparable to that of magnetic resonance microscopy. Spectral PACT studies indicate that structural contrasts mainly originate from cytochrome and lipid. The feasibility of imaging the structure of the brain in vivo has also been discussed. Our results demonstrate that PACT is a promising modality for both structural and functional brain imaging.

In this work, we introduce an improved prototype of the imaging system that combines three-dimensional optoacoustic tomography (3D-OAT) and laser ultrasound tomography slicer (2D-LUT) to obtain coregistered maps of tissue optical absorption and speed of sound (SOS). The imaging scan is performed by a 360 degree rotation of a phantom/mouse with respect to a static arc-shaped array of ultrasonic transducers. A Q-switched laser system is used to establish optoacoustic illumination pattern appropriate for deep tissue imaging with a tunable (730-840 nm) output wavelengths operated at 10 Hz pulse repetition rate. For the LUT slicer scans, the array is pivoted by 90 degrees with respect to the central transducers providing accurate registration of optoacoustic and SOS maps, the latter being reconstructed using waveform inversion with source encoding (WISE) technique. The coregistered OAT-LUT modality is validated by imaging a phantom and a live mouse. SOS maps acquired in the imaging system can be employed by an iterative optoacoustic reconstruction algorithm capable of compensating for acoustic wavefield aberrations. The most promising applications of the imaging system include 3D angiography, cancer research, and longitudinal studies of biological distributions of optoacoustic contrast agents (carbon nanotubes, metal plasmonic nanoparticles, fluorophores, etc.).

Osteoporosis is a widespread disease that has a catastrophic impact on patient's lives and overwhelming related healthcare costs. In recent works, we have developed a multi-spectral, frequency domain photoacoustic method for the evaluation of bone pathologies. This method has great advantages over pure ultrasonic or optical methods as it provides both molecular information from the bone absorption spectrum and bone mechanical status from the characteristics of the ultrasound propagation. These characteristics include both the Speed of Sound (SOS) and Broadband Ultrasonic Attenuation (BUA). To test the method's quantitative predictions, we have constructed a combined ultrasound and photoacoustic setup. Here, we experimentally present a dual modality system, and compares between the methods on bone samples in-vitro. The differences between the two modalities are shown to provide valuable insight into the bone structure and functional status.

Phase transition contrast agents were first introduced in ultrasound (US) in the form of perfluorocarbon droplets. When their size is reduced to the nanoscale, surface tension dominates their stability and high pressure is required to vaporize them using long US emissions at high frequencies. Our group recently showed that nanoemulsion beads (100-300 nm) coated with gold nanopsheres could be used as non-linear contrast agents. Beads can be vaporized with light only, inducing stronger photoacoustic signals by increasing thermal expansion. A photoacoustic cavitation threshold study (US: 1.2 MHz, Laser 750 nm and 10-ns pulse) shows that the vaporization thresholds of NEB-GNS can be greatly reduced using simultaneous light and US excitations. The resulting signal is driven only by the pressure amplitude for a fluence higher than 2.4 mJ/cm². At diagnostic exposures, it is possible to capture very high signals from the vaporized beads at concentrations reduced to 10 pM with optical absorption smaller than 0.01 cm^-1. A real-time imaging mode selectively isolating vaporization signals was implemented on a Verasonics system. A linear US probe (L74, 3 MHz) launched short US bursts before light was emitted from the laser. Vaporization of NEB-GNS resulted in a persistent 30-dB signal enhancement compared to a dye with the same absorption. Specific vaporization signals were retrieved in phantom experiments with US scatterers. This technique, called sonophotoacoustics, has great potential for targeted molecular imaging and therapy using compact nanoprobes with potentially high-penetrability into tissue.

Magnetomotive photoacoustic/ultrasound imaging has shown superior specificity in visualizing targeted objects at cellular and molecular levels. By detecting magnet-induced displacements, magnetic-particles-targeted objects can be differentiated from background signals insensitive to the magnetic field. Unfortunately, background physiologic motion interferes during measurement, such as cardiac-induced motion and respiration, greatly reducing the robustness of the technique. In this paper, we proposed cyclic magnetomotive imaging with narrowband magnetic excitation. By synchronizing magnetic motion with the excitations, targeted objects moving coherently can be distinguished from background static signals and signals moving incoherently. HeLa cells targeted with magnetic nanoparticle-polymer core-shell particles were used as the targets for an initial test. A linear ultrasound array was interfaced with a commercial scanner to acquire a photoacoustic/ultrasound image sequence (maximum 1000 frames per second) during multi-cycle magnetic excitation (0.5 – 40 Hz frequency range) with an electromagnet. An image mask defined by a threshold on the displacement-coherence map was applied to the original images for background suppression. The results show that contrast was increased by more than 60 dB in an in-vitro experiment with the tagged cells fixed in a polyvinyl-alcohol gel and sandwiched between porcine liver tissues. Using a single sided system, cells injected subcutaneously on the back of a mouse were successfully differentiated from the background, with less than 20 µm coherent magnetic induced displacements isolated from millimetric background breathing motion. These results demonstrate the technique’s motion robustness for highly sensitive and specific diagnosis.

We have successfully developed a fully-sheathed, flexible shaft-based, mechanical scanning photoacoustic endoscopy (PAE) system for imaging the human gastrointestinal tract via the instrument channel of a clinical video endoscope. The endoscopic system uses a single element ultrasonic transducer and flexible shaft-based proximal actuation mechanism, and it has a 2.5 m long and 3.2 mm diameter catheter section, which can be accommodated in the 3.7 mm diameter instrument channel of a clinical video endoscope. Here, we demonstrate the intra-instrument channel workability and in vivo imaging capability of the PAE system.

Laser-Scanning-Optical-Resolution Photoacoustic Microscopy (LSOR-PAM) requires an ultrasound detector with a low noise equivalent pressure (NEP) and a large angular detection aperture in order to image a large field of view (FOV). It is however challenging to meet these requirements when using piezoelectric receivers since using a small sensing element size (<100μm) in order to achieve a large angular detection aperture will inevitability reduce the sensitivity of the detector as it scales with decreasing element size. Fibre optic ultrasound sensors based on a Fabry Perot cavity do not suffer from this limitation and can provide high detection sensitivity (NEP<0.1kPa over a 20 MHz measurement bandwidth) with a large angular detection aperture due to their small active element size (~10μm). A LSOR-PAM system was developed and combined with this type of fibre optic ultrasound sensor. A set of phantom studies were undertaken. The first study demonstrated that a high resolution image over a large field of view (Ø11mm) could be obtained with a sampledetector separation of only 1.6mm. In the second study, a 12μm diameter tube filled with methylene blue whose absorption coefficient was similar to that of blood was visualised demonstrating that the fibre optic sensor could provide sufficient SNR for in-vivo microvascular OR-PAM imaging. These preliminary results suggest that the fibre optic sensor has the potential to outperform piezoelectric detectors for Laser-Scanning Optical Resolution Photoacoustic Microscopy (LSOR-PAM).

Optical diffusion in scattering media prevents focusing beyond shallow depths, causing optical imaging and sensing to suffer from low optical intensities, resulting in low signal-to-noise ratios (SNR). Here, we demonstrate focusing using a fast binary-amplitude digital micromirror device to characterize the transmission modes of the scattering medium. We then identify and selectively illuminate the transmission modes which contribute constructively to the intensity at the optical focus. Applying this method to photoacoustic flowmetry, we increased the optical intensity at the focus six-fold, and showed that the corresponding increase in SNR allows particle flow to be measured.

A range of miniature (125μm o.d.) fibre optic ultrasound sensors based on the use of interferometric polymer optical cavities has been developed for minimally invasive photoacoustic imaging and sensing applications. It was observed that by careful selection of both the fibre tip and cavity geometry it is possible to achieve exceptional acoustic performance. Specifically, rounding the tip of the fibre to remove the presence of sharp diffractive boundaries enables a well behaved frequency response along with a near omnidirectional response at frequencies in the tens of MHz range to be achieved. The use of a plano-convex rather than a planar cavity provides high finesse and therefore detection sensitivity. Thus, by using a plano-convex cavity formed at the tip of radiused single mode fibre it was possible to realise a miniature ultrasound detector with a bandwidth of 80MHz, a noise-equivalent pressure of 40Pa (over a 20MHz measurement bandwidth) and a near omnidirectional response at frequencies as high as 30MHz. These characteristics suggest this type of sensor could find applications in interventional medicine for guiding needles or catheters, as mechanically scanned photoacoustic imaging probes or in laser scanning OR-PAM.

Photoacoustic images of exquisite quality have previously been obtained using planar Fabry-Pérot ultrasound sensors, as they can synthesize detection arrays with small, highly sensitive, elements. However, their planarity prevents reconstruction of structures perpendicular to the sensor plane, which gives rise to limited-view artifacts. Here, a novel FP sensor array configuration is described that incorporates two orthogonal planar arrays in order to overcome this limitation. Three dimensional photoacoustic images of suitably structured phantoms, obtained using a time reversal reconstruction algorithm, are used to demonstrate the significant improvement in the reconstructed images.

Conventional photoacoustic computed tomography (PACT) image reconstruction methods assume that the object and surrounding medium are described by a constant speed-of-sound (SOS) value. In order to accurately recover fine structures, SOS heterogeneities should be quantified and compensated for during PACT reconstruction. To address this problem, several groups have proposed hybrid systems that combine PACT with ultrasound computed tomography (USCT). In such systems, a SOS map is reconstructed first via USCT. Consequently, this SOS map is employed to inform the PACT reconstruction method. Additionally, the SOS map can provide structural information regarding tissue, which is complementary to the functional information from the PACT image. We propose a paradigm shift in the way that images are reconstructed in hybrid PACT-USCT imaging. Inspired by our observation that information about the SOS distribution is encoded in PACT measurements, we propose to jointly reconstruct the absorbed optical energy density and SOS distributions from a combined set of USCT and PACT measurements, thereby reducing the two reconstruction problems into one. This innovative approach has several advantages over conventional approaches in which PACT and USCT images are reconstructed independently: (1) Variations in the SOS will automatically be accounted for, optimizing PACT image quality; (2) The reconstructed PACT and USCT images will possess minimal systematic artifacts because errors in the imaging models will be optimally balanced during the joint reconstruction; (3) Due to the exploitation of information regarding the SOS distribution in the full-view PACT data, our approach will permit high-resolution reconstruction of the SOS distribution from sparse array data.

We propose a new reconstruction algorithm for photoacoustic and laser-ultrasound imaging based on reverse time migration (RTM), a time reversal imaging algorithm originally developed for exploration seismology. RTM inherently handles strong velocity heterogeneity and complex propagation paths. A successful RTM analysis with appropriate handling of boundary conditions results in enhanced signal-to-noise, accurately located structures, and minimal artifacts. A laser-ultrasound experiment begins with a source wave field generated at the surface that propagates through the sample. Acoustic scatterers in the propagation path give rise to a scattered wave field, which travels to the surface and is recorded by acoustic detectors. To reconstruct the laser-ultrasound image, a synthetic source function is forward propagated and cross-correlated with the time-reversed and back-propagated recorded (scattered) wave field to image the scatterers at the correct location. Conversely, photoacoustic waves are generated by chromophores within the sample and propagate "one-way" to the detection surface. We utilize the velocity model validated by the laser-ultrasound reconstruction to accurately reconstruct the photoacoustic image with RTM. This approach is first validated with simulations, where inclusions behave both as a photoacoustic source and an acoustic scatterer. Subsequently, we demonstrate the capabilities of RTM with tissue phantom experiments using an all-optical, multi-channel acquisition geometry.

Iterative image reconstruction algorithms can model complicated imaging physics, compensate for imperfect data acquisition systems, and exploit prior information regarding the object. Hence, they produce higher quality images than do analytical image reconstruction algorithms. However, three-dimensional (3D) iterative image reconstruction is computationally burdensome, which greatly hinders its use with applications requiring a large field-of-view (FOV), such as breast imaging. In this study, an improved GPU-based implementation of a numerical imaging model and its adjoint have been developed for use with general gradient-based iterative image reconstruction algorithms. Both computer simulations and experimental studies are conducted to investigate the efficiency and accuracy of the proposed implementation for optoacoustic tomography (OAT). The results suggest that the proposed implementation is more than five times faster than the previous implementation.

Targeted contrast agents can improve the sensitivity of imaging systems for cancer detection and monitoring the treatment. In order to accurately detect contrast agent concentration from photoacoustic images, we developed a decomposition algorithm to separate photoacoustic absorption spectrum into components from individual absorbers. In this study, we evaluated novel prostate-specific membrane antigen (PSMA) targeted agents for imaging prostate cancer. Three agents were synthesized through conjugating PSMA-targeting urea with optical dyes ICG, IRDye800CW and ATTO740 respectively. In our preliminary PA study, dyes were injected in a thin wall plastic tube embedded in water tank. The tube was illuminated with pulsed laser light using a tunable Q-switch ND-YAG laser. PA signal along with the B-mode ultrasound images were detected with a diagnostic ultrasound probe in orthogonal mode. PA spectrums of each dye at 0.5 to 20 μM concentrations were estimated using the maximum PA signal extracted from images which are obtained at illumination wavelengths of 700nm-850nm. Subsequently, we developed nonnegative linear least square optimization method along with localized regularization to solve the spectral unmixing. The algorithm was tested by imaging mixture of those dyes. The concentration of each dye was estimated with about 20% error on average from almost all mixtures albeit the small separation between dyes spectrums.

Photoacoustic (PA) imaging is becoming an important tool for various clinical and pre-clinical applications. Acquiring pre-beamformed channel ultrasound data is essential to reconstruct PA images. Accessing these pre-beamformed channel data requires custom hardware to allow parallel beam-forming, and is available for only few research ultrasound platforms. However, post-beamformed radio frequency (RF) data is readily available in real-time and in several clinical and research ultrasound platforms. To broaden the impact of clinical PA imaging, our goal is to devise new PA reconstruction approach based on these post-beamformed RF data. In this paper, we propose to generate PA image by using a single receive focus beamformed RF data. These beamformed RF data are considered as pre-beamformed input data to a synthetic aperture beamforming algorithm, where the focal point per received RF line is a virtual element. The image resolution is determined by the fixed focusing depth as well as the aperture size used in fixed focusing. In addition, the signal-to-noise (SNR) improvement is expected because beamforming is performed twice with different noise distribution. The performance of the proposed method is analyzed through simulation, the practical feasibility is validated experimentally. The results indicate that the post-beamformed RF data has potential to be re-beamformed to a PA image using the proposed synthetic aperture beamformer.

Plano-convex optical microresonator detectors have been developed as an alternative to planar Fabry-Pérot (FP) sensors used in all-optical photoacoustic imaging systems with the potential to provide two or more orders-of-magnitude higher detection sensitivity. This study further characterises the performance of these detectors by investigating their normal incidence frequency response and frequency-dependent directivity. It is shown that sensors with thicknesses in the range ~50-320μm provide broadband, smooth frequency response characteristics and low directional sensitivity. This suggests that a photoacoustic imaging system based on microresonator detectors may be capable of imaging with similar performance to the FP system but with significantly higher sensitivity, paving the way to deep tissue imaging applications such as the clinical assessment of breast cancer and preclinical whole body small animal imaging.

The feasibility of an innovative biomedical diagnostic technique, thermal photoacoustic (TPA) measurement, for nonionizing and non-invasive assessment of bone health is investigated. Unlike conventional photoacoustic PA methods which are mostly focused on the measurement of absolute signal intensity, TPA targets the change in PA signal intensity as a function of the sample temperature, i.e. the temperature dependent Grueneisen parameter which is closely relevant to the chemical and molecular properties in the sample. Based on the differentiation measurement, the results from TPA technique is less susceptible to the variations associated with sample and system, and could be quantified with improved accurately. Due to the fact that the PA signal intensity from organic components such as blood changes faster than that from non-organic mineral under the same modulation of temperature, TPA measurement is able to objectively evaluate bone mineral density (BMD) and its loss as a result of osteoporosis. In an experiment on well established rat models of bone loss and preservation, PA measurements of rat tibia bones were conducted over a temperature range from 37⁰ C to 44⁰ C. The slope of PA signal intensity verses temperature was quantified for each specimen. The comparison among three groups of specimens with different BMD shows that bones with lower BMD have higher slopes, demonstrating the potential of the proposed TPA technique in future clinical management of osteoporosis.

We developed a simple and effective contrast for tissue characterization based on the recently proposed dual-pulse nonlinear photoacoustic technology. The new contrast takes advantage of the temperature dependence of Grüneisen parameter of tissue and involves a dual-pulse laser excitation process. A short pulse first heats the sample and causes a temperature jump, which then leads to the change of Grüneisen parameter and amplitude of the photoacoustic signal of the second pulse. For different tissues, the induced rate or trend of change is expected to be different, which constitutes the basis of the new contrast. Preliminary phantom experiment in blood and lipid mixtures and in vitro experiment in fatty rat liver have demonstrated that the proposed contrast has the capability of fast characterization of lipid-rich and blood-rich tissues.

It has previously been shown that photoacoustic imaging can interrogate lifetimes of exogenous agents by a sequence of pulses with varying pump-probe delay intervals. Rather than attempt to unmix molecules based on their composite lifetime profile, we introduce a technique called lifetime weighted imaging, which preferentially weights signals from chromophores with long lifetimes (including exogenous contrast agents such as methylene blue and porphyrins with microsecond-scale lifetimes) while nulling chromophores with short lifetimes (including hemoglobin with ps-ns-scale lifetimes). A probe beam is used to interrogate samples with or without a pump beam. By subtracting probe-beam photoacoustic signals with pump- from those without a pump excitation, we effectively eliminate probe signals from chromophores with short lifetimes while preserving excited-state photoacoustic signals from long-lifetimes. This differential signal will be weighted by a decaying exponential function of the pump-probe delay divided by the exogenous agent lifetime. This technique enabled the imaging of both triplet excited state lifetime and ground-state recovery lifetime. We demonstrate the oxygen-dependent lifetime of both methylene blue and porphyrins. Lifetimeweighted imaging could be used for photodynamic therapy dosimetry guidance, oxygen sensing, or other molecular imaging applications.

We introduce a new approach for multiplexing fiber-based ultrasound sensors using Optical Frequency Domain Reflectometry (OFDR). In the present demonstration of the method, each sensor was a short section of Polyimide-coated single-mode fiber. One end of the sensing fiber was pigtailed to a mirror and the other end was connected, via a fiber optic delay line, to a 1X4 fiber coupler. The multiplexing was enabled by using a different delay to each sensor. Ultrasonic excitation was performed by a 1MHz transducer which transmitted 4μs tone-bursts above the sensor array. The ultrasound waves generated optical phase variations in the fibers which were detected using the OFDR method. The ultrasound field at the sensors was successfully reconstructed without any noticeable cross-talk.

We report on an optoacoustic imaging system capable of acquiring volumetric multispectral optoacoustic data in real time. The system is based on simultaneous acquisition of optoacoustic signals from 256 different tomographic projections by means of a spherical matrix array. Thereby, volumetric reconstructions can be done at high frame rate, only limited by the pulse repetition rate of the laser. The developed tomographic approach presents important advantages over previously reported systems that use scanning for attaining volumetric optoacoustic data. First, dynamic processes, such as the biodistribution of optical biomarkers, can be monitored in the entire volume of interest. Second, out-of-plane and motion artifacts that could degrade the image quality when imaging living specimens can be avoided. Finally, real-time 3D performance can obviously save time required for experimental and clinical observations. The feasibility of optoacoustic imaging in five dimensions, i.e. real time acquisition of volumetric datasets at multiple wavelengths, is reported. In this way, volumetric images of spectrally resolved chromophores are rendered in real time, thus offering an unparallel imaging performance among the current bio-imaging modalities. This performance is subsequently showcased by video-rate visualization of in vivo hemodynamic changes in mouse brain and handheld visualization of blood oxygenation in deep human vessels. The newly discovered capacities open new prospects for translating the optoacoustic technology into highly performing imaging modality for biomedical research and clinical practice with multiple applications envisioned, from cardiovascular and cancer diagnostics to neuroimaging and ophthalmology.

Three-dimensional hand-held optoacoustic imaging comes with important advantages that prompt the clinical translation of this modality, with applications envisioned in cardiovascular and peripheral vascular disease, disorders of the lymphatic system, breast cancer, arthritis or inflammation. Of particular importance is the multispectral acquisition of data by exciting the tissue at several wavelengths, which enables functional imaging applications. However, multispectral imaging of entire three-dimensional regions is significantly challenged by motion artefacts in concurrent acquisitions at different wavelengths. A method based on acquisition of volumetric datasets having a microsecond-level delay between pulses at different wavelengths is described in this work. This method can avoid image artefacts imposed by a scanning velocity greater than 2 m/s, thus, does not only facilitate imaging influenced by respiratory, cardiac or other intrinsic fast movements in living tissues, but can achieve artifact-free imaging in the presence of more significant motion, e.g., abrupt displacements during handheld-mode operation in a clinical environment.

A system for dynamic mapping of broadband ultrasound fields has been designed, with high frame rate photoacoustic imaging in mind. A Fabry-Pérot interferometric ultrasound sensor was interrogated using a coherent light single-pixel camera. Scrambled Hadamard measurement patterns were used to sample the acoustic field at the sensor, and either a fast Hadamard transform or a compressed sensing reconstruction algorithm were used to recover the acoustic pressure data. Frame rates of 80 Hz were achieved for 32x32 images even though no specialist hardware was used for the on-the-fly reconstructions. The ability of the system to obtain photocacoustic images with data compressions as low as 10% was also demonstrated.

In quantitative photoacoustic imaging, the aim is to recover physiologically relevant tissue parameters such as chromophore concentrations or oxygen saturation. Obtaining accurate estimates is challenging due to the non-linear relationship between the concentrations and the photoacoustic images. Nonlinear least squares inversions designed to tackle this problem require a model of light transport, the most accurate of which is the radiative transfer equation. This paper presents a highly scalable Monte Carlo model of light transport that computes the radiance in 2D using a Fourier basis to discretise in angle. The model was validated against a 2D finite element model of the radiative transfer equation, and was used to compute gradients of an error functional with respect to the absorption and scattering coefficient. It was found that adjoint-based gradient calculations were much more robust to inherent Monte Carlo noise than a finite difference approach. Furthermore, the Fourier angular discretisation allowed very efficient gradient calculations as sums of Fourier coefficients. These advantages, along with the high parallelisability of Monte Carlo models, makes this approach an attractive candidate as a light model for quantitative inversion in photoacoustic imaging.

In this paper, we investigate the feasibility of high-frequency photoacoustic (PA) imaging to study the shear rate dependent relationship between red blood cell (RBC) aggregation and oxygen saturation (SO₂) in a simulated blood flow system. The PA signal amplitude increased during the formation of aggregates and cyclically varied at intervals corresponding to the beat rate (30, 60, 120, 180 and 240 bpm) for all optical wavelengths of illumination (750 and 850 nm).The SO₂ also cyclically varied in phase with the PA signal amplitude for all beat rates. In addition, the mean blood flow velocity cyclically varied at the same interval of beat rate, and the shear rate (i.e. the radial gradient of flow velocity) also cyclically varied. On the other hand, the phase of the cyclic variation in the shear rate was reversed compared to that in the PA signal amplitude. This study indicates that RBC aggregation induced by periodic changes in the shear rate can be correlated with the SO₂ under pulsatile blood flow. Furthermore, PA imaging of flowing blood may be capable of providing a new biomarker for the clinical application in terms of monitoring blood viscosity, oxygen delivery and their correlation.

Blood oxygen saturation (SO2) reflects the oxygenation level in blood transport and tissue. Previous studies have shown the capability of non-invasive quantitative measurements of SO2 by multi-wavelength photoacoustic (PA) spectroscopy for diagnosis of brain, tumor hemodynamics and other pathophysiological phenomena. However, those multi-wavelength methods require a tunable laser or multiple lasers which are relatively expensive and bulky for filed measurement environment and applications. Besides, the operation of multiple wavelengths, calibration procedures and data processing gets system complicated, which reduces the feasibility and flexibility for continuous real-time monitoring. Here we report a newly proposed method by combining PA and scattered light signals wherein imposing a hypothesis that scattering intensity is linear to the concentrations of oxygenated hemoglobin and deoxygenated hemoglobin weighed by blood scattering coefficients. A rigorous theoretical relationship between PA and scattering signals is thus established, making it possible that SO2 can be measured with only one excitation wavelength. To verify the theory basis, both dual-ink phantoms and fresh porcine blood sample have been employed in the experiments. The phantom experiment is able to quantify the concentration of mixed red-green ink that is in precise agreement with pre-set values. The ex vivo experiment with fresh porcine blood was conducted and the results of the proposed single-wavelength method achieved high accuracy of 1% - 4% errors. These demonstrated that the proposed single-wavelength SO2 detection is able to provide non-invasive, accurate measurement of blood oxygenation, and herein create potential for applying it to real clinical applications with low cost and high flexibility.

Ultrasound (US) guided biopsy is the standard procedure for evaluating the presence of prostate cancer (PCa). The biopsied tissues are evaluated by a Gleason scoring system which quantifies the severity of prostate cancer based on the clustering patterns of the cancer cells. The clustering patterns can be visualized by staining the cancer cells with optical contrast enhancing nanoparticles (NPs). This study investigates the feasibility of evaluating the NP stained Gleason patterns using photoacoustic spectrum analysis (PASA), aiming toward achieving minimally invasive prostate biopsy. The experiment on H&E stained human prostate tissue slices demonstrates that this approach is capable of identifying and potentially grading PCa.

Staging of cancers and selection of appropriate treatment requires histological examination of multiple dissected lymph nodes (LNs) per patient, so that a staggering number of nodes require histopathological examination, and the finite resources of pathology facilities create a severe processing bottleneck. Histologically examining the entire 3D volume of every dissected node is not feasible, and therefore, only the central region of each node is examined histologically, which results in severe sampling limitations. In this work, we assess the feasibility of using quantitative photoacoustics (QPA) to overcome the limitations imposed by current procedures and eliminate the resulting under sampling in node assessments. QPA is emerging as a new hybrid modality that assesses tissue properties and classifies tissue type based on multiple estimates derived from spectrum analysis of photoacoustic (PA) radiofrequency (RF) data and from statistical analysis of envelope-signal data derived from the RF signals. Our study seeks to use QPA to distinguish cancerous from non-cancerous regions of dissected LNs and hence serve as a reliable means of imaging and detecting small but clinically significant cancerous foci that would be missed by current methods. Dissected lymph nodes were placed in a water bath and PA signals were generated using a wavelength-tunable (680-950 nm) laser. A 26-MHz, f-2 transducer was used to sense the PA signals. We present an overview of our experimental setup; provide a statistical analysis of multi-wavelength classification parameters (mid-band fit, slope, intercept) obtained from the PA signal spectrum generated in the LNs; and compare QPA performance with our established quantitative ultrasound (QUS) techniques in distinguishing metastatic from non-cancerous tissue in dissected LNs. QPA-QUS methods offer a novel general means of tissue typing and evaluation in a broad range of disease-assessment applications, e.g., cardiac, intravascular, musculoskeletal, endocrine-gland, etc.

The fast heart rate (~7 Hz) of the mouse makes cardiac imaging and functional analysis difficult when studying mouse models of cardiovascular disease, and cannot be done truly in real-time and 3D using established imaging modalities. Optoacoustic imaging, on the other hand, provides ultra-fast imaging at up to 50 volumetric frames per second, allowing for acquisition of several frames per mouse cardiac cycle. In this study, we combined a recently-developed 3D optoacoustic imaging array with novel analytical techniques to assess cardiac function and perfusion dynamics of the mouse heart at high, 4D spatiotemporal resolution. In brief, the heart of an anesthetized mouse was imaged over a series of multiple volumetric frames. In another experiment, an intravenous bolus of indocyanine green (ICG) was injected and its distribution was subsequently imaged in the heart. Unique temporal features of the cardiac cycle and ICG distribution profiles were used to segment the heart from background and to assess cardiac function. The 3D nature of the experimental data allowed for determination of cardiac volumes at ~7-8 frames per mouse cardiac cycle, providing important cardiac function parameters (e.g., stroke volume, ejection fraction) on a beat-by-beat basis, which has been previously unachieved by any other cardiac imaging modality. Furthermore, ICG distribution dynamics allowed for the determination of pulmonary transit time and thus additional quantitative measures of cardiovascular function. This work demonstrates the potential for optoacoustic cardiac imaging and is expected to have a major contribution toward future preclinical studies of animal models of cardiovascular health and disease.

In photoacoustic tomography (PAT) the image contrast is due to optical absorption, and because of this PAT images are sensitive to changes in blood oxygen saturation (_sO₂). However, this is not a linear relationship due to the presence of a non-uniform light fluence distribution. In this paper we systematically evaluate the conditions in which an approximate linear inversion scheme–which assumes the internal fluence distribution is unchanged when the absorption coefficient changes–can give accurate estimates of _sO₂. A numerical phantom of highly vascularised tissue is used to test this assumption. It is shown that using multiple wavelengths over a broad range of the near-infrared spectrum yields inaccurate estimates of oxygenation, while a careful selection of wavelengths in the 620-920nm range is likely to yield more accurate oxygenation values. We demonstrate that a 1D fluence correction obtained by fitting a linear function to the average decay rate in the image can further improve the estimates. However, opting to use these longer wavelengths involves sacrificing signal-to-noise ratio in the image, as the absorption of blood is low in this range. This results in an inherent trade-off between error in the _sO₂ estimates due to fluence variation and error due to noise. This study shows that the depth to which _sO₂ can be estimated accurately using a linear approximation is limited in vivo, even with idealised measurements, to at most 3mm. In practice, there will be even greater uncertainties affecting the estimates, e.g., due to bandlimited or partial-view acoustic detection.

Quantification of the optical properties of the tissues and blood by noninvasive photoacoustic (PA) imaging may provide useful information for screening and early diagnosis of diseases. Linearized 2D image reconstruction algorithm based on PA wave equation and the photon diffusion equation (PDE) can reconstruct the image with computational cost smaller than a method based on 3D radiative transfer equation. However, the reconstructed image is affected by the differences between the actual and assumed light propagations. A quantitative capability of a linearized 2D image reconstruction was investigated and discussed by the numerical simulations and the phantom experiment in this study. The numerical simulations with the 3D Monte Carlo (MC) simulation and the 2D finite element calculation of the PDE were carried out. The phantom experiment was also conducted. In the phantom experiment, the PA pressures were acquired by a probe which had an optical fiber for illumination and the ring shaped P(VDF-TrFE) ultrasound transducer. The measured object was made of Intralipid and Indocyanine green. In the numerical simulations, it was shown that the linearized image reconstruction method recovered the absorption coefficients with alleviating the dependency of the PA amplitude on the depth of the photon absorber. The linearized image reconstruction method worked effectively under the light propagation calculated by 3D MC simulation, although some errors occurred. The phantom experiments validated the result of the numerical simulations.

We found and interpreted the universal temperature-dependent optoacoustic (photoacoustic) response (ThOR) in blood; the normalized ThOR is invariant with respect to hematocrit at the hemoglobin’s isosbestic point. The unique compartmentalization of hemoglobin, the primary optical absorber at 805 nm, inside red blood cells (RBCs) explains the effect. We studied the temperature dependence of Gruneisen parameter in blood and aqueous solutions of hemoglobin and for the first time experimentally observed transition through the zero optoacoustic response at temperature T₀, which was proved to be consistent for various blood samples. On the other hand, the hemoglobin solutions demonstrated linear concentration function of the temperature T₀. When this function was extrapolated to the average hemoglobin concentration inside erythrocytes, the temperature T₀ was found equivalent to that measured in whole and diluted blood. The obtained universal curve of blood ThOR was validated in both transparent and light scattering media. The discovered universal optoacoustic temperature dependent blood response provides foundation for future development of non-invasive in vivo temperature monitoring in vascularized tissues and blood vessels.

In vivo photoacoustic estimations of tumor oxygenation were used to assess the therapeutic efficacy of a thermosensitive liposome treatment in a pre-clinical mouse model. The treated group (n = 12) was administered doxorubicin-loaded, heat sensitive liposomes and exposed to mild hyperthermia (43°C) in order to deliver doxorubicin locally within the tumor micro-vessels. Control groups received systemic doxorubicin (n = 7) or saline (n = 12). The changes in tumor blood vessels after treatment were probed by analyzing the frequency content of the photoacoustic radiofrequency signals. Tumor oxygenation dropped by 15-20% during the first 30 minutes post-treatment when the tumors were exposed to encapsulated (Heat-Activated cyToxic – HaT-DOX) or free doxorubicin (DOX). The early (30 minutes to 5 hours) decrease in oxygen saturation strongly correlated to the reduction in tumor size assessed by caliper measurements. Control animals did not exhibit significant changes in tumor oxygenation at the early time points. The oxygenation at 7 days increased significantly for all groups. Measurements of the spectral slope from the normalized power spectra of the photoacoustic signals could also be used to differentiate between responder and non-responder mice. The results of this study suggest that photoacoustic imaging of tumors undergoing vascular-targeted cancer therapy can be used to assess treatment response early (hours) post-treatment through a combined analysis of oxygen saturation and photoacoustic radiofrequency spectroscopy.

The oxygen partial pressure (pO2), which results from the balance between oxygen delivery and its consumption, is a key component of the physiological state of a tissue. Images of oxygen distribution can provide essential information for identifying hypoxic tissue and optimizing cancer treatment. Previously, we have reported a noninvasive in vivo imaging modality based on photoacoustic lifetime. The technique maps the excited triplet state of oxygen-sensitive dye, thus reflects the spatial and temporal distribution of tissue oxygen. We have applied PALI on tumor on small animals to identify hypoxia area. We also showed that PALI is able monitor changes of tissue oxygen, in an acute ischemia and breathing modulation model. Here we present our work on developing a treatment/imaging modality (PDT-PALI) that integrates PDT and a combined ultrasound/photoacoustic imaging system. The system provides real-time feedback of three essential parameters namely: tissue oxygen, light penetration in tumor location, and distribution of photosensitizer. Tissue oxygen imaging is performed by applying PALI, which relies on photoacoustic probing of oxygen-dependent, excitation lifetime of Methylene Blue (MB) photosensitizer. Lifetime information can also be used to generate image showing the distribution of photosensitizer. The level and penetration depth of PDT illumination can be deduced from photoacoustic imaging at the same wavelength. All images will be combined with ultrasound B-mode images for anatomical reference.

Detection of tissue structures such as nerves and blood vessels is of critical importance during many needle-based minimally invasive procedures. For instance, unintentional injections into arteries can lead to strokes or cardiotoxicity during interventional pain management procedures that involve injections in the vicinity of nerves. Reliable detection with current external imaging systems remains elusive. Optical generation and reception of ultrasound allow for depth-resolved sensing and they can be performed with optical fibers that are positioned within needles used in clinical practice. The needle probe developed in this study comprised separate optical fibers for generating and receiving ultrasound. Photoacoustic generation of ultrasound was performed on the distal end face of an optical fiber by coating it with an optically absorbing material. Ultrasound reception was performed using a high-finesse Fabry-Pérot cavity. The sensor data was displayed as an M-mode image with a real-time interface. Imaging was performed on a biological tissue phantom.

Imaging the vasculature close around the finger joints is of interest in the field of rheumatology. Locally increased vasculature in the synovial membrane of these joints can be a marker for rheumatoid arthritis. In previous work we showed that part of the photoacoustically induced ultrasound from the epidermis reflects on the bone surface within the finger. These reflected signals could be wrongly interpreted as new photoacoustic sources. In this work we show that a conventional ultrasound reconstruction algorithm, that considers the skin as a collection of ultrasound transmitters and the PA tomography probe as the detector array, can be used to delineate bone surfaces of a finger. This can in the future assist in the localization of the joint gaps. This can provide us with a landmark to localize the region of the inflamed synovial membrane. We test the approach on finger mimicking phantoms.

We present spatially Fourier-encoded photoacoustic microscopy using a digital micromirror device (DMD). The spatial fluence distribution of laser pulses is Fourier-encoded by the DMD, and a series of such encoded photoacoustic (PA) measurements enables decoding of the spatial distribution of optical absorption. By imaging a chromium target, we demonstrated the throughput and Fellgett advantages, which increased the PA signal-to-noise ratio (SNR) compared to raster scanning. The system was used to image two biological targets, a monolayer of red blood cells, and melanoma cells. The enhanced SNR benefited PA images by increasing the image’s contrast-to-noise ratio and target identifiability.

We developed a reflection-mode, raster-scan optoacoustic mesoscopy system, based on a custom-made ultrasonic detector, with an ultra wide bandwidth of 20-180 MHz. To optimally use this bandwidth, we implemented multifrequency reconstruction. System characterization reveals a 4 μm axial, and 18 μm transverse resolution, at penetration depths reaching 5 mm. After characterization, the system was applied to image a zebrafish ex vivo, and an excised mouse ear. In the zebrafish, the lateral line, intestines, eyes, and melanocytes are seen, while in the mouse ear, multi-frequency reconstruction recovered the small vessels, otherwise not seen on the image.

Combining the absorption-based photoacoustic effect and intensity-dependent photobleaching effect, we demonstrate a simple method for super-resolution photoacoustic imaging of both fluorescent and non-fluorescent samples. Our method is based on a double-excitation process, where the first excitation pulse partially and inhomogeneously bleaches the molecules in the diffraction-limited excitation volume, thus biasing the signal contributions from a second excitation pulse striking the same region. By scanning the excitation beam, we performed three-dimensional sub-diffraction imaging of varied fluorescent and non-fluorescent species. A lateral resolution of 80 nm and an axial resolution of 370 nm have been demonstrated. This technique has the potential to enable label-free super-resolution imaging, and can be transferred to other optical imaging modalities or combined with other super-resolution methods.

Acoustic droplet vaporization has been proposed for sonoporation. In this study, we hypothesize that, by using gold nanodroplets (AuNDs), vaporization can be triggered with external application of laser irradiation. In addition, the vaporization assisted sonoporation can enhance delivery of gold nanoparticles (AuNPs) into the cells, thus potentially enhancing effects of plasmonic photothermal therapy. To test our hypothesis, in vitro studies were conducted. The delivery efficiency of AuNDs was also compared to that of AuNPs encapsulated in ultrasound microbubbles (AuMBs). The inertial cavitation dose (ICD), and optical density (OD) value of AuNPs were all measured under the applications of ultrasound only, laser only, and both ultrasound and laser. Results show that the cavitational effects and microbubble destruction were the highest with both ultrasound and laser being applied. In addition, destruction ratio of AuNDs was around 43%, compared to 35% microbubble destruction of AuMBs. Likewise, the OD value of AuNDs is 1.3 times higher than that of AuMBs under the same conditions, indicating that cavitation resulting from microbubble destruction did have the capability to assist the delivery of AuNPs into the cells. After the delivery, laser heating resulted in cell death. The cell viability with AuNDs was 45% left in the in vitro studies. Synergistic effects were also evident when combing laser with ultrasound.

Antibiotic drug resistance is a major worldwide issue. Development of new therapies against pathogenic bacteria requires appropriate research tools for replicating and characterizing infections. Previously fluorescence and bioluminescence modalities have been used to image infectious burden in animal models but scattering significantly limits imaging depth and resolution. We hypothesize that photoacoustic imaging, which has improved depth-toresolution ratio, could be useful for visualizing MelA-expressing bacteria since MelA is a bacterial tyrosinase homologue involved in melanin production. Using an inducible expression system, E. coli expressing MelA were visibly black in liquid culture. Phosphate buffered saline (PBS), MelA-expressing bacteria (at different dilutions in PBS), and chicken embryo blood were injected in plastic tubes which were imaged using a VisualSonics Vevo LAZR system. Photoacoustic imaging at 6 different wavelengths (680, 700, 750, 800, 850 and 900nm) enabled spectral de-mixing to distinguish melanin signals from blood. The signal to noise ratio of 9x diluted MelA bacteria was 55, suggesting that ~20 bacteria cells could be detected with our system. When MelA bacteria were injected as a 100 μL bolus into a chicken embryo, photoacoustic signals from deoxy- and oxy- hemoglobin as well as MelA-expressing bacteria could be separated and overlaid on an ultrasound image, allowing visualization of the bacterial location. Photoacoustic imaging may be a useful tool for visualizing bacterial infections and further work incorporating photoacoustic reporters into infectious bacterial strains is warranted.

Activatable photoacoustic probes hold great promise for in vivo imaging of enzyme activity as they exhibit high contrast and high selectivity at depths similar to that of ultrasound imaging. Here we report the synthesis and testing of a matrix metalloproteinase 2 (MMP-2) specific peptide probe capable of changing its transient photoacoustic lifetime from short to long after activation. The intact probe comprises an enzyme-specific cleavable sequence conjugated to a pair of methylene blue (MB) molecules that dimerize due to forced proximity, resulting in static quenching. Upon cleavage, the MB molecules dissociate and recover their intrinsic excited-state lifetime of more than 2 μs. We demonstrated using a pump-probe photoacoustic imaging approach that the cleaved probe exhibits a transient signal with lifetime comparable to that of MB monomers, whereas no lifetime was detected for the intact probe. This could allow for detection of the cleaved probe in the presence of high levels of intact probe as well as short transients due to endogenous tissue absorbers, thereby providing high contrast and low background noise. Finally, we have compared peptide sequences of varying length and structure using absorption spectrometry in order to select the probe best suited for our imaging needs. Our results suggest that both factors could potentially have an impact on dimerization efficiency and the cleavage rate of the peptide. However, all probes provided a high degree of dimerization and efficient separation after cleavage, indicating that a lifetime-based activatable peptide can be constructed for in vivo applications using a two-wavelength imaging approach.

Purpose: Photoacoustic imaging (PAI) enables one to visualize the distribution of hemoglobin and acquire a map of microvessels without using contrast agents. The purpose of our study is to develop a clinically applicable PAI system integrated with a clinical ultrasound (US) array system with handheld PAI probes providing coregistered PAI and US images. Clinical research trials were performed to evaluate the performance and feasibility of clinical value.

Materials and Methods: We developed two types of handheld PAI probes: a linear PAI probe combining a conventional linear-array US probe with optical illumination and a transrectal ultrasonography (TRUS)-type PAI probe. We performed experiments with Japanese white rabbits and conducted clinical research trials of urology and vascular medicine with the approval of the medical human ethics committee of the National Defense Medical College.

Results: We successfully acquired high-dynamic-range images of the vascular network ranging from capillaries to landmark arteries and identified the femoral vein, deep femoral vein, and great saphenous vein of rabbits. These major vessels in the rabbits groin are surrounded with microvessels connected to each other. Periprostatic microvessels were monitored during radical prostatectomy for localized prostate cancer and they were colocalized with nerve fibers, and their distribution was consistent with the corresponding PAI. The TRUS-type PAI probe clearly demonstrated the location and extent of the neurovascular bundle (NVB) better than does TRUS alone.

Conclusions: The system, which can obtain a PAI, a US image, and a merged image, was innovatively designed so that medical doctors can easily find the location without any prior knowledge or extended skills to analyze the obtained images. Our pilot feasibility study confirms that PAI could be an imaging modality useful in the screening study and diagnostic biopsy.

A method for simultaneously measuring the absorption coefficient μa and Grüneisen parameter Γ of biological absorbers in photoacoustics is designed and implemented using a coupled-integrating sphere system. A soft transparent tube with inner diameter of 0.58mm is used to mount the liquid absorbing sample horizontally through the cavity of two similar and adjacent integrating spheres. One sphere is used for measuring the sample’s μa using a continuous halogen light source and a spectrometer fiber coupled to the input and output ports, respectively. The other sphere is used for simultaneous photoacoustic measurement of the sample’s Γ using an incident pulsed light with wavelength of 750nm and a flat transducer with central frequency of 5MHz. Absolute optical energy and pressure measurements are not necessary. However, the derived equations for determining the sample’s μa and Γ require calibration of the setup using aqueous ink dilutions. Initial measurements are done with biological samples relevant to biomedical imaging such as human whole blood, joint and cyst fluids. Absorption of joint and cyst fluids is enhanced using a contrast agent like aqueous indocyanine green dye solution. For blood sample, measured values of μa = 0.580 ± 0.016 mm^-1 and Γ = 0.166 ± 0.006 are within the range of values reported in literature. Measurements with the absorbing joint and cyst fluid samples give Γ values close to 0.12, which is similar to that of water and plasma.

Multispectral photoacoustic (MPA) imaging is a promising tool for the diagnosis of atherosclerotic carotids. Excitation of different constituents of a plaque with different wavelengths of the light may provide morphological information to evaluate plaque vulnerability. Preclinical validation of in vivo photoacoustic (PA) imaging requires a comprehensive phantom study. In this study, the design of optically realistic vessel phantoms for photoacoustics was examined by characterizing their optical properties for different dye concentrations, and comparing those to PA measurements. Four different concentrations of Indian ink and molecular dye were added to a 15 wt% PVA and 1 wt% orgasol mixture. Next, the homogeneously mixed gels were subjected to five freeze - thaw cycles to increase the stiffness of vessel phantoms (r_inner = 2:5mm, r_outer = 4mm). For each cycle, the optical absorbance was measured between 400 nm 990 nm using a plate reader. Additionally, photoacoustic responses of each vessel phantom at 808 nm were tested with a novel, hand-held, integrated PA probe. Measurements show that the PA signal intensity increases with the optical absorber concentration (0.3 to 0.9) in close agreement with the absorbance measurements. The freeze - thaw process has no significant effect on PA intensity. However, the total attenuation of optical energy increases after each freeze-thaw cycle, which is primarily due to the increase in the scattering coefficient. In future work, the complexity of these phantoms will be increased to examine the feasibility of distinguishing different constituents with MPA imaging.

We present an ultrahigh resolution dual modality optical resolution photoacoustic microsopy (OR-PAM) and spectral domain optical coherence microscopy (SD-OCM) system. The ultrahigh sub-micron lateral resolution is provided by the high numerical aperture of the objective lens used while the ultrahigh axial resolution is provided by the broadband OCT laser that covers 107 nm with a central wavelength of 840 nm. The synchronized simultaneous acquisition for the two modalities is achieved using a 40MHz FPGA. 2D-scanning is realized by two orthogonal translation stages (PI, 400 nm resolution). The transversal resolution of the system is 0.5 μm, the axial resolutions are 30 μm (PAM) and 4 μm (OCM), respectively. The values have been determined experimentally using nanospheres (diameter 10-200nm). For a demonstration of the imaging capability we present images from thin slices of different biological samples as well as in vivo imaging in the zebrafish embryo.

Photoacoustic Computed Tomography (PACT) records signals from a wide range of angles to achieve uniform, highresolution images. A high-power laser is generally used for PACT, but the long acquisition time with a single probe is a problem due to the low pulse-repetition frequency (PRF). For PACT, this degrades image resolution and contrast because it is hard to scan with a small step interval. Moreover, in vivo measurement requires a fast image acquisition system to avoid motion artifacts. The problem can be resolved by using a high PRF laser, which provides only weak energy. Averaging measured signals many times can mitigate the low signal-to-noise issue, but the PRF is restricted by the acoustic time of flight, so this is a new source of measurement time increase. Here, we present the coded-excitation approach, which we previously proposed for linear scanning, to increase the PACT frame rate. Coded excitation irradiates temporally encoded pulses and enhances the signal amplitude through decoding. The PRF is thus not restricted to acoustic time of flight. Consequently, acquisition time can be shortened by increasing PRF, and the SNR increases for the same measurement time. To validate the proposed idea, we conducted experiments using a high PRF laser with a revolving motor and compared the performance of coded excitation to that of averaging. Results demonstrated that the contamination of a signal acquired from different angles was negligible, and that the scanning pitch was remarkably improved because the start point of decoding can be set in any code in the periodic sequence.

The speed of sound (SoS) in the imaged sample and in the coupling medium is an important parameter in optoacoustic tomography that must be specified in order to accurately restore maps of local optical absorbance. In this work, several hybrid focusing functions are described that successfully determine the most suitable SoS based on post-reconstruction images. The SoS in the coupling medium (water) can be determined from temperature readings. Thereby, this value is suggested to be used as an initial guess for faster SoS calibration in the reconstruction of tissues having a different SoS than water.

Laser-induced thermotherapy (LITT), i.e. tissue destruction induced by a local increase of temperature by means of laser light energy transmission, has been frequently used for minimally invasive treatments of various diseases such as benign thyroid nodules and liver cancer. The emerging photoacoustic (PA) imaging, when integrated with ultrasound (US), could contribute to LITT procedure. PA can enable a good visualization of percutaneous apparatus deep inside tissue and, therefore, can offer accurate guidance of the optical fibers to the target tissue. Our initial experiment demonstrated that, by picking the strong photoacoustic signals generated at the tips of optical fibers as a needle, the trajectory and position of the fibers could be visualized clearly using a commercial available US unit. When working the conventional US Bscan mode, the fibers disappeared when the angle between the fibers and the probe surface was larger than 60 degree; while working on the new PA mode, the fibers could be visualized without any problem even when the angle between the fibers and the probe surface was larger than 75 degree. Moreover, with PA imaging function integrated, the optical fibers positioned into the target tissue, besides delivering optical energy for thermotherapy, can also be used to generate PA signals for on-line evaluation of LITT. Powered by our recently developed PA physio-chemical analysis, PA measurements from the tissue can provide a direct and accurate feedback of the tissue responses to laser ablation, including the changes in not only chemical compositions but also histological microstructures. The initial experiment on the rat liver model has demonstrated the excellent sensitivity of PA imaging to the changes in tissue temperature rise and tissue status (from native to coagulated) when the tissue is treated in vivo with LITT.

Elastography is implemented by applying a mechanical force to a specimen and visualizing the resulting displacement. As a basis of elastographic imaging typically ultrasound, optical coherence tomography or magnetic resonance imaging are used. Photoacoustics has not been viewed as a primary imaging modality for elastography, but only as a complementary method to enhance the contrast in ultrasound elastography. The reason is that photoacoustics is considered speckle free [3], which hinders application of speckle tracking algorithms. However, while conventional ultrasound only uses a single frequency, photoacoustics utilizes a broad frequency spectrum. We are therefore able to generate artificial texture by using a frequency band limited part of the recorded data. In this work we try to assess the applicability of this technique to photoacoustic tomography. We use Agar phantoms with predefined Young's moduli and laterally apply a 50μm static compression. Pre- and post compression data are recorded via a Fabry Pérot interferometer planar sensor setup and reconstructed via a non-uniform-FFT reconstruction algorithm. A displacement vector field, between pre- and post compressed data is then determined via optical flow algorithms. While the implementation of texture generation during post processing reduces image quality overall, it turns out that it improves the detection of moving patterns and is therefore better suited for elastography.

Malignant melanoma is a form of skin cancer, with increasing incidence worldwide. Early diagnosis is crucial for the prognosis and treatment of the disease. The objective of this study is to develop a novel animal model of melanoma and apply a combination of the non-invasive imaging techniques acoustic microscopy, infrared (IR) and Raman spectroscopies, for the detection of developing tumors. Acoustic microscopy provides information about the 3D structure of the tumor, whereas, both spectroscopic modalities give qualitative insight of biochemical changes during melanoma development. In order to efficiently set up the final devices, propagation of ultrasonic and electromagnetic waves in normal skin and melanoma simulated structures was performed. Synthetic and grape-extracted melanin (simulated tumors), endermally injected, were scanned and compared to normal skin. For both cases acoustic microscopy with central operating frequencies of 110MHz and 175MHz were used, resulting to the tomographic imaging of the simulated tumor, while with the spectroscopic modalities IR and Raman differences among spectra of normal and melanin- injected sites were identified in skin depth. Subsequently, growth of actual tumors in an animal melanoma model, with the use of human malignant melanoma cells was achieved. Acoustic microscopy and IR and Raman spectroscopies were also applied. The development of tumors at different time points was displayed using acoustic microscopy. Moreover, the changes of the IR and Raman spectra were studied between the melanoma tumors and adjacent healthy skin. The most significant changes between healthy skin and the melanoma area were observed in the range of 900-1800cm^-1 and 350-2000cm^-1, respectively.

We compare scanned-mosaicking and blanket illumination schemes for wide-field photoacoustic tomography with potential applications to breast imaging. For each illumination, a locally high-SNR image patch is reconstructed then mosaicked with image patches from other illuminations. Because the beam is not diffused over the entire area, the fluence of the beam can be maximized, therefore maximizing the signal generated. Moreover, the imaging can potentially still be done fast enough within a breath-hold. A Monte Carlo simulation as a function of beam-spot size and depth is performed to quantify this signal gain. We experimentally test both schemes using a 256-element Imasonic ring array on a tissue-mimicking phantom. We were able to verify the simulated signal gain of 2.9x under 0.5 cm of tissue with the experimental data, and measured the signal gain decrease expected when imaging deeper into the tissue. We also measured the effectiveness of averaging the diffused beam versus the scanned-mosaicking approach, and observed that for the same scan times and limited laser power output, scanned-mosaicking was able to produce a higher SNR than the blanket illumination approach. We have shown that this technique will allow wide-area PAT to utilize the maximum SNR available from any system while minimizing the number of acquisitions to reach this SNR.

The development of technologies capable of non-invasive characterization of tissue has the potential to significantly improve diagnostic and therapeutic medical interventions. In this study we investigated the feasibility of a noninvasive photoacoustic (PA) approach for characterizing biological tissues. The measurement was performed in the transmission mode with a wideband hydrophone while a tunable Q-switched Nd:YAG pulsed laser was used for illumination. The power spectrum of photoacoustic signal induced by a pulsed laser light from tissue was analyzed and features were extracted to study their correlation with tissue biomechanical properties. For a controlled study, tissue mimicking gelatin phantoms with different densities and equivalent optical absorptions were used as targets. The correlation between gelatin concentration of such phantoms and their mechanical properties were validated independently with a dynamic mechanical analyzer capable of calculating complex loss and storage moduli between two compression plates. It was shown that PA spectrums were shifted towards higher frequencies by increasing gelatin concentration. In order to quantify this effect, signal energy in two intervals of low and high frequency ranges were calculated. Gelatin concentration was correlated with PA energy in high frequency range with R²=0.94. Subsequently, PA signals generated from freshly resected human thyroid specimens were measured and analyzed in a similar fashion. We found that in aggregate, malignant thyroid tissue contains approximately 1.6 times lower energy in the high frequency range in comparison to normal thyroid tissue (p<0.01). This ratio increased with increasing illumination wavelength from 700 nm to 900nm. In summary, this study demonstrated the feasibility of using photoacoustic technique for characterizing tissue on the basis of viscoelastic properties of the tissue.

Pulse-echo ultrasound and optoacoustic imaging possess very different, yet highly complementary, advantages of mechanical and optical contrast in living tissues. Integration of pulse-echo ultrasound with optoacoustic imaging may therefore significantly enhance the potential range of clinical applications. Nonetheless, efficient integration of these modalities remains challenging owing to the fundamental differences in the underlying physical contrast, optimal signal acquisition and image reconstruction approaches. We report on a new method for hybrid three-dimensional optoacoustic and pulse-echo ultrasound imaging based on passive generation of ultrasound with a spherical optical absorber, thus avoiding the hardware complexity of active ultrasound generation. The proposed approach allows for acquisition of complete hybrid datasets with a single laser interrogation pulse, resulting in simultaneous rendering of ultrasound and optoacoustic images at a rate of 10 volumetric frames per second. Real time image rendering for both modalities is enabled by using parallel GPU-based implementation of the reconstruction algorithms. Performance is first characterized in tubing phantoms followed by in vivo measurements in healthy human volunteers, confirming general clinical applicability of the method.

We investigated a novel needle visualization using the PA effect to enhance needle-tip tracking. An optical fiber and laser source are used to generate acoustic waves inside the needle with the PA effect. Acoustic waves are generated along the needle. Some amount of acoustic energy leaks into the surrounding material. The leakage of acoustic waves is captured by a conventional US transducer and US channel data collection system. Then, the collected data are converted to a PA image. The needle-tip can be visualized more clearly in this PA image than a general US brightness mode image.

To achieve real-time photoacoustic tomography (PAT), massive transducer arrays and data acquisition (DAQ) electronics are needed to receive the PA signals simultaneously, which results in complex and high-cost ultrasound receiver systems. To address this issue, we have developed a new PA data acquisition approach using acoustic time delay. Optical fibers were used as parallel acoustic delay lines (PADLs) to create different time delays in multiple channels of PA signals. This makes the PA signals reach a single-element transducer at different times. As a result, they can be properly received by single-channel DAQ electronics. However, due to their small diameter and fragility, using optical fiber as acoustic delay lines poses a number of challenges in the design, construction and packaging of the PADLs, thereby limiting their performances and use in real imaging applications. In this paper, we report the development of new silicon PADLs, which are directly made from silicon wafers using advanced micromachining technologies. The silicon PADLs have very low acoustic attenuation and distortion. A linear array of 16 silicon PADLs were assembled into a handheld package with one common input port and one common output port. To demonstrate its real-time PAT capability, the silicon PADL array (with its output port interfaced with a single-element transducer) was used to receive 16 channels of PA signals simultaneously from a tissue-mimicking optical phantom sample. The reconstructed PA image matches well with the imaging target. Therefore, the silicon PADL array can provide a 16× reduction in the ultrasound DAQ channels for real-time PAT.

Photoacoustic microscopy with linear array transducers enables fast two-dimensional, cross-sectional photoacoustic imaging. Unfortunately, most ultrasound transducers are only sensitive to a very narrow angular acceptance range and preferentially detect signals along the main axis of the transducer. This often limits photoacoustic microscopy from detecting blood vessels which can extend in any direction. Rotational compounded photoacoustic imaging is introduced to overcome the angular-dependency of detecting acoustic signals with linear array transducers. An integrate system is designed to control the image acquisition using a linear array transducer, a motorized rotational stage, and a motorized lateral stage. Images acquired at multiple angular positions are combined to form a rotational compounded image. We found that the signal-to-noise ratio improved, while the sidelobe and reverberation artifacts were substantially reduced. Furthermore, the rotational compounded images of excised kidneys and hindlimb tumors of mice showed more structural information compared with any single image collected.

Intravital microscopy techniques have become increasingly important in biomedical research because they can provide unique microscopic views of various biological or disease developmental processes in situ. Here we present an optical-resolution photoacoustic endomicroscopy (OR-PAEM) system that visualizes internal organs with a much finer resolution than conventional acoustic-resolution photoacoustic endoscopy systems. By combining gradient index (GRIN) lens-based optical focusing and ultrasonic ring transducer-based acoustic focusing, we achieved a transverse resolution as fine as ~10 μm at an optical working distance of 6.5 mm. The OR-PAEM system’s high-resolution intravital imaging capability is demonstrated through animal experiments.

Photoacoustic Tomography (PAT) systems based on commercial ultrasound instruments have the benefit of dualmodality imaging, which increases their appeal from a clinical standpoint. However, factors that influence PAT system performance have not been thoroughly investigated and standardized test methods have not been established for image quality evaluation. To address these issues we have adapted phantom-based approaches from ultrasound imaging standards and implemented them to assess a PAT system developed for vascular imaging. Our system comprises a tunable near-infrared pulsed laser and a commercial ultrasound imaging system, including four interchangeable linear array clinical ultrasound transducers with varying center frequencies, acoustic bandwidths and geometries. Phantoms consisted of a customized polyvinyl chloride (PVC) plastisol gel that simulates both optical and acoustic properties of breast tissue. One phantom incorporates a sub-resolution filament array suitable for bimodal ultrasound-photoacoustic imaging, while another contains an array of hemoglobin-filled cylindrical inclusions at various depths. Key performance characteristics were evaluated, including spatial resolution, signal uniformity, contrast, and penetration depth. These characteristics were evaluated at 750 nm at radiant exposures below ANSI safety limits. Effects of transducer properties on imaging performance were evaluated. Axial and lateral resolution ranged from 0.27-0.83 mm and 0.28-1.8 mm, respectively, and penetration depths from 1.9-4.2 cm were achieved. These results demonstrate variation in PAT system performance based on clinical transducer selection, as well as the utility of realistic phantom-based test methods in performing benchtop evaluations of system performance.

The addition of photoacoustic endoscopy to conventional endoscopic ultrasound offers imaging capabilities that may improve diagnosis and clinical care of gastrointestinal tract diseases. In this study, using a 3.8-mm diameter dual-mode photoacoustic and ultrasonic endoscopic probe, we investigated photoacoustic and ultrasonic image features of rabbit esophagi. Specifically, we performed ex vivo imaging of intact rabbit esophagi and correlated the acquired images with histology. Without motion artifact-based limitations, we were able to utilize the full resolving power of the endoscopic device and acquire the first three-dimensional vasculature map of the esophagus and mediastinum, along with coregistered tissue density information. Here, we present the experimental results and discuss potential clinical applications of the technique.

We used photoacoustic microscopy (PAM) to assist diagnoses and monitor the progress and treatment outcome of complex regional pain syndrome type 1 (CRPS-1). Blood vasculature and oxygen saturation (sO₂) were imaged by PAM in eight adult patients with CRPS-1. Patients’ hands and cuticles were imaged both before and after stellate ganglion block (SGB) for comparison. For all patients, both the vascular structure and sO₂ could be assessed by PAM. In addition, more vessels and stronger signals were observed after SGB.

A methodology for simulating photoacoustic (PA) images of samples with solid spherical absorbers acquired using linear array transducer is described. Two types of numerical phantoms (i.e., polystyrene beads suspended in agar medium) of two different size regimes were imaged with a 40 MHz linear array transducer utilizing this approach. The frequency domain features and statistics of the simulated signals were quantified for tissue characterization. The midband fit at 40 MHz was found to be about 35 dB higher for the sample with larger beads (radius ~7.36 μm) than that of the sample with smaller particles (radius ~ 1.77 μm). The scale parameter of the generalized gamma distribution function was found to be nearly 51 times greater for the former sample compared to the latter sample. The method developed here shows potential to be used a s a fast simulation tool for the PA imaging of collection of absorbers mimicking biological tissue.

Conventional photoacoustic computed tomography (PACT) image reconstruction methods assume that the object and surrounding medium are described by a constant speed-of-sound (SOS) value. In order to accurately recover fine structures, SOS heterogeneities should be quantified and compensated for during PACT reconstruction. To address this problem, several groups have proposed hybrid systems that combine PACT with ultrasound computed tomography (USCT). In such systems, a SOS map is reconstructed first via USCT. Consequently, this SOS map is employed to inform the PACT reconstruction method. Additionally, the SOS map can provide structural information regarding tissue, which is complementary to the functional information from the PACT image. We propose a paradigm shift in the way that images are reconstructed in hybrid PACT-USCT imaging. Inspired by our observation that information about the SOS distribution is encoded in PACT measurements, we propose to jointly reconstruct the absorbed optical energy density and SOS distributions from a combined set of USCT and PACT measurements, thereby reducing the two reconstruction problems into one. This innovative approach has several advantages over conventional approaches in which PACT and USCT images are reconstructed independently: (1) Variations in the SOS will automatically be accounted for, optimizing PACT image quality; (2) The reconstructed PACT and USCT images will possess minimal systematic artifacts because errors in the imaging models will be optimally balanced during the joint reconstruction; (3) Due to the exploitation of information regarding the SOS distribution in the full-view PACT data, our approach will permit high-resolution reconstruction of the SOS distribution from sparse array data.

Photoacoustic computed tomography (PACT) holds great promise for transcranial brain imaging. However, the strong reflection, scattering, attenuation, and mode-conversion of photoacoustic waves in the skull pose serious challenges to establishing the method. The lack of an appropriate model of solid media in conventional PACT imaging models, which are based on the canonical scalar wave equation, causes a significant model mismatch in the presence of the skull and thus results in deteriorated reconstructed images. The goal of this study was to develop an image reconstruction algorithm that accurately models the skull and thereby ameliorates the quality of reconstructed images. The propagation of photoacoustic waves through the skull was modeled by a viscoelastic stress tensor wave equation, which was subsequently discretized by use of a staggered grid fourth-order finite-difference time-domain (FDTD) method. The matched adjoint of the FDTD-based wave propagation operator was derived for implementing a back-projection operator. Systematic computer simulations were conducted to demonstrate the effectiveness of the back-projection operator for reconstructing images in a realistic three-dimensional PACT brain imaging system. The results suggest that the proposed algorithm can successfully reconstruct images from transcranially-measured pressure data and readily be translated to clinical PACT brain imaging applications.

The P(VDF-TrFE) sensor which had uniform sensitivity in a frequency range of 2.9 – 19.6 MHz was developed for multispectral photoacoustic imaging (MS-PAI). A small organic molecule-based PA probe synthesized by our group had the absorption maximum at 530 nm and was used as a contrast agent. The PA probe was designed to have low quantum yield. Therefore, the PA probe efficiently converted absorbed optical energies to PA signals. The probe was injected in subcutaneous tumor of mice. Then, the subcutaneous tumor was imaged in vivo by using P(VDF-TrFE) sensor. MS-PAI successfully discriminated the probe signals from background signals produced from endogenous optical absorbers such as hemoglobin. The probe detectability of the P(VDF-TrFE) sensor was evaluated and then compared with that of lead zirconium titanate (PZT) sensors. The P(VDF-TrFE) sensor imaged the tumor more clearly than the PZT sensor with central frequency of 20 MHz, especially when the probe was accumulated in the tumor with low concentration. That was because the low-concentrated probe generated PA signals with low frequency. MS-PAI using P(VDF-TrFE) sensor which can detect PA signals with wide range of frequency is able to image various distribution of the probe and is superior to that using PZT sensor which detects PA signals with narrow frequency range.

Fluorophores, such as exogenous dyes and genetically expressed proteins, exhibit radiative relaxation with long excited state lifetimes. This can be exploited for PA detection based on dual wavelength excitation using pump and probe wavelengths that coincide with the absorption and emission spectra, respectively. While the pump pulse raises the fluorophore to a long-lived excited state, simultaneous illumination with the probe pulse reduces the excited state lifetime due to stimulated emission (SE).This leads to a change in thermalized energy, and hence PA signal amplitude, compared to single wavelength illumination. By introducing a time delay between pump and probe pulses, the change in PA amplitude can be modulated. Since the effect is not observed in endogenous chromophores, it provides a contrast mechanism for the detection of fluorophores via PA difference imaging. In this study, a theoretical model of the PA signal generation in fluorophores was developed and experimentally validated. The model is based on a system of coupled rate equations, which describe the spatial and temporal changes in the population of the molecular energy levels of a fluorophore as a function of pump-probe energy and concentration. This allows the prediction of the thermalized energy distribution, and hence the time-resolved PA signal amplitude. The model was validated by comparing its predictions to PA signals measured in solutions of rhodamine 6G, a well-known laser dye, and Atto680, a NIR fluorophore.

We propose using noninvasive longitudinal optical-resolution photoacoustic microscopy (L-ORPAM) to quantify blood flow flux, oxygen saturation (sO₂), and thereby the metabolic rate of oxygen (MRO₂), for a renal tumor model in the same mouse over weeks to months. Experiments showed that the sO2 difference between the artery and vein decreased greatly due to the arteriovenous shunting effect during tumor growth. Moreover, hypermetabolism was exhibited by an increase in MRO₂.

Optoacoustic (photoacoustic) imaging is being adopted for monitoring tissue temperature during hypothermic and hyperthermic cancer treatments. The technique’s accuracy benefits from the knowledge of speed of sound (SoS) and acoustic coefficient of attenuation (AcA) as they change with temperature in biological tissues, blood, and acoustic lens of an ultrasound probe. In these studies we measured SoS and AcA of different ex vivo tissues and blood components (plasma and erythrocyte concentrates) in the temperature range from 5°C to 60°C. We used the technique based on measurements of time-delay and spectral amplitude of pressure pulses generated by wideband planar acoustic waves propagating through the interrogated medium. Water was used as a reference medium with known acoustic properties. In order to validate our experimental technique, we measured the temperature dependence of SoS and AcA for aqueous NaCl solution of known concentration and obtained the results in agreement with published data. Similar to NaCl solution and pure water, SoS in blood and plasma was monotonously increasing with temperature. However, SoS of erythrocyte concentrates displayed abnormalities at temperatures above 45°C, suggesting potential effects from hemoglobin denaturation and/or hemolysis of erythrocytes. On the contrary to aqueous solutions, the SoS in polyvinyl-chloride (plastisol) – a material frequently used for mimicking optical and acoustic properties of tissues – decreased with temperature. We also measured SoS and AcA in silicon material of an acoustic lens and did not observe temperature-related changes of SoS.

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.

Various causes can lead to methemoglobinemia, and it has the potential to be confused with other diseases. In vivo measurements of methemoglobin have significant applications in the clinics. We quantified the average and the distributed percentage of methemoglobin both in vitro and in vivo using photoacoustic microscopy (PAM). Based on the absorption spectra of methemoglobin, oxyhemoglobin, and deoxyhemoglobin, three wavelengths were chosen to differentiate methemoglobin from the others. We imaged the methemoglobin percentage in microtubes that mimicked blood vessels as a phantom experiment. The methemoglobin concentrations calculated from the photoacoustic signals were in accordance with the preset concentrations. We also demonstrated the ability of PAM to quantitatively image methemoglobin distribution in vivo in a mouse ear.

Optoacoustic tomography (OAT) is a promising imaging modality for human breast cancer imaging, with higher resolution and deeper penetration compared to other optical imaging modalities such as diffuse optical tomography or optical coherence tomography. It yields a resolution of 1 mm at depth up to 2 cm. But there is an inherent conflict between the limitations imposed on laser power and the need to adequately penetrate a substantial portion of the breast. To achieve sufficient penetration at every view angle, instead of illuminating the whole breast all at once, sometimes illumination is focused onto a small region of the breast and rotated along with the transducer array to cover the entire object. This paper evaluates the effect of this rotating partial illumination design on OAT image reconstruction. The optical process is simulated by conducting Monte Carlo simulations on a numerical phantom mimicking a real breast, with various specially designed illumination schemes. The acoustic process is simulated by incorporating the transducer's spatial impulse response. Iterative reconstruction is applied to estimate the OAT image. We conclude that rotating partial illumination introduces inconsistency into the system equation, and the degree of inconsistency determines the reconstruction quality.

Early diagnosis of intracranial hematomas is necessary to improve outcome in patients with traumatic brain injury (TBI). CT and MRI can diagnose intracranial hematomas, but cannot be used until the patient arrives at a major healthcare facility, resulting in delayed diagnosis. Near infrared spectroscopy may suggest the presence of unilateral intracranial hematomas, but provides minimal information on hematoma type and location due to limitations associated with strong light scattering. We have used optoacoustics (which combines high endogenous optical contrast with the resolution of ultrasound) to diagnose hematomas and monitor cerebral oxygenation. We performed animal and clinical studies on detection and characterization of hematomas and on monitoring cerebral hypoxia by probing the superior sagittal sinus (SSS). Recently, we built a medical grade, multi-wavelength, OPO-based optoacoustic system tunable in the near infrared spectral range. We developed new patient interfaces for noninvasive, transcranial measurements in the transmission mode in the presence of dense hair and used it in patients with TBI. The optoacoustic system was capable of detecting and characterizing intra- and extracranial hematomas. SSS blood oxygenation was measured as well with the new interface. The obtained results indicate that the optoacoustic system in the transmission mode provides detection and characterization of hematomas in TBI patients, as well as cerebral venous blood oxygenation monitoring. The transmission mode approach can be used for optoacoustic brain imaging, tomography, and mapping in humans.

Optical-resolution photoacoustic microscopy (OR-PAM) has been studied to improve its imaging resolution and functional imaging modality without labeling on biology sample. However the use of high numerical aperture (NA) objective lens confines the field of view or the axial imaging range of OR-PAM. In order to obtain images at different layers, one needs to change either the sample position or the focusing position by mechanical scanning. This mechanical movement of the sample or the objective lens limits the scanning speed and the positioning precision. In this study, we propose a multi-depth PAM with a focus tunable lens. We electrically adjusted the focal length in the depth direction of the sample, and twice extended the axial imaging range up to 660 μm with the objective lens (20X, NA 0.4). The proposed approach can increase scanning speed and avoid step motor induced distortions during PA signal acquisitions without mechanical scanning in the depth direction. To investigate the performance of the multi-depth PAM system, we scanned a black human hair and the ear of a living nude mouse (BALB/c Nude). The obtained PAM images presented the volumetric rendering of black hair and the vasculature of the nude mouse.

Atherosclerotic plaque at the carotid bifurcation is the underlying cause of the majority of ischemic strokes. Noninvasive imaging and quantification of the compositional changes preceding gross anatomic changes within the arterial wall is essential for diagnosis of disease. Current imaging modalities such as duplex ultrasound, computed tomography, positron emission tomography are limited by the lack of compositional contrast and the detection of flow-limiting lesions. Although high-resolution magnetic resonance imaging has been developed to characterize atherosclerotic plaque composition, its accessibility for wide clinical use is limited. Here, we demonstrate a fiber-based multispectral photoacoustic tomography system for excitation of lipids and external acoustic detection of the generated ultrasound. Using sequential ultrasound imaging of ex vivo preparations we achieved ~2 cm imaging depth and chemical selectivity for assessment of human arterial plaques. A multivariate curve resolution alternating least squares analysis method was applied to resolve the major chemical components, including intravascular lipid, intramuscular fat, and blood. These results show the promise of detecting carotid plaque in vivo through esophageal fiber-optic excitation of lipids and external acoustic detection of the generated ultrasound. This imaging system has great potential for serving as a point-ofcare device for early diagnosis of carotid artery disease in the clinic.

In this paper we present multimodal non-contact photoacoustic and optical coherence tomography (OCT) imaging using a galvanometer scanner. Photoacoustic signals are acquired without contact on the surface of a specimen using an interferometric technique. The interferometer is realized in a fiber-optic network using a fiber laser at 1550 nm as source. In the same fiber-optic network a spectral-domain OCT system is realized, using a broadband light source at 1300 nm. Light from the fiber laser and the OCT source are multiplexed into the same fiber and the same objective is used for both imaging modalities. Fast non-contact photoacoustic and OCT imaging is demonstrated by scanning the detection spot utilizing a galvanometer scanner. Multimodal photoacoustic and OCT imaging is shown on agarose phantoms. As the same fiber network and optical components are used for non-contact photoacoustic and OCT imaging the obtained images are co-registered intrinsically.

In this work we present photoacoustic projection imaging with a 64-channel integrating line detector array, which average the pressure over cylindrical surfaces. For imaging, the line detectors are arranged parallel to each other on a cylindrical surface surrounding a specimen. Thereby, the three-dimensional imaging problem is reduced to a twodimensional problem, facilitating projection imaging. After acquisition of a dataset of pressure signals, a twodimensional photoacoustic projection image is reconstructed. The 64 channel line detector array is realized using optical fibers being part of interferometers. The parts of the interferometers used to detect the ultrasonic pressure waves consist of graded-index polymer-optical fibers (POFs), which exhibit better sensitivity than standard glass-optical fibers. Ultrasonic waves impinging on the POFs change the phase of light in the fiber-core due to the strain-optic effect. This phase shifts, representing the pressure signals, are demodulated using high-bandwidth balanced photo-detectors. The 64 detectors are optically multiplexed to 16 detection channels, thereby allowing fast imaging. Results are shown on a Rhodamine B dyed microsphere.

We demonstrate the first time a widely tunable MOPA (Master Oscillator Power Amplifier) laser system for Optical resolution photoacoustic microscopy (OR-PAM). This unique laser is capable of tuning the repetition rate (0.1- 120MHz), the wavelength (1030-1080nm), the pulse-width (100ps-5ns) and power (up to 1.1W). It is also capable of programmable dithering and synchronization with other laser systems to within ~ps jitter. To apply it to OR-PAM we take advantage of frequency doubling to convert IR light into green light. For doubling, a periodically poled magnesium doped lithium niobate (MgO:LiNbO₃) crystal was chosen. Wavelength tuning was accomplished by tuning the output wavelength of the MOPA then tuning the temperature of the (MgO:LiNbO₃) crystal to generate tunable visible light. Characterization of the system performance revealed a lateral resolution of ~7μm and a SNR of ~21dB when imaging 7μm carbon fibers.

We present a fiber-based dual-modal imaging system that combines non-contact photoacoustic tomography (NCPAT) and fluorescence imaging by using double cladding fiber (DCF). The NCPAT system utilizing an all-fiber heterodyne interferometer as an ultrasound detector measures the photoacoustic signal at the sample surface without physical contact. Fluorescence imaging system is composed of fiber-optics to deliver the excitation light and the emission light. For combined system the probe consists of a specially fabricated DCF coupler and a lensed fiber so that we can simultaneously acquire the signals of two systems with the same probe. The DCF has a core and two claddings, inner and outer, which allows two concentric light-guiding channels via the core and the inner cladding. The lensed fiber of the DCF probe is compactly fabricated to focus the interferometer light and the excitation light, and to efficiently collect the fluorescence signal. To demonstrate the feasibility of the proposed dual-modal system, we have conducted phantom experiments using tissue mimicking phantoms which contained a couple of tubes filled with fluorescein solution and black ink, respectively. The proposed imaging system is implanted with fiber-optic configurations so that it has the potential for minimally invasive and improved diagnosis and guided treatment of diseases.

We’ve successfully measured photoacoustic signal by NIR-LED array that has very small power, approx. 1/1500 of light amount compared with Nd:YAG OPO light. In order to achieve high output power, we drove NIR-LED array with unusual amount of electric current. The experiment results showed that the photoacoustic signal strength was about 1/40 of the laser, which suggests NIR-LED array has good photoacoustic reception efficiency versus the ultrasound transducer bandwidth. NIR-LED array photoacoustic system may be able to achieve high-speed imaging which cannot be obtained by the solid-state laser. NIR-LED system can be a game changer for photoacoustic imaging.

When we consider the needle visualization in the field of point of care by utilizing the photoacoustic imaging system, and using the conventional solid state laser light source, the issue arises such as device size and not a green system due to the high power consumption. Therefore, we aimed at an environmentally friendly and compact system with low power consumption by using a NIR-LED array light source. The intensity of NIR-LED light is weak, but, by averaging photoacoustic signals with multiple pulse, we have improved S/N of the photoacoustic signal. As a result, we’ve achieved penetration depth of 30mm.

Optical-resolution photoacoustic microscopy (OR-PAM) has demonstrated the capability of mapping microvasculature and is promising for biomedical applications. However, the popularity of the PAM system is limited due to the use of expansive and bulky laser sources. In this paper, a compact and cost-efficient OR-PAM system with a laser diode excitation has been developed. The black threads, phantom made of polyethylene tubes filled with rat blood, and a mouse ear were imaged to evaluate the PAM and demonstrate its capability of imaging biological tissue.

Ultrasound-modulated optical tomography is an emerging biomedical imaging modality which uses the spatially localised acoustically-driven modulation of coherent light as a probe of the structure and optical properties of biological tissues. In this work we model the first-harmonic flux generated by the coupled physics using a simple linearised diffusion-style forward model. We derive analytical expressions for the sensitivity of this measurement type with respect to the optical absorption and scattering coefficients. These correlation measurement density functions can be employed as part of an image-reconstruction procedure capable of reconstructing quantitative images of the optical properties of a medium under investigation.

Ultrasound-based measurements using Doppler, contrast, and more recently photoacoustics (PA), have emerged as techniques for tissue perfusion measurements. In this study, the feasibility of in vitro perfusion measurements with a fully integrated, hand-held, photoacoustic probe was investigated and compared to Power Doppler (PD).

Three cylindrical polyvinyl alcohol (PVA) phantoms were made (diameter = 15 mm) containing 100, 200 and 400 parallel polysulfone tubes (diameter = 0.2 mm), resulting in a perfused cross-sectional area of 1.8, 3.6 and 7.1% respectively. Each phantom was perfused with porcine blood (15 mL/min). Cross-sectional PA images (λ = 805nm, frame rate = 10Hz) and PD images (PRF = 750Hz) were acquired with a MyLab One and MyLab 70 scanner (Esaote, NL), respectively. Data were averaged over 70 frames. The average PA signal intensity was calculated in a region-of-interest of 4 mm by 6 mm. The percentage of colored PD pixels was measured in the entire phantom region.

The average signal intensity of the PA images increased linearly with perfusion density, being 0.54 (± 0.01), 0.56 (± 0.01), 0.58 (± 0.01) with an average background signal of 0.53 in the three phantoms, respectively. For PD, the percentage of colored pixels in the phantom area (1.5% (± 0.2%), 4.4% (± 0.2%), 13.7% (± 0.8%)) also increased linearly. The preliminary results suggest that PA, like PD, is capable of detecting an increase of blood volume in tissue. In the future, in vivo measurements will be explored, although validation will be more complex.

Their intense optical absorbance in the near-infrared window and chemical versatility make gold nanorods attractive for biomedical applications, such as photothermal therapies and photoacoustic imaging. However, their limited photostability remains a drawback of practical concern. In fact, when gold nanorods are irradiated with nanosecond laser pulses in resonance with their plasmon oscillations, there may occur reshaping into spherical particles or even fragmentation at higher optical fluences, which cause substantial modifications of their optical features with a loss of photoacoustic conversion efficiency. In this contribution, we focus on how the gold nanorods photostability is affected when these particles are modified for cellular uptake, by investigating their stability and photoacoustic conversion efficiency under near infrared pulsed irradiation at different laser fluences.

Crohn’s disease (CD) is an autoimmune disease affecting 700,000 people in the United States. This condition may cause obstructing intestinal narrowings (strictures) due to inflammation, fibrosis (deposition of collagen), or a combination of both. Utilizing the unique strong optical absorption of hemoglobin at 532 nm and collagen at 1370 nm, this study investigated the feasibility of non-invasively characterizing intestinal strictures using photoacoustic imaging (PAI). Three normal controls, ten pure inflammation and 9 inflammation plus fibrosis rat bowel wall samples were imaged. Statistical analysis of the PA measurements has shown the capability of discriminating the purely inflammatory from mixed inflammatory and fibrotic strictures.

Imaging complicated structures with photoacoustic (PA) modality when the field of view is limited can result in significant imaging artifacts or missing structures. Approaches to solve this problem include new reconstruction algorithms and specific transducer structures, such as hemi-spherical transducer arrays for breast cancer detection. However, most existing PA imaging techniques require either fullview complete projection data collection or complex and computationally-intensive reconstructions. Such approaches are not only time-consuming but also unsuitable for many clinical applications, because most clinical imaging hardware is constrained to limited reconstruction angles. In this paper, we present a method of using two commercial linear array transducers at different orientations to increase the view angle and thus improve the reconstruction of PA imaging. The method involves a two-step process. First, a calibration phantom is imaged to calibrate the relative position of these two linear transducers. Second, two PA images are obtained by a simple back projection algorithm and these images are registered using the information from the calibration process. The final registered image contains more detailed structures without the requirements of a specialized transducer or long processing time. Experimental results show that this method has the potential to provide good image quality using standard low-cost transducers.

We describe the ongoing development of laser systems for advanced photoacoustic imaging (PAI). We discuss the characteristics of these laser systems and their particular benefits for soft tissue imaging and next-generation breast cancer diagnostics. We provide an overview of laser performance and compare this with other laser systems that have been used for early-stage development of PAI. These advanced systems feature higher pulse energy output at clinically relevant repetition rates, as well as a novel wavelength-cycling output pulse format. Wavelength cycling provides pulse sequences for which the output repeatedly alternates between two wavelengths that provide differential imaging. This capability improves co-registration of captured differential images. We present imaging results of phantoms obtained with a commercial ultrasound detector system and a wavelength-cycling laser source providing ~500 mJ/pulse at 755 and 797 nm, operating at 25 Hz. The results include photoacoustic images and corresponding pulse-echo data from a tissue mimicking phantom containing inclusions, simulating tumors in the breast. We discuss the application of these systems to the contrast-enhanced detection of various tissue types and tumors.

Stiffness of arteries, especially small arteries, is an important marker for many diseases and a good parameter to evaluate the risks of cardiovascular problems. In this research, we proposed a new method for measurement of local arterial distensibility by using photoacoustic microscopy (PAM) technology. Taking advantages from its excellent sensitivity and high spatial resolution, PAM can evaluate the morphology and volume change of a small artery accurately without involving any contrast agent. When working in the linear elastic range of a vessel, measuring the initial and the distended diameters of the vessel before and after pressure change facilitates quantitative assessment of vessel distensibility. The preliminary experiment on well-controlled gel phantoms demonstrates the feasibility of this technology.

Photoacoustic imaging is emerging as a bioimaging technique. The development of contrast agents extend the potential towards novel application. The design of stable phantoms is needed to achieve a semi-quantitative evaluation of the performance of contrast agents.

The aim of this study was to investigate the PA signal generated from gold nanorods (GNRs) loaded in custom made phantoms. VevoLAZR (VisualSonics Inc., Toronto) was used with custom made agar phantom, with 5 parallel polyethylene tubes (with 0.58mm internal and 0.99mm external diameter), and a PDMS phantom, with six parallel channels with sizes from 50 μm to 500 μm, loaded with two different types of GNRs: PEGGNRs (53nm length and 11nm axial diameter, plasmon resonance at 840nm, 87nM (15mM Au equivalent)); and gold nanorods (NPZ) coated in a dense layer of hydrophilic polymers by Nanopartz Inc., Loveland, CO (41nm length and 10nm axial diameter, plasmon resonance at 808nm, 83 nM (14mM Au equivalent)).

The absorption spectra acquired with the PA system and the spectrophotometer were compared. The reproducibility and stability of the PA signal were evaluated at different dilutions. The dynamic variation of the PA signal was evaluated as function of the number of the GNRs. The SNR and the contrast were measured across the range of concentrations studied. The custom made agar phantom demonstrated suitable for the characterization of PA contrast agents such as PEG-GNRs and NPZ. The PDMS phantom is promising in the field of photoacoustics, therefore future works will conducted exploiting its precise and controlled geometry.

Gold nanorods (GNRs) offer a tunable optical absorption in the near infra-red wavelength region due to their plasmon resonance, which results in strong photoacoustic (PA) signal and make them suitable as contrast agent by means of PA imaging. The aim of this study was to examine the performance of synthesized polyethilene glicol (PEG)-GNRs as contrast agent for in vivo PA imaging and to evaluate their accumulation and distribution real time. Two-three month old FVB female mice were enrolled for the study, a bolus of 200μL of synthesized PEG-GNRs (53 nm length and 11 nm axial diameter, plasmon resonance at 840 nm, 1 mM Au concentration) solution was injected intravenously and detected with PA imaging. The accumulation of GNRs in the spleen was studied by means of the amplitude dynamic variation of the PA signal during time. GNRs contrast was clearly distinguished from endogenous background thanks to the nanoparticle spectroscopic specificity. Our results suggest that PA imaging could allow an efficient and noninvasive tool for in vivo detection of GNRs content and for the assessment of the kinetic parameters in target organs. The coregistration of μ-ultrasound and PA imaging is crucial for the real time evaluation of the GNRs distribution in different organs.

Photoacoustic spectral analysis (PASA), i.e., systemically analyzing the power spectrum of photoacoustic (PA) radio-frequency signals, has demonstrated the capability of characterizing the histological microstructures in biological tissues. The purpose of this study is to theoretically validate the method of PASA

. The analytical solution to the power spectrum of PA signals generated by identical microspheres following discrete uniform random distribution in space was derived. The power spectrum was decomposed into several independent factors, of which the explicit analytical expressions were individually derived. The analytical expressions in combination allow the quantification of the power spectrum profiles and the PASA parameters. The simulation and experiment validation of analytical solution include: 1) the power spectrum profile of a single microsphere with a diameter of 300 μm; and 2) the PASA parameters of the PA signals generated by randomly distributed microspheres of diameters of 100, 200, 300, 400 and 500 μm, and at concentrations of 30, 60, 120, 240, 480 per 1.53 cubic centimeter in the observation range of [0.5, 13 MHz].

The single microsphere experiment confirmed our hypothesis that the periodical fluctuations of the PA signal power spectrum can be used to quantify the dimension of the microspheres. The multiple microsphere experiment validated that the PASA could quantify the dimensions and concentrations of the optical absorbers. In addition, slope, in comparison with other PASA parameters, is more robust as it is less affected by the uncertainties brought in by the optical illumination.

Photoacoustic (PA) measurements encode the information associated with both physical microstructures and chemical contents in biological tissues. A two-dimensional physio-chemical spectrogram (PCS) can be formulated by combining the power spectra of PA signals acquired at a series of optical wavelengths. The analysis of PCS, or namely PA physio-chemical analysis (PAPCA), enables the quantification of the relative concentrations and the spatial distributions of a variety of chemical components in the tissue. This study validated the feasibility of PAPCA in characterizing liver conditions during the progression of non-alcoholic fatty liver disease. A catheter based setup facilitating measurement in deep tissues was also tested.

Osteoporosis is a progressive bone disease that is characterized by a decrease in bone mass and deterioration in microarchitecture. This study investigates the feasibility of characterizing bone microstructure by analyzing the frequency spectrum of the photoacoustic signals from the bone. Modeling and numerical simulation of photoacoustic signals and their frequency-domain analysis were performed on trabecular bones with different mineral densities. The resulting quasilinear photoacoustic spectra were fit by linear regression, from which spectral parameter slope can be quantified. The modeling demonstrates that, at an optical wavelength of 685 nm, bone specimens with lower mineral densities have higher slope. Preliminary experiment on osteoporosis rat tibia bones with different mineral contents has also been conducted. The finding from the experiment has a good agreement with the modeling, both demonstrating that the frequency-domain analysis of photoacoustic signals can provide objective assessment of bone microstructure and deterioration. Considering that photoacoustic measurement is non-ionizing, non-invasive, and has sufficient penetration in both calcified and noncalcified tissues, this new technology holds unique potential for clinical translation.

Low frequency alternating magnetic field (AMF) had been advocated for thermoacoustic imaging to exploit their inherent deeper penetrations. AMF induced thermoacoustic imaging of magnetic nanoparticles is particularly appealing since the system setup is inherently compatible with nanoparticle hyperthermia therapy. More importantly, owing to the capacity of thermoacoustics for accurate temperature measurement, the integration of AMF induced thermoacoustic imaging into nanoparticle hyperthermia therapy will potentially enable a theranostic platform with imaging guidance and temperature monitoring capabilities. We present herein the AMF induced thermoacoustic process of magnetic nanoparticles experimentally and then investigate furthermore its utilization in temperature monitoring for the nanoparticle hyperthermia. To demonstrate the concept of an integrated theranostic system with minimal overhead, a single coil is used for both the hyperthermia heating and thermoacoustic imaging by interleaving the two processes in time domain. In thermoacoustic imaging mode, the power is set at the amplifier's maximum value whereas to avoid excess heating of the coil in hyperthermia-mode, the power is switched to a lower value and the coil is further cooled by static water. Phantom imaging results of the magnetic nanoparticles and the self temperature monitoring with sub-degree accuracy during hyperthermia process are demonstrated. These proof-of-concept experiments showcase the potential to integrate thermoacoustic imaging with nanoparticle hyperthermia system.

Inflammatory arthritis is often manifested in finger joints. The growth of new or withdrawal of old blood vessels can be a sensitive marker for these diseases. Photoacoustic (PA) imaging has great potential in this respect since it allows the sensitive and highly resolved visualization of blood. We systematically investigated PA imaging of finger vasculature in healthy volunteers using a newly developed PA tomographic system. We present the PA results which show excellent detail of the vasculature. Vessels with diameters ranging between 100 μm and 1.5 mm are visible along with details of the skin, including the epidermis and the subpapillary plexus. The focus of all the studies is at the proximal and distal interphalangeal joints, and in the context of ultimately visualizing the inflamed synovial membrane in patients. This work is important in laying the foundation for detailed research into PA imaging of the phalangeal vasculature in patients suffering from rheumatoid arthritis.

High resolution optical imaging technologies, such as optical coherence tomography or multiphoton microscopy has given us an opportunity to do in vivo imaging noninvasively. However, due to the high laser scattering, these optical imaging techniques were prohibited from obtaining high resolution in the diffusive regime. Photoacoustic microscopy (PAM) can overcome this soft depth limit and maintain high resolution at the same time. In the past, PAM was limited to using an Nd:YAG laser, which requires an optical parametric oscillator (OPO) to obtain wavelengths selectively other than the second harmonic. However, OPO is unstable and cumbersome to control. We replaced the Nd:YAG laser and the OPO with a nanosecond pulsed Ti:Sapphire laser to give PAM more flexibility in the speed and the input wavelength while reducing the footprint of our system. This also increased our stability by removing OPO. Using a Ti:Sapphire laser allowed us to increase the pulse repetition rate to 100-500 kHz. Normally, micro-lasers with this pulse repetition rate will suffer from a significant decrease in pulse energy, but we were able to maintain stable pulses with a few hundreds nJ. Also, a well-known advantage of a Ti:Sapphire laser is its tunability from 650 to 1100 nm. For our PAM application, we used a range from 700 to 900 nm to obtain significant functional images. This added flexibility can help acquire functional images such as the angiogenesis process with better contrast. Here, we present a nanosecond Ti:Sapphire laser designated for PAM applications with increased contrast imaging.

Photoacoustic tomography is expected to impact biology and medicine broadly by providing multiscale in vivo functional and molecular imaging of structures ranging from subcellular organelles to organs, enabling a noninvasive look at subcutaneous tissue at a deep level. Lihong Wang holds the Gene K. Beare Distinguished Professorship of Biomedical Engineering at Washington University in St. Louis, and is Editor-in-Chief of the Journal of Biomedical Optics. Wang was awarded the 2015 Britton Chance Biomedical Optics Award for his pioneering technical contributions and visionary leadership in the development and application of photoacoustic tomography, photoacoustic microscopy, and photon transport modeling.