This PDF file contains the front matter associated with SPIE Proceedings Volume 10064, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

Genetically encoded probes with absorbance and fluorescence spectra within a near-infrared tissue transparency window are preferable for deep-tissue imaging. On the basis of bacterial phytochromes we engineered several types of near-infrared absorbing probes for photoacoustic tomography and fluorescent probes for purely optical imaging. They can be used as protein and cell labels and as building blocks for biosensors. The probes enabled imaging of tumors and metastases, protein-protein interactions, RNA visualization, detection of apoptosis, cellular metabolites, signaling pathways and cell proliferation. The developed probes allow non-invasive visualization of biological processes across scales, from super-resolution microscopy to tissue and whole-body animal imaging.

The brain has been likened to a great stretch of unknown territory consisting of a number of unexplored continents. Small animal brain imaging plays an important role charting that territory. By using 1064 nm illumination from the side, we imaged the full coronal depth of rat brains in vivo. The experiment was performed using a real-time full-ring-array photoacoustic computed tomography (PACT) imaging system, which achieved an imaging depth of 11 mm and a 100 μm radial resolution. Because of the fast imaging speed of the full-ring-array PACT system, no animal motion artifact was induced. The frame rate of the system was limited by the laser repetition rate (50 Hz). In addition to anatomical imaging of the blood vessels in the brain, we continuously monitored correlations between the two brain hemispheres in one of the coronal planes. The resting states in the coronal plane were measured before and after stroke ligation surgery at a neck artery.

Monitoring of the membrane potential is possible using voltage sensitive dyes (VSD), where fluorescence intensity changes in response to neuronal electrical activity. However, fluorescence imaging is limited by depth of penetration and high scattering losses, which leads to low sensitivity in vivo systems for external detection. In contrast, photoacoustic (PA) imaging, an emerging modality, is capable of deep tissue, noninvasive imaging by combining near infrared light excitation and ultrasound detection. In this work, we develop the theoretical concept whereby the voltage-dependent quenching of dye fluorescence leads to a reciprocal enhancement of PA intensity. Based on this concept, we synthesized a novel near infrared photoacoustic VSD (PA-VSD) whose PA intensity change is sensitive to membrane potential. In the polarized state, this cyanine-based probe enhances PA intensity while decreasing fluorescence output in a lipid vesicle membrane model. With a 3-9 μM VSD concentration, we measured a PA signal increase in the range of 5.3 % to 18.1 %, and observed a corresponding signal reduction in fluorescence emission of 30.0 % to 48.7 %. A theoretical model successfully accounts for how the experimental PA intensity change depends on fluorescence and absorbance properties of the dye. These results not only demonstrate the voltage sensing capability of the dye, but also indicate the necessity of considering both fluorescence and absorbance spectral sensitivities in order to optimize the characteristics of improved photoacoustic probes. Together, our results demonstrate photoacoustic sensing as a potential new modality for sub-second recording and external imaging of electrophysiological and neurochemical events in the brain.

Thyroid cancer is one of the most prevalent cancers. About 3-8% of the people in the United States have thyroid nodules, and 5-15% of these nodules are malignant. Fine-needle aspiration biopsy (FNAB) is a standard procedure to diagnose malignity of nodules. However, about 10-20% of FNABs produce indeterminable results, which leads to repeat biopsies and unnecessary surgical operations. We have explored photoacoustic (PA) imaging as a new method to identify cancerous nodules. In a pilot study to test its feasibility, we recruited patients with thyroid nodules (currently 36 cases with 21 malignant and 15 benign nodules), acquired in vivo PA and ultrasound (US) images of the nodules in real time using a recently-developed clinical PA/US imaging system, and analyzed the acquired data offline. The preliminary results show that malignant and benign nodules could be differentiated by utilizing their PA amplitudes at different excitation wavelengths. This is the first in vivo PA analysis of thyroid nodules. Although a larger-scale study is needed for statistical significance, the preliminary results show the good potential of PA imaging as a non-invasive tool for triaging thyroid cancer.

Photoacoustic (PA) imaging combined with ultrasonography (US) holds promise to offer a novel and powerful tool for clinical management of inflammatory arthritis, including early detection and treatment monitoring. As a complement to US, PA imaging can assess additional hemodynamic changes in inflammatory synovium, including hyperemia and hypoxia, both important and early physiological biomarkers of synovitis reflecting the increased metabolic demand and the relatively inadequate oxygen delivery of the inflammatory synovial tissue. In this study on arthritis patients and normal volunteers, the targeted metacarpophalangeal (MCP) joints were imaged using our real-time US-PA dual-modality imaging system. The blood volume and the blood oxygenation in the segmented synovium were quantified, and the results from the arthritis patients were compared to those from the normal volunteers. This initial study on human subjects demonstrated that PA imaging, by working at the optical wavelengths that are sensitive to oxygenated and deoxygenated hemoglobin, is capable of identifying and characterizing inflammation in joints based on the detection of hemodynamic changes.

Noninvasive, transcranial mapping, monitoring, and imaging are highly important for detection and management of cerebral abnormalities and neuroscience research. Mapping, imaging, and monitoring of cerebral blood oxygenation are necessary for diagnostics and management of patients with traumatic brain injury, stroke, and other neurological conditions. We proposed to use optoacoustic technology for noninvasive, transcranial monitoring and imaging. In this work, we developed optoacoustic systems for mapping of cerebral blood oxygenation in humans and tested them in adults and neonates. The systems provide noninvasive, transcranial optoacoustic measurements in the transmission (forward) and reflection (backward) modes in the near infrared spectral range. Novel, ultra-sensitive probes were built for detection of optoacoustic signals and measurement of blood oxygenation in neonates and adults. Cerebral oxygenation was measured at different lateral sites from the superior sagittal sinus (SSS), a large central cerebral vein, located immediately beneath the midline of the human skull. In neonates, cerebral oxygenation was measured through open anterior and posterior fontanelles. Optoacoustic signal detection at different locations allowed for mapping of cerebral blood oxygenation. Our future studies will be focused on 3D mapping of cerebral blood oxygenation.

Elastography can noninvasively map the elasticity distribution of biological tissue, which is often altered in pathological states. In this work, we report quantitative photoacoustic elastography (QPAE), capable of measuring Young’s modulus of human tissue in vivo. By combining photoacoustic elastography with a stress sensor having known stress-strain behavior, QPAE can simultaneously measure strain and stress, from which Young’s modulus is calculated. We first applied QPAE to quantify the Young’s modulus of tissue-mimicking agar phantoms with different concentrations. The measured values fitted well with both the empirical expectations based on the agar concentrations and those measured in independent standard compression tests. We then demonstrated the feasibility of QPAE by measuring the Young’s modulus of human skeletal muscle in vivo. The data showed a linear relationship between muscle stiffness and loading. The results proved that QPAE can noninvasively quantify the absolute elasticity of biological tissue, thus enabling longitudinal imaging of tissue elasticity. QPAE can be exploited for both preclinical biomechanics studies and clinical applications.

Ocular chemical damage may induce limbal vessel ischemia and neovascularization, but the pathophysiology of the disease is not completely known. To observe changes in blood vessels after alkaline burn, we monitored the anterior segment and choroidal vasculature using a photoacoustic microscope (OR-PAM). We were able to observe not only the iris blood vessels but also the choroidal vessels under the sclera, which were difficult to be observed with conventional photographs. After alkali burning, we observed neovascularization and limbal ischemia and successfully tracked changes in vasculature during the 7-day healing process. We also used the RANdom SAmple Consensus (RANSAC) method to segment the abnormally generated blood vessels in the cornea by detecting the eyeball surface and successfully visualize the distance from each PA signal to the center of the eye. We believe that photoacoustic imaging has an important potential to reveal the pathophysiology of limb ischemia and neovascularization.

Identifying and delineating invisible anatomical and pathological details during surgery guides surgical procedures in real time. Various intraoperative imaging modalities have been increasingly employed to minimize such surgical risks as anatomical changes, damage to normal tissues, and human error. However, current methods provide only structural information, which cannot identify critical structures such as blood vessels. The logical next step is an intraoperative imaging modality that can provide functional information. Here, we have successfully developed a photoacoustic (PA) image-guided navigation system for surgery by integrating a position tracking system and a real-time clinical photoacoustic/ultrasound (PA/US) imaging system. PA/US images were acquired in real time and overlaid on pre-acquired cross-sectional magnetic resonance (MR) images. In the overlaid images, PA images represent the optical absorption characteristics of the surgical field, while US and MR images represent the morphological structure of surrounding tissues. To test the feasibility of the system, we prepared a tissue mimicking phantom which contained two samples, methylene blue as a contrast agent and water as a control. We acquired real-time overlaid PA/US/MR images of the phantom, which were well-matched with the optical and morphological properties of the samples. The developed system is the first approach to a novel intraoperative imaging technology based on PA imaging, and we believe that the system can be utilized in various surgical environments in the near future, improving the efficacy of surgical guidance.

Photoacoustic (PA) imaging of tumor oxygenation can be used to monitor vascular-targeted novel therapies. This study examines how a combination treatment, ultrasound-microbubbles (USMB)/radiation-therapy (XRT) alters oxygen saturation (sO2) estimates, which are then compared to power Doppler (PD) assessments of tumor vascularity. SCID mice were inoculated with subcutaneous, hind-leg PC3 tumors. The treatment consisted of XRT/MB (XRT: 8Gy/single-fraction; USMB: 3%/500 kHz/570kPa; n=3), USMB (n=3) and XRT (n=5) alone and untreated control (n=5). PA/PD imaging was acquired pre-treatment and 2h/24h post-treatment using the VevoLAZR (21 MHz, 750/850 nm). The volumetric tumor sO2 was quantified using histogram distributions and the average mode was computed. The vascularization index (VI), a PD metric of tumor vessel density, was studied along with the sO2 mode by comparing changes at 2h with pre-treatment. Mice whose pre-treatment sO2 levels were over 65%, exhibited a 15% drop in oxygenation at 2h, remaining unchanged by 24h. Examining the sO2 and VI relationships revealed differences between the groups. All groups (except control) exhibited a positive correlation when the ∆VI was plotted as a function of ∆sO2 (r2≥0.85). Mice in the XRT/MB group had the largest slope (11.7) suggesting that a change in sO2 was accompanied by the largest change in vessel density. The slope of the USMB and XRT treatments was 5.6 and 2.9, respectively. The combination treatment induced the largest changes in vessel density and sO2. Early PA estimates of tumor oxygenation appear to correlate with the treatment-induced vascular changes. Such measure could potentially be used for predicting treatment outcome.

Reflection artifacts caused by the high signal from the optical fiber/ needle tip reflecting off the seed is an important problem in minimally invasive photoacoustic imaging of brachytherapy seeds. The presence of these artifacts confounds the interpretation of images and reduces contrast. We apply a new method called PAFUSion (Photoacoustic-guided focused ultrasound) to identify and reduce reflection artifacts generated in interstitial illumination imaging of brachytherapy seeds. We present the system comprising of a US imager and linear array, with illumination provided via a cutting needle. Non-radioactive brachytherapy seeds are implanted in a tissue mimicking phantom and ex vivo porcine tissue. The PAFUSion-corrected imaging results successfully demonstrate that our approach can identify and strongly reduce reflection artifacts in the context of photoacoustic needle. The phantom result also shows that multi-spectral photoacoustics can separate signals between the seeds and other optical absorbers.

Lack of haptic feedback during laser surgery hampers controlling the incision depth, leading to a high risk of undesired tissue damage. Here we present a new feedback sensing method that accomplishes non-contact realtime monitoring of laser ablation procedures by detecting shock waves emanating from the ablation spot with air-coupled transducers. Experiments in soft and hard tissue samples attained high reproducibity in real-time depth estimation of the laser-induced cuts. The advantages derived from the non-contact nature of the suggested monitoring approach are expected to greatly promote the general applicability of laser-based surgeries.

Radio-frequency ablation (RFA) creates a thermal lesion in the atrial wall, with clearly recognizable optical and structural changes to the tissue. This can be detected by photoacoustic (PA) imaging, and used for monitoring of lesion depth, lesion functionality, and limiting excessive ablation. Porcine left atrium tissue can be split into three visually distinguishable regions, a thick white endocardium, pinkish myocardium and a thin gelatinous epicardium. In this study, we characterize the layered left atrium tissue in terms of the relevant photoacoustic parameters (wavelength, frequency content, imaging depth, lesion contrast). Previous studies in the literature targeted the photoacoustic characterization of fresh and ablated ventricular myocardium in the range of 650nm to 900nm. In this study we target the characterization of fresh and ablated left atrial tissue from 410nm to 1000nm, including the endocardium and epicardium. We generate the photoacoustic signals using a tunable pulsed laser source, and record those signals using either a broadband 1 mm hydrophone or a L12-3v transducer connected to the Verasonics machine for more realistic conditions. Initial experiments on fresh porcine tissue show that the presence of the endocardium and epicardium layers do affect the photoacoustic signal received. The signal recorded is representative of the difference in optical and mechanical properties between the layers. Ablated and non-ablated tissue also present differences in spectra. The determined optical contrast could be used in the PA monitoring of RFA lesion to monitor the extension of the lesion to the edge of the myocardium-epicardium border avoiding complications related to over ablation.

This work explores light delivery optimization for a photoacoustic surgical system previously proposed to provide real-time, intraoperative visualization of the internal carotid arteries hidden by bone during minimally invasive neurosurgeries. Monte Carlo simulations were employed to study 3D light propagation in tissue. For a 2.4 mm diameter drill shaft and 2.9 mm spherical drill tip, the optimal fiber distance from the drill shaft was 2 mm, determined from the maximum normalized fluence seen by the artery. A single fiber was insufficient to deliver light to arteries separated by a minimum of 8 mm. Using similar drill geometry and the optimal 2 mm fiber-to-drill shaft distance, Zemax ray tracing simulations were employed to propagate a 950 nm wavelength Gaussian beam through one or more 600 μm core diameter optical fibers, and the resulting optical beam profile was detected on the representative bone surface. For equally spaced fibers, a single merged optical profile formed with 7 or more fibers, determined by thresholding the resulting light profile images at 1/e times the maximum intensity. The corresponding spot size was larger than that of a single fiber transmitting the same input energy, thus reducing the fluence delivered to the sphenoid bone and enabling higher energies within safety limits. A prototype was designed and built based on these optimization parameters. The methodology we used to optimize our light delivery system to surround surgical tools is generalizable to multiple interventional photoacoustic applications.

Glaucoma is associated with dysfunction of the trabecular meshwork (TM), a fluid drainage tissue in the anterior eye. A promising treatment involves delivery of stem cells to the TM to restore tissue function. Currently histology is the gold standard for tracking stem cell delivery and differentiation. To expedite clinical translation, non-invasive longitudinal monitoring in vivo is desired. Our current research explores a technique combining ultrasound (US) and photoacoustic (PA) imaging to track mesenchymal stem cells (MSCs) after intraocular injection. Adipose-derived MSCs were incubated with gold nanospheres to label cells (AuNS-MSCs) for PA imaging. Successful labeling was first verified with in vitro phantom studies. Next, MSC delivery was imaged ex vivo in porcine eyes, while intraocular pressure was hydrostatically clamped to maintain a physiological flow rate through the TM. US/PA imaging was performed before, during, and after AuNS-MSC delivery. Additionally, spectroscopic PA imaging was implemented to isolate PA signals from AuNS-MSCs. In vitro cell imaging showed AuNS-MSCs produce strong PA signals, suggesting that MSCs can be tracked using PA imaging. While the cornea, sclera, iris, and TM region can be visualized with US imaging, pigmented tissues also produce PA signals. Both modalities provide valuable anatomical landmarks for MSC localization. During delivery, PA imaging can visualize AuNS-MSC motion and location, creating a unique opportunity to guide ocular cell delivery. Lastly, distinct spectral signatures of AuNS-MSCs allow unmixing, with potential for quantitative PA imaging. In conclusion, results show proof-of-concept for monitoring MSC ocular delivery, raising opportunities for in vivo image-guided cell delivery.

Heparin is used broadly in cardiac, pulmonary, surgical, and vascular medicine to treat thrombotic disorders with over 500 million doses per year globally. Despite this widespread use, it has a narrow therapeutic window and is one of the top three medication errors. The active partial thromboplastin time (PTT) monitors heparin, but this blood test suffers from long turnaround times, a variable reference range, and limited utility with low molecular weight heparin. Here, we describe an imaging technique that can monitor heparin concentration and activity in real time using photoacoustic spectroscopy via methylene blue as a simple and Federal Drug Agency-approved contrast agent. We found a strong correlation between heparin concentration and photoacoustic signal measured in phosphate buffered saline (PBS) and blood (R²>0.90). Clinically relevant concentrations were detected in blood with a heparin detection limit of 0.28 U/mL and a low molecular weight heparin (enoxaparin) detection limit of 72 μg/mL. We validated this imaging approach by correlation to the PTT (Pearson’s r = 0.86; p<0.05) as well as with protamine sulfate treatment. To the best of our knowledge, this is the first report to use imaging data to monitor anticoagulation.

Imaging of small animals, especially rodents provides physiological, pathological, and phenotypical insights into the most relevant milieu—an intact, living system. Currently, non-optical small-animal wholebody imaging approaches lack either spatiotemporal resolution or functional contrasts, whereas pure optical imaging suffers from either shallow penetration (up to ~1 mm) or a poor resolution-to-depth ratio (~1/3). Here, we present a standalone system that breaks all the above limitations. Our system features high spatiotemporal resolution and deep penetration, and can capture anatomical and functional contrasts. We imaged mouse wholebody dynamics in real time with clear sub-organ anatomical and functional details.

The ability to evaluate tumor oxygenation in the clinic could indicate prognosis and enable treatment monitoring, since oxygen deficient cancer cells are often more resistant to chemotherapy and radiotherapy. MultiSpectral Optoacoustic Tomography (MSOT) is a hybrid technique combining the high contrast of optical imaging with spatial resolution and penetration depth similar to ultrasound. We hypothesized that MSOT could reveal both tumor vascular density and function based on modulation of blood oxygenation.

We performed MSOT on nude mice (n=8) bearing subcutaneous xenograft PC3 tumors using an inVision 256 (iThera Medical). The mice were maintained under inhalation anesthesia during imaging and respired oxygen content was modified from 21% to 100% and back. After imaging, Hoechst 33348 was injected to indicate vascular perfusion and permeability. Tumors were then extracted for histopathological analysis and fluorescence microscopy. The acquired data was analyzed to extract a bulk measurement of blood oxygenation (SO₂^MSOT) from the whole tumor using different approaches. The tumors were also automatically segmented into 5 regions to investigate the effect of depth on SO₂^MSOT.

Baseline SO₂^MSOT values at 21% and 100% oxygen breathing showed no relationship with ex vivo measures of vascular density or function, while the change in SO₂^MSOT showed a strong negative correlation to Hoechst intensity (r=- 0.92, p=0.0016). Tumor voxels responding to oxygen challenge were spatially heterogeneous. We observed a significant drop in SO2 MSOT value with tumor depth following a switch of respiratory gas from air to oxygen (0.323±0.017 vs. 0.11±0.05, p=0.009 between 0 and 1.5mm depth), but no such effect for air breathing (0.265±0.013 vs. 0.19±0.04, p=0.14 between 0 and 1.5mm depth).

Our results indicate that in subcutaneous prostate tumors, baseline SO₂^MSOT levels do not correlate to tumor vascular density or function while the magnitude of the response to oxygen challenge provides insight into these parameters. Future work will include validation using in vivo imaging and protocol optimization for clinical application.

Noninvasive, accurate, continuous monitoring of multiple variables, including blood oxygenation, i.e. oxyhemoglobin saturation (SO₂) and total hemoglobin concentration (THb) in both high acuity and low acuity environments would greatly facilitate prompt diagnosis and treatment of physiologic derangements. However, most of the existing techniques for patient monitoring are invasive, while noninvasive techniques often fail to provide accurate measurements. We built a compact, multi-wavelength, nanosecond, fiber-coupled laser diode-based optoacoustic system for noninvasive, accurate monitoring of blood SO₂ and THb in veins and arteries. We tested the system by probing the radial artery of healthy volunteers. Using blood samples obtained by venipuncture, we also measured a reference THb for each volunteer. Moreover, the optoacoustic data were compared with that obtained from a commercially available noninvasive monitor for measurements of these variables. The optoacoustic system provided rapid, simultaneous, and continuous measurement of THb and SO₂ with high precision. The obtained results are promising and we plan to further test the system in clinical studies and at conditions simulating circulatory shock.

Cerebral hypoxia is a major contributor to neonatal/infant mortality and morbidity including severe neurological complications such as mental retardation, cerebral palsy, motor impairment, and epilepsy. Currently, no technology is capable of accurate monitoring of neonatal cerebral oxygenation. We proposed to use optoacoustics for this application by probing the superior sagittal sinus (SSS), a large central cerebral vein. We developed and built a multi-wavelength, optical parametric oscillator (OPO) and laser diode optoacoustic systems for measurement of SSS blood oxygenation in the reflection mode through open anterior or posterior fontanelles and in the transmission mode through the skull in the occipital area. In this paper we present results of initial tests of the laser diode system for neonatal cerebral oxygenation measurements. First, the system was tested in phantoms simulating neonatal SSS. Then, using the data obtained in the phantoms, we optimized the system’s hardware and software and tested it in neonates admitted in the Neonatal Intensive Care Unit. The laser diode system was capable of detecting SSS signals in the reflection mode through the open anterior and posterior fontanelles as well as in the transmission mode through the skull with high signal-to-noise ratio. Using the signals measured at different wavelengths and algorithms developed for oxygenation measurements, the laser diode system provided real-time, continuous oxygenation monitoring with high precision at all these locations.

We will present reflection-mode bioimaging system providing complementary optical, photoacsoutic and acoustic measurements by acoustic detector after each laser pulse with 2kHz repetition rate. The photons absorbed within the biological tissue provide optoacoustic (OA) signals, the photons absorbed by the external electrode of a detector provide the measurable diffuse reflectance (DR) from the sample and the probing ultrasonic (US) pulse. To demonstrate the in vivo capabilities of the system we performed complementary DR/OA/US imaging of small laboratory animals and human palm with 3.5mm/50μm/35μm lateral resolution at up to 3 mm diagnostic depth. Functional OA and DR imaging demonstrated the levels of tissue vascularization and blood supply. Structural US imaging was essential for understanding the position of vessels and zones with different perfusion. Before BiOS-2017 we plan to accomplish more in vivo experiments validating the developed triple-modality system as diagnostic tool to detect vascularization as well as mechanisms of vascular changes when monitoring response to therapy.

Intravascular photoacoustic-ultrasound (IVPA-US) imaging is an emerging hybrid modality for the detection of lipidladen plaques by providing simultaneous morphological and lipid-specific chemical information of an artery wall. The clinical utility of IVPA-US technology requires real-time imaging and display at speed of video-rate level. Here, we demonstrate a compact and portable IVPA-US system capable of imaging at up to 25 frames per second in real-time display mode. This unprecedented imaging speed was achieved by concurrent innovations in excitation laser source, rotary joint assembly, 1 mm IVPA-US catheter, differentiated A-line strategy, and real-time image processing and display algorithms. By imaging pulsatile motion at different imaging speeds, 16 frames per second was deemed to be adequate to suppress motion artifacts from cardiac pulsation for in vivo applications. Our lateral resolution results further verified the number of A-lines used for a cross-sectional IVPA image reconstruction. The translational capability of this system for the detection of lipid-laden plaques was validated by ex vivo imaging of an atherosclerotic human coronary artery at 16 frames per second, which showed strong correlation to gold-standard histopathology.

Arterial tissue imaging and characterization is important for disease diagnosis, treatment planning and monitoring, and research into disease processes. The high optical contrast of photoacoustic imaging can distinguish molecules with unique optical spectra from surrounding arterial tissue, while ultrasound is sensitive to variations in acoustic properties. Combining photoacoustics with ultrasonics provides more comprehensive diagnostic information by extracting molecular information from photoacoustics and structural information from ultrasound. Furthermore, ultrasound may be able to distinguish molecules with indistinct optical spectra but strong acoustic properties, such as calcification. In this work we will present our results applying our recently developed all-optical, multi-channel photoacoustic and laser-ultrasound imaging techniques to arterial tissue ex-vivo. We first apply redatuming techniques to remove reverberation artifacts, and subsequently image with time-reversal.

Ultrasound (US) imaging is widely used to guide vascular access procedures such as arterial and venous cannulation. As needle visualisation with US imaging can be very challenging, it is easy to misplace the needle in the patient and it can be life threating. Photoacoustic (PA) imaging is well suited to image medical needles and catheters that are commonly used for vascular access. To improve the success rate, a certain level of proficiency is required that can be gained through extensive practice on phantoms. Unfortunately, commercial training phantoms are expensive and custom-made phantoms usually do not replicate the anatomy very well. Thus, there is a great demand for more realistic and affordable ultrasound and photoacoustic imaging phantoms for vasculature access procedures training. Three-dimensional (3D) printing can help create models that replicate complex anatomical geometries. However, the available 3D printed materials do not possess realistic tissue properties. Alternatively, tissue-mimicking materials can be employed using casting and 3D printed moulds but this approach is limited to the creation of realistic outer shapes with no replication of complex internal structures. In this study, we developed a realistic vasculature access phantom using a combination of mineral oil based materials as background tissue and a non-toxic, water dissolvable filament material to create complex vascular structure using 3D printing. US and PA images of the phantoms comprising the complex vasculature network were acquired. The results show that 3D printing can facilitate the fabrication of anatomically realistic training phantoms, with designs that can be customized and shared electronically.

CCD camera based optical ultrasound detection is a promising alternative approach for high resolution 3D photoacoustic imaging (PAI). To fully exploit its potential and to achieve an image resolution <50 μm, it is necessary to incorporate variations of the speed of sound (SOS) in the image reconstruction algorithm. Hence, in the proposed work the idea and a first implementation are shown how speed of sound imaging can be added to a previously developed camera based PAI setup. The current setup provides SOS-maps with a spatial resolution of 2 mm and an accuracy of the obtained absolute SOS values of about 1%. The proposed dual-modality setup has the potential to provide highly resolved and perfectly co-registered 3D photoacoustic and SOS images.

Improved techniques for breast cancer screening are critically needed as current methods lack diagnostic accuracy. Using spectroscopic photoacoustic (sPA) molecular imaging with a priori knowledge of optical absorption spectra allows suppression of endogenous background signal, increasing the overall sensitivity and specificity of the modality to exogenous contrast agents. Here, sPA imaging was used to monitor antibody-indocyanine green (ICG) conjugates as they undergo optical absorption spectrum shifts after cellular endocytosis and degradation to allow differentiation between normal murine mammary glands from breast cancer by enhancing molecular imaging signal from target (B7-H3)-bound antibody-ICG. First, B7-H3 was shown to have highly specific (AUC of 0.93) expression on both vascular endothelium and tumor stroma in malignant lesions through quantitative immunohistochemical staining of B7-H3 on 279 human samples (normal (n=53), benign lesions (11 subtypes, n=182), breast cancers (4 subtypes, n=97)), making B7-H3 a promising target for sPA imaging. Second, absorption spectra of intracellular and degraded B7-H3-ICG and Isotype control (Iso-ICG) were characterized through in vitro and in vivo experiments. Finally, a transgenic murine breast cancer model (FVB/N-Tg(MMTVPyMT)634Mul) was imaged, and sPA imaging in found a 3.01 (IQR 2.63, 3.38, P<0.001) fold increase in molecular B7-H3-ICG signal in tumors (n=80) compared to control conditions (B7-H3-ICG in tumor negative animals (n=60), Iso-ICG (n=30), blocking B7-H3+B7-H3-ICG (n=20), and free ICG (n=20)) despite significant tumor accumulation of Iso-ICG, confirmed through ex vivo histology. Overall, leveraging anti-B7-H3 antibody-ICG contrast agents, which have dynamic optical absorption spectra representative of molecular interactions, allows for highly specific sPA imaging of murine breast cancer.

A key step in staging cancer is the diagnosis of metastasis that spreads through lymphatic system. For this reason, researchers develop various methods of sentinel lymph node mapping that often use a radioactive tracer. This study introduces a safe, cost-effective, high-resolution, high-sensitivity, and real-time method of visualizing the sentinel lymph node: ultrasound-guided photoacoustic (US/PA) imaging augmented by a contrast agent. In this work, we use clearable gold nanoparticles covered by a biocompatible polymer (glycol chitosan) to enhance cellular uptake by macrophages abundant in lymph nodes. We incubate macrophages with glycol-chitosan-coated gold nanoparticles (0.05 mg Au/ml), and then fix them with paraformaldehyde solution for an analysis of in vitro dark-field microscopy and cell phantom. The analysis shows enhanced cellular uptake of nanoparticles by macrophages and strong photoacoustic signal from labeled cells in tissue-mimicking cell phantoms consisting gelatin solution (6 %) with silica gel (25 μm, 0.3%) and fixed macrophages. The in-vivo US/PA imaging of cervical lymph nodes in healthy mice (nu/nu, female, 5 weeks) indicates a strong photoacoustic signal from a lymph node 10 minutes post-injection (2.5 mg Au/ml, 80 μl). The signal intensity and the nanoparticle-labeled volume of tissue within the lymph node continues to increase until 4 h post-injection. Histological analysis further confirms the accumulation of gold nanoparticles within the lymph nodes. This work suggests the feasibility of molecular/cellular US/PA imaging with biocompatible gold nanoparticles as a photoacoustic contrast agent in the diagnosis of lymph-node-related diseases.

Model organisms such as zebrafish play an important role for developmental biologists and experimental geneticists. Still, as they grow into their post-embryonic stage of development it becomes more and more difficult to image them because of high light scattering inside biological tissue. Optoacoustic mesoscopy based on spherically focused, high frequency, ultrasound detectors offers an alternative, where it relies on the focusing capabilities of the ultrasound detectors in generating the image rather than on the focusing of light. Nonetheless, because of the limited numerical aperture the resolution is not isotropic, and many structures, especially elongated ones, such as blood vessels and other organs, are either invisible, or not clearly identifiable on the final image. Herein, based on high frequency ultrasound detectors at 100 MHz and 50 MHz we introduce multi orientation (view) optoacoustic mesoscopy. We collect a rich amount of signals from multiple directions and combine them using a weighted sum in the Fourier domain and a Wiener deconvolution into a single high resolution three-dimensional image. The new system achieves isotropic resolutions on the order of 10 μm in-plane, 40 μm axially, and SNR enhancement of 15 dB compared to the single orientation case. To showcase the system we imaged a juvenile zebrafish ex vivo, which is too large to image using optical microscopic techniques, the reconstructed images show unprecedented performance in terms of SNR, resolution, and clarity of the observed structures. Using the system we see the inner organs of the zebrafish, the pigmentation, and the vessels with unprecedented clarity.

Nearly 800,000 people in the U.S. experience an incident of stroke each year; ~80% of these are first time occurrences and ~87% are ischemic in nature. Someone dies of a stroke every few minutes in the U.S. but despite its prevalence there have been minimal advances in the early detection and screening of thromboembolic events, especially during patient post-operative periods or in genetically predisposed individuals. Environmental or genetic factors may disrupt the balance between coagulation and lysis of micro-thrombi in circulation and increase the risk of stroke. We introduced here a novel in vivo multicolor negative-contrast photoacoustic (PA) flow cytometry (PAFC ) platform with many innovations including customized high pulse repetition rate 1064 laser from IPG Photonics Corporation, powerful laser diode array, multichannel optical schematic, and time-resolved recording system. Using animal models, we verified the potential of this technology to detect small clots in relatively large vessels in vivo. If future clinical trials using a cost-effective, easy-to-use, safe, watch-like, wearable PA probe are successful, PAFC could provide breakthroughs in early monitoring of the growth in size and number of small clots that may predict and potentially prevent fatal thromboembolic complications. We also believe that this technology could be utilized to assess therapeutic benefits of anticoagulants and develop more efficient dosage in treatments by analyzing changes in the composition and frequency of micro-thrombi

Intravascular photoacoustics (IVPA) can complement intravascular ultrasound (IVUS) morphological information adding specificity. A practical IVUS/IVPA catheter is mandatory to have a wideband ultrasonic detector with enough sensitivity despite the necessary miniaturization to be fitted in less than 1 mm to pass through thin vasculature. The optical detection of ultrasound provides the compactness, sensitivity and bandwidth required for this application. In this work we compare the ultrasonic detection performance of two FBG sensors inscribed in singlemode microstructured PMMA and TOPAS polymer optical fibers to be integrated as ultrasonic sensor in an IVUS/IVPA catheter. The outer diameters of the fibers are 140 μm. The NEPs obtained were 1.31 kPa and 1. 98 kPa for the 5 mm long PMMA FBG and the 2 mm long TOPAS FBG respectively at 55 MHz detection bandwidth. The directivity and the spectral response are characterized as well. The intrinsic acoustic sensitivity of the TOPAS optical fiber is comparable to the PMMA optical fiber but without any water swelling effects associated to the last one what cause a slow FBG central wavelength drift over several hours when the sensor is immersed in water. The use of TOPAS mPOF is an improvement in terms of robustness and usability of this kind of ultrasound sensors based on FBGs in polymer optical fibers.

While molecular and cellular imaging can be used to visualize the conventional morphology characteristics of vulnerable plaques, there is a need to monitor other physiological factors correlated with high rupture rates; a high M1 activated macrophage concentration is one such indicator of high plaque vulnerability. Here, we present a molecularly targeted contrast agent for intravascular photoacoustic (IVPA) imaging consisting of liposomes loaded with indocyanine green (ICG) J-aggregates with high absorption at 890 nm, allowing for imaging in the presence of blood. This “Lipo-ICG” was targeted to a biomarker of M1 activated macrophages in vulnerable plaques: folate receptor beta (FRβ). The targeted liposomes accumulate in plaques through areas of endothelial dysfunction, while the liposome encapsulation prevents nonspecific interaction with lipids and endothelium. Lipo-ICG specifically interacts with M1 activated macrophages, causing a spectral shift and change in the 890/780 nm photoacoustic intensity ratio upon breakdown of J-aggregates. This sensing mechanism enables assessment of the M1 activated macrophage concentration, providing a measure of plaque vulnerability. In a pilot in vivo study utilizing ApoE deficient mouse models of atherosclerosis, diseased mice showed increased uptake of FRβ targeted Lipo-ICG in the heart and arteries vs. normal mice. Likewise, targeted Lipo-ICG showed increased uptake vs. two non-targeted controls. Thus, we successfully synthesized a contrast agent to detect M1 activated macrophages in high risk atherosclerotic plaques and exhibited targeting both in vitro and in vivo. This biocompatible agent could enable M1 macrophage detection, allowing better clinical decision making in treatment of atherosclerosis.

Photoacoustic tomography can, in principle, provide quantitatively accurate, high-resolution, images of chromophore distributions in 3D in vivo. However, achieving this goal requires not only dealing with the optical fluence-related spatial and spectral distortion but also having access to high quality, calibrated, measurements and using image reconstruction algorithms free from inaccurate assumptions. Furthermore, accurate knowledge of experimental parameters, such as the positions of the ultrasound detectors and the illumination pattern, is necessary for the reconstruction step. A meticulous and rigorous experimental phantom study was conducted to show that highly-resolved 3D estimation of chromophore distributions can be achieved: a crucial step towards in vivo implementation. The phantom consisted of four 580 μm diameter tubes with different ratios of copper sulphate and nickel sulphate as hemoglobin analogues, submersed in a background medium of intralipid and india ink. The optical absorption, scattering, photostability, and Grüneisen parameter were characterised for all components independently. A V-shaped imaging scanner enabled 3D imaging with the high resolution, high sensitivity, and wide bandwidth characteristic of Fabry-Pérot ultrasound sensors, but without the limited-view disadvantage of single-plane scanners. The optical beam profile and position were determined experimentally. Nine wavelengths between 750 and 1110 nm were used. The images of the chromophore concentrations were obtained using a model-based, two-step, procedure, that did not require image segmentation. First, the acoustic reconstruction was solved with an iterative time-reversal algorithm to obtain images of the initial acoustic pressure at each of the nine wavelengths for an 18×17×13 mm³ volume with 50μm voxels. Then, 3D high resolution estimates of the chromophore concentrations were obtained by using a diffusion model of light transport in an iterative nonlinear optimisation scheme. Among the lessons to be drawn from this study, one is fundamental: in order to obtain accurate estimates of chromophores (or their ratios) it is not only necessary to model the light fluence accurately, but it is just as crucial to obtain accurate estimates of the initial acoustic pressure distributions, and to account for variations in the thermoelastic efficiency (Grüneisen parameter).

Quantitative photoacoustic tomography (qPAT) aims to extract physiological parameters, such as blood oxygen saturation (sO₂), from measured multi-wavelength image data sets. The challenge of this approach lies in the inherently nonlinear fluence distribution in the tissue, which has to be accounted for by using an appropriate model, and the large scale of the inverse problem. In addition, the accuracy of experimental and scanner-specific parameters, such as the wavelength dependence of the incident fluence, the acoustic detector response, the beam profile and divergence, needs to be considered. This study aims at quantitative imaging of blood sO₂, as it has been shown to be a more robust parameter compared to absolute concentrations. We propose a Monte-Carlo–based inversion scheme in conjunction with a reduction in the number of variables achieved using image segmentation. The inversion scheme is experimentally validated in tissue-mimicking phantoms consisting of polymer tubes suspended in a scattering liquid. The tubes were filled with chromophore solutions at different concentration ratios. 3-D multi-spectral image data sets were acquired using a Fabry-Perot based PA scanner. A quantitative comparison of the measured data with the output of the forward model is presented. Parameter estimates of chromophore concentration ratios were found to be within 5 % of the true values.

Spectral analysis of photoacoustic (PA) signals in the ultrasound frequency domain is a method that analyzes the power spectrum of PA signals to quantify tissue microstructures. PA spectral analysis has been correlated to changes in the size, morphology and concentration of absorbers that are smaller than the system spatial resolution. However, the calculated spectral parameters are still not system independent due to difficulty in eliminating variations in the light distribution for different optical wavelengths. Changes in spectral parameters for the same absorber geometry but different optical illumination wavelengths needs to be carefully examined. A gelatin vessel phantom is used. The vessels contain red blood cells comprised of oxy, deoxy and methemoglobin induced using oxygen, sodium hydrosulfite and sodium nitrite, respectively. The samples were imaged using the VevoLAZR system at wavelengths 680 – 905 nm in steps of 15 nm. The radiofrequency (RF) signals were analyzed to calculate the spectral slope. The results were compared to simulated RF signals acquired using the mcxyz Monte Carlo package coupled to the solution of the PA wave equation using the Green’s function approach. Changes in the spectral slope as a function of optical wavelength were detected. For longer optical wavelengths, the spectral slope increased for deoxyhemoglobin, but decreased for oxyhemoglobin and methemoglobin. The changes in the spectral slope were correlated to changes in the fluence distribution as optical properties change for different wavelengths. The change in the spectral slope as a function of optical wavelength and chromophore content can potentially be used in spectral unmixing for better estimation of hemoglobin content.

Light fluence inside turbid media can be experimentally mapped by measuring ultrasonically modulated light (Acousto-optics). To demonstrate the feasibility of fluence corrected Photoacoustic (PA) imaging, we have realized a tri-modality (i.e. photoacoustic, acousto-optic and ultrasound) tomographic small animal imaging system. Wherein PA imaging provides high resolution map of absorbed optical energy density, Acousto-optics yields the fluence distribution map in the corresponding PA imaging plane and Ultrasound provides morphological information. Further, normalization of the PA image with the acousto-optically measured fluence map results in an image that directly represents the optical absorption. Human epidermal growth factor receptor 2 (HER2) is commonly found overexpressed in human cancers, among which breast cancers, resulting in a more aggressive tumor phenotype. Identification of HER2-expression is clinically relevant, because cancers overexpressing this marker are amenable to HER2-directed therapies, among which antibodies trastuzumab and pertuzumab. Here, we investigate the feasibility and advantage of acousto-optically assisted fluence compensated PA imaging over PA imaging alone in visualizing and quantifying HER2 expression. For this experiment, nude mice were xenografted with human breast cancer cell lines SKBR3 and BT474 (both HER2 overexpressing), as well as HER2-negative MDA-MB-231. To visualize HER2 expression in these mice, HER2 monoclonal antibody pertuzumab (Perjeta®, Roche), was conjugated to near-infrared dye IRDye 800CW (800CW, LICOR Biosciences) at a ratio of 1∶2 antibody to 800CW. When xenograft tumors measured ≥ 100 mm3, mice received 100 µg 800CW-pertuzumab intravenously. Three days post injection, mice were scanned for fluorescence signal with an IVIS scanner. After fluorescence scans, mice were euthanized and imaged in our PA tomographic imaging system.

To unlock the full capability of photoacoustic tomography as a quantitative, high resolution, molecular imaging modality, the problem of quantitative photoacoustic tomography must be solved. The aim in this is to extract clinically relevant functional information from photoacoustic images by finding the concentrations of the chromophores in the tissue. This is a challenging task due to the effect of the unknown but spatially and spectrally varying light fluence within the tissue. Many inversion schemes that include a model of the fluence have been proposed, but these have yet to make an impact in pre-clinical or clinical imaging. In this study, the statistical independence of the chromophore's distributions is proposed as a means of improving the robustness and hence the usefulness of the model-based inversion methods. This was achieved by minimising the mutual information between the estimated chromophore distributions in addition to the least squares data error within a gradient-based optimisation scheme. By applying the proposed inversion scheme to simulated multiwavelength photoacoustic images, it was shown that more accurate estimates for the concentrations of independent chromophores could be obtained in the presence of errors in the model parameters.

In order to achieve real-time image rendering, optoacoustic tomography reconstructions are commonly done with back-projection algorithms due to their simplicity and low computational complexity. However, model-based algorithms have been shown to attain more accurate reconstruction performance due to their ability to model arbitrary detection geometries, transducer shapes and other experimental factors. The high computational complexity of the model-based schemes makes it challenging to be implemented for real time inversion. Herein, we introduce a novel discretization method for model-based optoacoustic tomography that enables its efficient parallel implementation on graphics processing units with extremely low memory overhead. We demonstrate that, when employing a tomographic scanner with 256 detectors, the new method achieves model-based optoacoustic inversion at 20 frames per second for a 200 × 200 image grid.

Due to modeling and experimental imperfections, multispectral optoacoustic tomography images are often afflicted with negative values, which are further amplified when propagating into the spectrally unmixed images of chromophore concentrations. Since negative values have no physical meaning, accuracy can potentially be improved by imposing non-negativity constraints on the initial reconstructions and the unmixing steps. Herein, we compare several non-negative constrained approaches with reconstruction and spectral unmixing performed separately or combined in a single inverse step. The quantitative performance and sensitivity of the different approaches in detecting small amounts of spectrally-distinct chromophores are studied in tissue-mimicking phantoms and mouse experiments.

A hybrid optical and acoustic resolution optoacoustic endoscopy is proposed. Laser light is transmitted to tissue by two types of illumination for optical and acoustic resolution imaging respectively. An unfocused ultrasound detector is used for recording optoacoustic signals. The endoscopy probe attains 3.6 mm diameter and is fully encapsulated into a catheter system. We examine the performance of the hybrid endoscope with phantoms and tissue sample, which shows that the hybrid endoscopy can obtain optical resolution in superficial microscopic imaging and ultrasonic tomography reconstruction resolution when imaging at greater depths.

Extraction of murine cardiac functional parameters on a beat-by-beat basis remains challenging with the existing imaging modalities. Novel methods enabling in vivo characterization of functional parameters at a high temporal resolution are poised to advance cardiovascular research and provide a better understanding of the mechanisms underlying cardiac diseases. We present a new approach based on analyzing contrast-enhanced optoacoustic (OA) images acquired at high volumetric frame rate without using cardiac gating or other approaches for motion correction. Acute myocardial infarction was surgically induced in murine models, and the method was modified to optimize for acquisition of artifact-free optoacoustic data. Infarcted hearts could be differentiated from healthy controls based on a significantly higher pulmonary transit time (PTT: infarct 2.07 s vs. healthy 1.34 s), while no statistically significant difference was observed in the heart rate (318 bpm vs. 309 bpm). In combination with the proven ability of optoacoustics to track targeted probes within the injured myocardium, our method is capable of depicting cardiac anatomy, function, and molecular signatures on a beat-by-beat basis, both with high spatial and temporal resolution, thus providing new insights into the study of myocardial ischemia.

Metastasis is responsible for as many as 90% of cancer-related deaths, and the deadliest skin cancer, melanoma, has a high propensity for metastasis. Since hematogenous spread of circulating tumor cells (CTCs) is cancer’s main route of metastasis, detecting and destroying CTCs can impede metastasis and improve patients’ prognoses. Extensive studies employing exogenous agents to detect tumor-specific biomarkers and guide therapeutics to CTCs have achieved promising results, but biosafety remains a critical concern. Taking another approach, physical detection and destruction of CTCs is a safer way to evaluate and reduce metastasis risks. Melanoma cells strongly express melanosomes, providing a striking absorption contrast with the blood background in the red to near-infrared spectrum. Exploiting this intrinsic optical absorption contrast of circulating melanoma cells, we coupled dual-wavelength photoacoustic flow cytography with a nanosecond-pulsed laser killing mechanism that specifically targets melanoma CTCs. We have successfully achieved in vivo label-free imaging of rare single CTCs and CTC clusters in mice. Further, the photoacoustic signal from a CTC immediately hardware-triggers a lethal pinpoint laser irradiation that lyses it on the spot in a thermally confined manner. Our technology can facilitate early inhibition of metastasis by clearing circulating tumor cells from vasculature.

Planar Fabry-Pérot (FP) ultrasound sensor arrays have been used to produce in-vivo photoacoustic images of high quality due to their broad detection bandwidth, small element size, and dense spatial sampling. However like all planar arrays, FP sensors suffer from the limited view problem. Here, a multi-angle FP sensor system is described that mitigates the partial view effects of a planar FP sensor while retaining its detection advantages. The possibility of improving data acquisition speed through the use of sub-sampling techniques is also explored. The capabilities of the system are demonstrated with 3D images of pre-clinical targets

Non-invasive observation of spatio-temporal neural activity of large neural populations distributed over the entire brain of complex organisms is a longstanding goal of neuroscience [1,2]. Recently, genetically encoded calcium indicators (GECIs) have revolutionized neuroimaging by enabling mapping the activity of entire neuronal populations in vivo [3]. Visualization of these powerful sensors with fluorescence microscopy has however been limited to superficial regions while deep brain areas have so far remained unreachable [4]. We have developed a volumetric multispectral optoacoustic tomography platform for imaging neural activation deep in scattering brains [5]. The developed methodology can render 100 volumetric frames per second across scalable fields of view ranging between 50-1000 mm3 with respective spatial resolution of 35-150µm. Experiments performed in immobilized and freely swimming larvae and in adult zebrafish brains expressing the genetically-encoded calcium indicator GCaMP5G demonstrated, for the first time, the fundamental ability to directly track neural dynamics using optoacoustics while overcoming the depth barrier of optical imaging in scattering brains [6]. It was further possible to monitor calcium transients in a scattering brain of a living adult transgenic zebrafish expressing GCaMP5G calcium indicator [7]. Fast changes in optoacoustic traces associated to GCaMP5G activity were detectable in the presence of other strongly absorbing endogenous chromophores, such as hemoglobin. The results indicate that the optoacoustic signal traces generally follow the GCaMP5G fluorescence dynamics and further enable overcoming the longstanding optical-diffusion penetration barrier associated to scattering in biological tissues [6]. The new functional optoacoustic neuroimaging method can visualize neural activity at penetration depths and spatio-temporal resolution scales not covered with the existing neuroimaging techniques. Thus, in addition to the well-established capacity of optoacoustics to resolve vascular anatomy and multiple hemodynamic parameters deep in scattering tissues, the newly developed methodology offers unprecedented capabilities for functional whole brain observations of fast calcium dynamics.

Prostate cancer (PCa) is the most commonly diagnosed cancer in American men for the past decades. PCa has a relatively low progression rate but the 5 year survival rate decreases dramatically once the cancer has metastasized. Differentiating aggressive from indolent PCa is critical for improving PCa patient outcomes and preventing metastasis and death. Prostate biopsy is the standard procedure for evaluating the presence and aggressiveness of PCa. The microarchitecture of the biopsied tissues visualized by histology process is evaluated by pathologists and assigned a Gleason score as a quantification of the aggressiveness. In our previous study, we have shown that photoacoustic spectral analysis (PASA) is capable of quantifying the Gleason scores of the H&E stained human prostate tissues. In this study, we attempt to assess the Gleason scores without any staining by taking advantage of the strong optical absorption of nucleic acid at ultraviolet wavelengths. PA signals were generated by wide field illumination at 266 nm and received by a hydrophone with a bandwidth of 0-20 MHz. DU145 prostate cancer cells at the concentrations of 0.8, 0.4, 0.05, 0.025 and 0.0125 million per cm3 simulating those in cancerous and normal tissues were first attempted. The measurements were repeated for 10 times at each concentration. A correlation of 0.86 was observed between the PA signal intensities and the cell concentrations. Human PCa tissues with Gleason score 6, 7 and 8 and normal tissues were assessed. With 11 samples, a correlation of 0.89 was found between the Gleason scores and PASA slopes.

Limited-view artefacts affect most optoacoustic (photoacoustic) imaging systems due to geometrical constraints that impede achieving full tomographic coverage as well as limited light penetration into scattering and absorbing objects. Indeed, it has been theoretically established and experimentally verified that accurate optoacoustic images can only be obtained if the imaged sample is fully enclosed (< π angular coverage) by the measuring locations. Since in many cases full angular coverage cannot be achieved, the visibility of structures along certain orientations is hampered. These effects are of particular relevance in the case of hand-held scanners with the imaged volume only accessible from one side. Herein, a new approach termed dynamic particle-enhanced optoacoustic tomography (DPOT) is described for accurate structural imaging in limited-view scenarios. The method is based on the non-linear combination of a sequence of tomographic reconstructions representing sparsely distributed moving particles. Good performance of the method is demonstrated in experiments consisting of dynamic visualization of flow of suspended microspheres in three-dimensions. The method is expected to be applicable for improving accuracy of angiographic optoacoustic imaging in living organisms.

In vivo photoacoustic (PA) imaging with high spatial resolution and depth penetration has remained a challenge due to the high background signal from chromophores in biological tissue. In this study, we introduce a novel imaging technique called triplet differential (TD) imaging which allows for the substantial reduction of background signals from tissue. TD imaging uses the ability of molecules to enter a triplet state via intersystem crossing from an excited singlet state. Molecules in the triplet state exhibit a spectral shift in their optical absorption spectra, creating two separate absorption peaks for each molecule’s singlet and triplet states. Since the PA signal is proportional to optical absorption, a differential signal can be obtained by comparing the PA signal for molecules raised to the triplet state to those in the singlet state. We worked with methylene blue conjugated polyacrylamide nanoparticles with a polyethylene glycol dimethacrylate cross-linker (MBNP) which has a singlet peak absorption at 660nm and triplet peak absorption at 840nm. Since only certain molecules such as methylene blue can enter the triplet state efficiently, the difference in the PA signal before and after excitation of the MBNP to the triplet state is largely independent of the background noise and mainly contributed by the MBNP in the triplet state. Preliminary results have shown that up to an 8-fold increase in the PA signal of the MBNP in the triplet state can be achieved.

Optical resolution photoacoustic tomography (OR-PAM) is a non-invasive imaging method that uses endogenous contrast agents in the body, such as hemoglobin in the blood. OR-PAM has a resolution equivalent to a microscope, and has high optical contrast. OR-PAM has been made to expand the application to the medical field by increasing the speed of imaging and minimizing the size of the system. In this research, we accomplished these two specifications by using MEMS technology to integrate a fast scan functionality into a handheld probe. Using MEMS technology, beam guides, ultrasound guidance, and mechanical scanning subsystems were integrated into a single small probe. The measured lateral resolution is 16 μm, and the measured B-scan and volume imaging rate are 35 and 0.2 Hz to obtain a photoacoustic (PA) image with 200 and 700 pixels. We imaged a variety of phantom and in vivo samples such as carbon fibers, mouse ears, eyes, and brains.

Optical-resolution photoacoustic microscopy offers exquisite and specific contrast to optical absorption. Conventional approaches generally involves raster scanning a focused spot over the sample. Here, we demonstrate that a full-field illumination approach with multiple speckle illumination can also provide diffraction-limited optical-resolution photoacoustic images. Two different proof-of-concepts are demonstrated with micro-structured test samples. The first approach follows the principle of correlation/ghost imaging,^{1, 2} and is based on cross-correlating photoacoustic signals under multiple speckle illumination with known speckle patterns measured during a calibration step. The second approach is a speckle scanning microscopy technique, which adapts the technique proposed in fluorescence microscopy by Bertolotti and al.:³ in our work, spatially unresolved photoacoustic measurements are performed for various translations of unknown speckle patterns. A phase-retrieval algorithm is used to reconstruct the object from the knowledge of the modulus of its Fourier Transform yielded by the measurements. Because speckle patterns naturally appear in many various situations, including propagation through biological tissue or multi-mode fibers (for which focusing light is either very demanding if not impossible), speckle-illumination-based photoacoustic microscopy provides a powerful framework for the development of novel reconstruction approaches, well-suited to compressed sensing approaches.²

Circulating tumor cell (CTC) clusters arise from multicellular grouping in the primary tumor and elevate the metastatic potential by 23 to 50 fold compared to single CTCs. High throughout detection and quantification of CTC clusters is critical for understanding the tumor metastasis process and improving cancer therapy. In this work, we report a linear-array-based photoacoustic tomography (LA-PAT) system capable of label-free high-throughput CTC cluster detection and quantification in vivo. LA-PAT detects CTC clusters and quantifies the number of cells in them based on the contrast-to-noise ratios (CNRs) of photoacoustic signals. The feasibility of LA-PAT was first demonstrated by imaging CTC clusters ex vivo. LA-PAT detected CTC clusters in the blood-filled microtubes and computed the number of cells in the clusters. The size distribution of the CTC clusters measured by LA-PAT agreed well with that obtained by optical microscopy. We demonstrated the ability of LA-PAT to detect and quantify CTC clusters in vivo by imaging injected CTC clusters in rat tail veins. LA-PAT detected CTC clusters immediately after injection as well as when they were circulating in the rat bloodstreams. Similarly, the numbers of cells in the clusters were computed based on the CNRs of the photoacoustic signals. The data showed that larger CTC clusters disappear faster than the smaller ones. The results prove the potential of LA-PAT as a promising tool for both preclinical tumor metastasis studies and clinical cancer therapy evaluation.

Achieving a good signal-to-noise ratio at increased depths remains a challenge, even for photoacoustic imaging, which stimulates the search for possible contrast improvements. Both double-pulse and pulse burst excitation are shown beneficial for increasing the signal-to-noise ratio or acquiring additional information about the sample. We use the advantage of semiconductor laser diodes offering great opportunities regarding both number of pulses in the burst and inter-pulse delay times to study the dynamics of the pulse burst excitation responses of the single-element PZT transducers, looking for possibilities towards contrast improvement. We concentrate on inter-pulse delay ranges of few hundred nanoseconds and low central frequency transducers as they are mainly used for clinical applications We show that using pulse burst excitation with up to five pulses per burst and transducer-matched inter-pulse delays can increase the registered maximum amplitude, leading to signal-to-noise ratio improvement. The multi-pulse response amplitude increase amounted to 20% of the amplitude of a single-pulse response in the performed measurement.

Roughly 0.6 million people die each year from malaria due to lack of early diagnosis and well-timed treatment. Our previous study demonstrated great potential of in vivo photoacoustic (PA) flow cytometry (PAFC) for early diagnosis of deadly diseases with focus on cancer and thromboembolic complications. Here we demonstrate potential of advanced PAFC platforms using new laser, ultrasound transducer array and recording system to detect infected red blood cells (iRBCs) with malaria-associated pigment hemozoin which has a higher PA contrast than blood background. Mature parasites of human infecting species such as P. falciparum characteristically sequester mature iRBCs in the capillary bed and display synchrony in their reproductive cycle. To address this issue prior to clinical application, new PAFC platform was verified in a pre-clinical study using new animal models. Specifically, we used P. chabaudi (a rodent malaria species that mimics the characteristics of the most virulent human counterpart) to estimate the detection sensitivity with immature ring-stage parasites in peripheral blood, compared PA signals from the differing species, and examined the relationship between PA signal amplitudes and level of blood oxygenation. Based on previous successful trials on melanoma patients with melanin as an intrinsic PA marker, which has similar absorption as hemozoin, we believe that after additional malaria–related clinical trials, PAFC with a small 1064 nm laser and wearable a cost-effective, easy-to-use, watch-like, safe PA probe will provide malaria diagnosis in humans at parasitemia levels 10e4 -times lower than the current gold standard of diagnosis, the Giemsa-stained blood smear. It can reduce malaria-related mortality by well-timed treatment, especially in children in malaria-endemic countries.

Arkansas Nanomedicine Center at the University of Arkansas for Medical Sciences in collaboration with other Arkansas Universities and the FDA-based National Center of Toxicological Research in Jefferson, AR is developing novel techniques for rapid quantification of graphene-based nanomaterials (GBNs) in various biological samples. All-carbon GBNs have wide range of potential applications in industry, agriculture, food processing and medicine; however, quantification of GBNs is difficult in carbon reach biological tissues. The accurate quantification of GBNs is essential for research on material toxicity and the development of GBNs-based drug delivery platforms. We have developed microscopy and cytometry platforms for detection and quantification of GBNs in single cells, tissue and blood samples using photoacoustic contrast of GBNs. We demonstrated PA quantification of individual graphene uptake by single cells. High-resolution PA microscopy provided mapping of GBN distribution within live cells to establish correlation with intracellular toxic phenomena using apoptotic and necrotic assays. This new methodology and corresponding technical platform provide the insight on possible toxicological risks of GBNs at singe cells levels. In addition, in vivo PA image flow cytometry demonstrated the capability to monitor of GBNs pharmacokinetics in mouse model and to map the resulting biodistribution of GBNs in mouse tissues. The integrated PA platform provided an unprecedented sensitivity toward GBNs and allowed to enhance conventional toxicology research by providing a direct correlation between uptake of GBNs at a single cell level and cell viability status.

Imaging modalities utilize contrast agents to improve morphological visualization and to assess functional and molecular/cellular information. Here we present a new type of nanometer scale multi-functional particle that can be used for multi-modal imaging and therapeutic applications. Specifically, we synthesized monodisperse 20 nm Prussian Blue Nanocubes (PBNCs) with desired optical absorption in the near-infrared region and superparamagnetic properties. PBNCs showed excellent contrast in photoacoustic (700 nm wavelength) and MR (3T) imaging. Furthermore, photostability was assessed by exposing the PBNCs to nearly 1,000 laser pulses (5 ns pulse width) with up to 30 mJ/cm2 laser fluences. The PBNCs exhibited insignificant changes in photoacoustic signal, demonstrating enhanced robustness compared to the commonly used gold nanorods (substantial photodegradation with fluences greater than 5 mJ/cm2). Furthermore, the PBNCs exhibited superparamagnetism with a magnetic saturation of 105 emu/g, a 5x improvement over superparamagnetic iron-oxide (SPIO) nanoparticles. PBNCs exhibited enhanced T2 contrast measured using 3T clinical MRI. Because of the excellent optical absorption and magnetism, PBNCs have potential uses in other imaging modalities including optical tomography, microscopy, magneto-motive OCT/ultrasound, etc. In addition to multi-modal imaging, the PBNCs are multi-functional and, for example, can be used to enhance magnetic delivery and as therapeutic agents. Our initial studies show that stem cells can be labeled with PBNCs to perform image-guided magnetic delivery. Overall, PBNCs can act as imaging/therapeutic agents in diverse applications including cancer, cardiovascular disease, ophthalmology, and tissue engineering. Furthermore, PBNCs are based on FDA approved Prussian Blue thus potentially easing clinical translation of PBNCs.

Super-resolution ultrasound imaging techniques have shown promising potential in non-invasive imaging of deep-lying tissue. However, these methods utilize microbubbles, limiting its utility to visualization of vasculature with moving bubbles. To resolve extravascular targets, our group previously introduced a method for super-resolution ultrasound imaging based on laser-activated nanodroplets (LANDs) that repeatedly vaporize and recondense in response to optical irradiation. The method resolves the location of LANDs from the difference between two imaging frames capturing vaporization and recondensation of individual LANDs. However, since only two neighboring frames are used to produce a difference frame, this method is sensitive to noise-related errors limiting the improvement in spatial resolution. In this study, we introduce a new approach to super-resolution imaging. In our approach, ultrafast imaging, which typically captures images at over several thousand frames per second, was used for spatio-temporal compounding. Specifically, multiple successive ultrasound frames were used to obtain the difference frame with improved reliability and repeatability thus enhanced spatial resolution. To evaluate our approach, we imaged a phantom containing uniformly-distributed LANDs using an ultrasound system equipped with a linear array transducer and interfaced with pulsed laser. An ultrafast plane-wave compounding approach was used to capture ultrasound images at 6 kHz frame rate. We achieved a four-fold improvement in spatial resolution over the previous approach. In addition, three-dimensional super-resolution imaging of a phantom with microcapillaries containing LANDs was performed illustrating the robustness of our method. These results suggest that our approach has the potential for high-resolution molecular imaging of intravascular and extravascular targets.