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.

The planar Fabry-Pérot (FP) sensor provides high quality photoacoustic (PA) images but beam walk-off limits sensitivity and thus penetration depth to ≈1 cm. Planoconcave microresonator sensors eliminate beam walk-off enabling sensitivity to be increased by an order-of-magnitude whilst retaining the highly favourable frequency response and directional characteristics of the FP sensor. The first tomographic PA images obtained in a tissue-realistic phantom using the new sensors are described. These show that the microresonator sensors provide near identical image quality as the planar FP sensor but with significantly greater penetration depth (e.g. 2-3cm) due to their higher sensitivity. This offers the prospect of whole body small animal imaging and clinical imaging to depths previously unattainable using the FP planar sensor.

A novel all-optical forward-viewing photoacoustic probe using a flexible coherent fibre-optic bundle and a Fabry- Perot (FP) ultrasound sensor has been developed. The fibre bundle, along with the FP sensor at its distal end, synthesizes a high density 2D array of wideband ultrasound detectors. Photoacoustic waves arriving at the sensor are spatially mapped by optically scanning the proximal end face of the bundle in 2D with a CW wavelength-tunable interrogation laser. 3D images are formed from the detected signals using a time-reversal image reconstruction algorithm. The system has been characterized in terms of its PSF, noise-equivalent pressure and field of view. Finally, the high resolution 3D imaging capability has been demonstrated using arbitrary shaped phantoms and duck embryo.

Although ultrasound transducers based on commercial piezoelectric-material have been widely used, they generally have limited bandwidth centered at the resonant frequency. Currently, several pure-optical ultrasonic detection methods have gained increasing interest due to their wide bandwidth and high sensitivity. However, most of them require customized components (such as micro-ring, SPR, Fabry-Perot film, etc), which limit their broad implementations. In this study, we presented a simple pure-optical ultrasound detection method, called “Polarization-dependent Reflection Ultrasonic Detection” (PRUD). It detects the intensity difference between two polarization components of the probe beam that is modulated by ultrasound waves. PRUD detect the two components by using a balanced detector, which effectively suppressed much of the unwanted noise. We have achieved the sensitivity (noise equivalent pressure) to be 1.7kPa, and this can be further improved. In addition, like many other pure-optical ultrasonic detection methods, PRUD also has a flat and broad bandwidth from almost zero to over 100MHz. Besides theoretical analysis, we did a phantom study by imaging a tungsten filament to demonstrate the performance of PRUD. We believe this simple and economic method will attract both researchers and engineers in optical and ultrasound fields.

The planar Fabry Perot (FP) photoacoustic scanner provides exquisite high resolution 3D images of soft tissue structures for sub-cm penetration depths. However, as the FP sensor is optically addressed by sequentially scanning an interrogation laser beam over its surface, the acquisition speed is low. To address this, a novel scanner architecture employing 8 interrogation beams and an optimised sub-sampling framework have been developed that increase the data acquisition speed significantly. With a 200Hz repetition rate excitation laser, full 3D images can be obtained within 10 seconds. Further increases in imaging speed with only minor decreases in image quality can be obtained by applying sub-sampling techniques with rates as low as 12.5%. This paper shows 3D images reconstructed from sub-sampled data for an ex vivo dataset, and results from a dynamic phantom imaging experiment.

A novel all-optical non-contact photoacoustic microscopy system is introduced. The confocal configuration is used to ensure detection of initial pressure shock wave-induced intensity reflections at the subsurface origin where pressures are largest. Phantom studies confirm signal dependence on optical absorption, index-contrast, and excitation fluence. Taking advantage of a focused1310 nm interrogation beam, the penetration depth of the system is improved to ~ 2mm for an optical resolution system. High signal-to-noise ratios (>60dB) with ~ 2.5 cm working distance from the objective lens to the sample is achieved. Real-time in-vivo imaging of microvasculature and melanoma tumors are demonstrated.

Laser-generated focused ultrasound has shown great promise in precisely treating cells and tissues by producing controlled micro-cavitation within the acoustic focal volume (<100 um). However, the previous demonstration used cells and tissues cultured on glass substrates. The glass substrates were found to be critical to cavitation, because ultrasound amplitude doubles due to the reflection from the substrate, thus allowing for reaching pressure amplitude to cavitation threshold. In other words, without the sound reflecting substrate, pressure amplitude may not be strong enough to create cavitation, thus limiting its application to only cultured biomaterials on the rigid substrates.

By using laser-generated focused ultrasound without relying on sound-reflecting substrates, we demonstrate free-field cavitation in water and its application to high-precision cutting of tissue-mimicking gels. In the absence of a rigid boundary, strong pressure for cavitation was enabled by recently optimized photoacoustic lens with increased focal gain (>30 MPa, negative pressure amplitude). By moving cavitation spots along pre-defined paths through a motorized stage, tissue-mimicking gels of different elastic moduli were cut into different shapes (rectangle, triangle, and circle), leaving behind the same shape of holes, whose sizes are less than 1 mm. The cut line width is estimated to be less than 50 um (corresponding to localized cavitation region), allowing for accurate cutting. This novel approach could open new possibility for in-vivo treatment of diseased tissues in a high-precision manner (i.e., high-precision invisible sonic scalpel).

We recently discovered that strong reflectivity modulations occur when a pulsed laser excites an absorption interface with an existing refractive index contrast. These modulations are observed using a low-coherence interrogation beam co-focused and co-scanned with an excitation beam to form high-resolution all-optical photoacoustic images. We call this new form of microscopy Photoacoustic Remote Sensing (PARS). To better understand the mechanism, analytical models were created of the time-evolution of these PARS signals. Shock waves propagating from the absorption interface create refractive index steps that form a time-varying multi-layer etalon. Besides an initial-pressure reflectivity change, GHz-modulations are predicted due to the propagating etalon effect. The characteristics of these modulations are related to the optical coherence length of the probe beam and the intrinsic optical properties of the sample. 1D plane-wave and 3D Mie-theory-based analytical models are compared with finite-difference time-domain simulations and experiments involving phantoms with different absorption- and refractive-index interfaces. Experimentally-observed modulations are detected with extremely high signal-to-noise ratios in phantoms and animal models. The newly predicted modulation mechanism offers a promising signature for deep all-optical absorption-contrast imaging with high fidelity.

We present an optoacoustic (photoacoustic) microscopy (OAM) imaging system that uses a pi-shifted Fiber Bragg Grating (pi-FBG) as ultrasound (US) sensor. The sensor has an ultra-small footprint and hence allows for the detection of optoacoustic signals in close proximity to their origin. The interrogation of the pi-FBG is performed by a broadband pulsed laser, enabling a high sensitivity of the sensor as well as the elimination of ambient noise. We characterize the pi-FBG in terms of axial and lateral resolution as well as its bandwidth and find that its performance is comparable to US sensors that are based on the piezoelectric effect. We demonstrate the system’s capabilities by images taken from ex vivo zebrafish and mouse ear samples. The results presented herein highlight that pi-FBGs are a promising tool for the comprehensive label-free optoacoustic imaging of biomedical samples.

Optical-resolution photoacoustic microscopy (OR-PAM), has been widely used and studied as noninvasive and in-vivo imaging technique, can achieve a high resolution and high contrast image. OR-PAM is combined with optical absorption contrast and detection of acoustic wave generated by thermal expansion. Recently, nanoparticles and dyes have been used as contrast agents of OR-PAM. To obtain functional OR-PAM image such as a distribution image of blood vessels and nanoparticles, a tunable dye laser or optical parametric oscillator (OPO) should be needed at more two wavelength. However, because these lasers have a low pulse repetition rate (10 Hz ~ 10 kHz), a functional OR-PAM image with real-time display has been limited. In our previous study, we demonstrated high-speed OR-PAM using an Ytterbium fiber laser and a graphics processing unit (GPU) technique at 300 kHz-pulse repetition rates. Although this Ytterbium fiber laser has a high pulse repetition rate, it is not comfortable for functional imaging owing to lasing at only single wavelength. Therefore, in this study, we used a high-speed interlaced illumination method at 532 nm and 1064 nm for real-time display functional OR-PAM. For high-speed interlaced illumination of two wavelength, we applied second harmonic generation effect and a high-speed optical switching using an electro-optic modulator. Therefore, we could obtain maximum amplitude projection (MAP) images about distributions of blood vessels and nanoparticles, simultaneously, with 500 x 500 pixels and a real-time display of approximately 0.5 fps.

Optical microscopy (OM) and photoacoustic microscopy (PAM) have previously been used to image the optical absorption of intercellular features of biological cells. However, the optical diffraction limit (~200 nm) makes it difficult for these modalities to image nanoscale inner cell structures and the distribution of internal cell components. Although super-resolution fluorescence microscopy, such as stimulated emission depletion microscopy (STED) and stochastic optical reconstruction microscopy (STORM), has successfully performed nanoscale biological imaging, these modalities require the use of exogenous fluorescence agents, which are unfavorable for biological samples. Our newly developed atomic force photoactivated microscopy (AFPM) can provide optical absorption images with nanoscale lateral resolution without any exogenous contrast agents. AFPM combines conventional atomic force microscopy (AFM) and an optical excitation system, and simultaneously provides multiple contrasts, such as the topography and magnitude of optical absorption. AFPM can detect the intrinsic optical absorption of samples with ~8 nm lateral resolution, easily overcoming the diffraction limit. Using the label-free AFPM system, we have successfully imaged the optical absorption properties of a single melanoma cell (B16F10) and a rosette leaf epidermal cell of Arabidopsis (ecotype Columbia (Col-0)) with nanoscale lateral resolution. The remarkable images show the melanosome distribution of a melanoma cell and the biological structures of a plant cell. AFPM provides superior imaging of optical absorption with a nanoscale lateral resolution, and it promises to become widely used in biological and chemical research.

In this paper a multimodal optical-resolution photoacoustic and fluorescence microscope in frequency domain is presented. Photoacoustic waves and modulated fluorescence are generated in chromophores by using a modulated diode laser. The photoacoustic waves, recorded with a hydrophone, and the fluorescence signals, acquired with an avalanche photodiode, are simultaneously measured using a lock-in technique. Two possibilities to optimize the signal-to-noise ratio are discussed. The first method is based on the optimization of the excitation waveform and it is argued why square-wave excitation is best. The second way to enhance the SNR is to optimize the modulation frequency. For modulation periods that are much shorter than the relaxation times of the excited chromophores, the photoacoustic signal scales linearly with the modulation frequency. We come to the conclusion that frequency-domain photoacoustic microscopy performed with modulation frequencies in the range of 100 MHz can compete with time-domain photoacoustic microscopy regarding the signal-to-noise ratio. The theoretical predictions are confirmed by experimental results. Additionally, images of stained and unstained biological samples are presented in order to demonstrate the capabilities of the multimodal imaging system.

Acoustic-resolution photoacoustic microscopy (ARPAM) is a promising tool for deep imaging of biological tissues. Synthetic aperture focusing technique (SAFT) can improve the degraded lateral resolution in the out-of-focus region of ARPAM when using a high numerical aperture acoustic transducer. We previously reported a three-dimensional (3D) deconvolution technique to improve both lateral and axial resolutions in the focus region of ARPAM. In this work, we extended resolution enhancement of ARPAM to the out-of-focus region based on two dimensional SAFT combined with the 3D deconvolution (SAFT+Deconv). In both the focus and out-of-focus regions, depth-independent lateral and axial resolution after SAFT ensures a depth-independent point spread function for 3D deconvolution algorithm. In an extended depth of focus (DOF) of ∼2 mm, SAFT+Deconv ARPAM improves the −6 dB lateral resolutions from 65–700 μm to 20–29 μm, and the −6 dB axial resolutions from 35–42 μm to 12–19 μm. The signal-to-noise ratio is also increased by 6–30 dB. The enhanced resolution in extended DOF by SAFT+Deconv ARPAM may enable important applications in biomedical photoacoustic imaging.

Photoacoustic microscopy (PAM) has been extensively applied in biomedical study because of its ability to visualize tissue morphology and physiology in vivo in three dimensions (3D). However, conventional PAM suffers from a rapidly decreasing resolution away from the focal plane because of the limited depth of focus of an objective lens, which deteriorates the volumetric imaging quality inevitably. Here, we propose a novel method to synthesize an ultra-long light needle to extend a microscope’s depth of focus beyond its physical limitations with wavefront engineering method. Furthermore, it enables an improved lateral resolution that exceeds the diffraction limit of the objective lens. The virtual light needle can be flexibly synthesized anywhere throughout the imaging volume without mechanical scanning. Benefiting from these advantages, we developed a synthetic light needle photoacoustic microscopy (SLN-PAM) to achieve an extended depth of field (DOF), sub-diffraction and motionless volumetric imaging. The DOF of our SLN-PAM system is up to 1800 µm, more than 30-fold improvement over that gained by conventional PAM. Our system also achieves the lateral resolution of 1.8 µm (characterized at 532 nm and 0.1 NA objective), about 50% higher than the Rayleigh diffraction limit. Its superior imaging performance was demonstrated by 3D imaging of both non-biological and biological samples. This extended DOF, sub-diffraction and motionless 3D PAM will open up new opportunities for potential biomedical applications.

Photoacoustic physio-chemical analysis (PAPCA) is a recently developed technology capable of simultaneously quantifying the content of molecular components and the corresponding microarchitectures in biological tissue. We have successfully quantified the diagnostic information in livers with PAPCA. In this study, we implemented PAPCA to the diagnosis of prostate cancers. 4 human prostates were scanned ex vivo. The PA signals from normal and cancerous regions in the prostates were acquired by an interstitial needle PA probe. A total of 14 interstitial measurements, including 6 within the normal regions and 8 in the cancerous regions, were acquired. The observed changes in molecular components, including lipid, collagen and hemoglobin were consistent with the findings by other research groups. The changes were quantified by PA spectral analysis (PASA) at wavelengths where strong optical absorption of the relevant molecular components was found. Statistically significant differences among the PASA parameters were observed (p=0.025 at significance of 0.05). A support vector machine model for differentiating the normal and cancerous tissue was established. With the limited number of samples, an 85% diagnostic accuracy was found. The diagnostic information in the PCPCA can be further enriched by targeted optical contrast agents visualizing the microarchitecture in PCa tissues. F3 PAA-PEG nanoparticles was employed to stain the PCa cells in a transgenic mouse model, in which the microarchitectures of normal and cancerous prostate tissues are comparable to that in human. Statistically significant differences were observed between the contrast-enhanced normal and cancerous regions (p=0.038 at a significance of 0.05).

Molecular photoacoustic imaging is hindered by hemoglobin background signal. Photoswitchable chromoproteins can be used to obtain images with significantly reduced background signal. Molecular imaging of multiple biological processes via multiple chromoprotiens is difficult due to overlapping imaging spectra. Using a new rate-of-change imaging methodology, we can obtain molecular images with multiple chromoprotiens with overlapping imaging spectra. We also present a new photoswitchable chromoprotein, GAF2, which is significantly smaller than the BphP1 which has shown promise for photoswitchable photoacoustic imaging [Yao et al., Nat. Meth. 13, 67–73 (2016)]. We use BphP1 and GAF2 with photoacoustic (Vevo LAZR, Fujifilm Visualsonics Inc) and fluorescence (In vivo Xtreme, Bruker) imaging systems to show background-free multiplexed images. We image before, after, and during photoconversion to obtain background-free rate-of-change images and compare our results to difference imaging and spectral demixing. After phantom imaging, we inject mice with different chromoprotein-expressing E. coli bacteria to show multiplexed images of bacterial infections. We show distinguishable differences in the rate-of-change between GAF2 and BphP1. We obtain rate-of-change feasibility images and in vivo images in mice showing the ability to differentiate between GAF2 and BphP1 even though they are spectrally similar. We photoconvert both GAF2 and BphP1 using 550nm and 735nm light. Phantom studies suggest a 10-20dB improvement in the rate-of-change and difference images in comparison to images with background. Multiplexed background-free molecular imaging using chromoproteins could prove to be a promising new imaging methodology especially when combined with spectral demixing.

The pathology of Crohn’s disease (CD) is characterized by obstructing intestinal strictures because of inflammation (with high levels of hemoglobin), fibrosis (high levels of collagen), or a combination of both. Inflammatory strictures are medically treated. Fibrotic strictures have to be removed surgically. The accurate characterization of the strictures is therefore critical for the management of CD. Currently the comprehensive assessment of a stricture is difficult, as the standard diagnostic procedure, endoscopic biopsy, is superficial and with limited locations as well as depth. In our previous studies, photoacoustic imaging (PAI) has recovered the layered architectures and the relative content of the molecular components in human and animal tissues ex vivo. This study will investigate the capability of multispectral PAI in resolving the architecture and the molecular components of intestinal strictures in rats in vivo. PA images at 532, 1210 and 1310 nm targeting the strong optical absorption of hemoglobin, lipid and collagen were acquired using two approaches. A compact linear array, CL15-7, was used to transcutaneously acquire PA signals generated by the a fiber optics diffuser positioned within the inner lumen of the strictures. Another approach was to use an endoscopic capsule probe for acoustic resolution PA microscopy. The capsule probe is designed for human and therefore cannot fit into rat colon. The inner surface of the intestinal stricture was exposed and the probe was attached to the diseased location for imaging. The findings in PA images were confirmed by histology results.

Photoacoustic imaging is an emerging technology capable of both functional and structural biological imaging. Absorption and scattering in tissue limit the penetration depth of conventional microscopy techniques to <1mm. Photoacoustic imaging however, can offer high-resolution and contrast at depths of several centimeters. Though functional imaging of endogenous contrast agents, such as hemoglobin, is widely implemented, currently photoacoustic imaging is unable to functionally report electrophysiological changes within cells. We aim to develop photoacoustic contrast agents to fulfill this need. Cells throughout the brain and body create electrical signals using ion channel proteins. These proteins undergo structural changes to regulate the flux of salt ions into the cell. We have recently developed ion channel activity tracers that dissociate from ion channels after the protein changes structure. By conjugating the tracer to dyes that are sensitive to changes in their chemical environment, we can detect tracer dissociation and therefore ion channel activity. We are exploring whether a similar mechanism can create photoacoustic signal intensity changes. To test if the environmental sensitivity of the dye is photoacoustically distinguishable, we imaged the dye in different solvent backgrounds. We report that manipulation of the chemical environment of the contrast dye results in robust changes in photoacoustic properties. We are working to capture photoacoustic signal changes that occur when ion channel proteins activate using live cell imaging. This technology could permit photoacoustic imaging of electrophysiological dynamics in deep tissue, such as the brain. Further optimization of this technology could lead to concurrent imaging of neural activity and hemodynamic responses, a crucial step towards understanding neurovascular coupling in the brain.

Ultrasound is broadly used in the clinics yet is limited in early cancer detection because of its poor contrast between healthy and diseased tissues. Photoacoustic imaging can improve this limitation and has been extensively studied in pre-clinical models. Contrast agents can help improve the accuracy of diagnosis. We recently reported a novel copper sulfide (CuS) nanodisk with strong directionally-localized surface plasmon resonance in the near infrared region. This plasmonic resonance of nanodisks is tunable by changing the size and aspect ratio of CuS nanodisk. Here, we demonstrate this CuS nanodisk is a strong photoacoustic contrast agent. We prepared CuS nanodisks via a solvent-based synthesis followed by surface modification of poly(ethylene glycol) methyl ether thiol for in vivo applications. These CuS nanodisks can be detected at a concentration as low as 26 pM at 920 nm. Their nanosize and strong photoacoustic response make this novel CuS nanodisk a strong candidate for photoacoustic cancer imaging.

For emerging tissue-engineering applications, transplants, and cell-based therapies it is important to assess cell viability and function in vivo in deep tissues. Bioluminescence and fluorescence methods are poorly suited to deep monitoring applications with high resolution and require genetically-engineered reporters which are not always feasible. We report on a method for imaging cell viability using deep, high-resolution photoacoustic imaging. We use an exogenous dye, Resazurin, itself weakly fluorescent until it is reduced from blue to a pink color with bright red fluorescence. Upon cell death fluorescence is lost and an absorption shift is observed. The irreversible reaction of resazurin to resorufin is proportional to aerobic respiration. We detect colorimetric absorption shifts using multispectral photoacoustic imaging and quantify the fraction of viable cells. SKOV-3 cells with and without ±80oC heat treatment were imaged after Resazurin treatment. High 575nm:620nm ratiometric absorption and photoacoustic signals in viable cells were observed with a much lower ratio in low-viability populations.

A new light and sound sensitive nanoemulsion contrast agent is presented. The agents feature a low boiling point liquid perfluorocarbon core and a broad light spectrum absorbing polypyrrole (PPy) polymer shell. The PPy coated nanoemulsions can reversibly convert from liquid to gas phase upon cavitation of the liquid perfluorocarbon core. Cavitation can be initiated using a sufficiently high intensity acoustic pulse or from heat generation due to light absorption from a laser pulse. The emulsions can be made between 150 and 350 nm in diameter and PPy has a broad optical absorption covering both the visible spectrum and extending into the near-infrared spectrum (peak absorption ~1053 nm). The size, structure, and optical absorption properties of the PPy coated nanoemulsions were characterized and compared to PPy nanoparticles (no liquid core) using dynamic light scattering, ultraviolet-visible spectrophotometry, transmission electron microscopy, and small angle X-ray scattering. The cavitation threshold and signal intensity were measured as a function of both acoustic pressure and laser fluence. Overlapping simultaneous transmission of an acoustic and laser pulse can significantly reduce the activation energy of the contrast agents to levels lower than optical or acoustic activation alone. We also demonstrate that simultaneous light and sound cavitation of the agents can be used in a new sono-photoacoustic imaging method, which enables greater sensitivity than traditional photoacoustic imaging.

We present an imaging method that uses the random optical speckle patterns that naturally emerge as light propagates through strongly scattering media as a structured illumination source for photoacoustic imaging. Our approach, termed blind structured illumination photoacoustic microscopy (BSIPAM), was inspired by recent work in fluorescence microscopy where super-resolution imaging was demonstrated using multiple unknown speckle illumination patterns. We extend this concept to the multiple scattering domain using photoacoustics (PA), with the speckle pattern serving to generate ultrasound. The optical speckle pattern that emerges as light propagates through diffuse media provides structured illumination to an object placed behind a scattering wall. The photoacoustic signal produced by such illumination is detected using a focused ultrasound transducer. We demonstrate through both simulation and experiment, that by acquiring multiple photoacoustic images, each produced by a different random and unknown speckle pattern, an image of an absorbing object can be reconstructed with a spatial resolution far exceeding that of the ultrasound transducer. We experimentally and numerically demonstrate a gain in resolution of more than a factor of two by using multiple speckle illuminations. The variations in the photoacoustic signals generated with random speckle patterns are utilized in BSIPAM using a novel reconstruction algorithm. Exploiting joint sparsity, this algorithm is capable of reconstructing the absorbing structure from measured PA signals with a resolution close to the speckle size.

Fast data acquisition is a central aspect of photoacoustic imaging. Increasing the imaging speed is especially crucial for optical detection schemes where an optical interrogation beam is scanned along a planar detection surface and the ultrasonic waves are recorded at each position sequentially. In this work, we demonstrate that the number of measurements in photoacoustic imaging can significantly be reduced by using techniques of compressed sensing. A main requirement in compressed sensing is the sparsity of the unknowns to be recovered. Sparsity of the pressure wave as a function of space and time is not valid directly. Therefore, we introduce the concept of sparsifying temporal transforms for three-dimensional photoacoustic imaging. We present reconstruction results for simulated data verifying that the proposed compressed sensing scheme allows a significant reduction of the number of spatial measurements without sacrificing the spatial resolution.

Bioluminescence imaging (BLI) is a commonly used imaging modality in biology to study cancer in vivo in small animals. Images are generated using a camera to map the optical fluence emerging from the studied animal, then a numerical reconstruction algorithm is used to locate the sources and estimate their sizes. However, due to the strong light scattering properties of biological tissues, the resolution is very limited (around a few millimetres). Therefore obtaining accurate information about the pathology is complicated. We propose a combined ultrasound/optics approach to improve accuracy of these techniques. In addition to the BLI data, an ultrasound probe driven by a scanner is used for two main objectives. First, to obtain a pure acoustic image, which provides structural information of the sample. And second, to alter the light emission by the bioluminescent sources embedded inside the sample, which is monitored using a high speed optical detector (e.g. photomultiplier tube). We will show that this last measurement, used in conjunction with the ultrasound data, can provide accurate localisation of the bioluminescent sources. This can be used as a priori information by the numerical reconstruction algorithm, greatly increasing the accuracy of the BLI image reconstruction as compared to the image generated using only BLI data.

Imaging dynamics in living organisms is essential for the understanding of biological complexity. While multiple imaging modalities are often required to cover both microscopic and macroscopic spatial scales, dynamic phenomena may also extend over different temporal scales, necessitating the use of different imaging technologies based on the trade-off between temporal resolution and effective field of view. Optoacoustic (photoacoustic) imaging has been shown to offer the exclusive capability to link multiple spatial scales ranging from organelles to entire organs of small animals. Yet, efficient visualization of multi-scale dynamics remained difficult with state-of-the-art systems due to inefficient trade-offs between image acquisition and effective field of view. Herein, we introduce a spiral volumetric optoacoustic tomography (SVOT) technique that provides spectrally-enriched high-resolution optical absorption contrast across multiple spatio-temporal scales. We demonstrate that SVOT can be used to monitor various in vivo dynamics, from video-rate volumetric visualization of cardiac-associated motion in whole organs to high-resolution imaging of pharmacokinetics in larger regions. The multi-scale dynamic imaging capability thus emerges as a powerful and unique feature of the optoacoustic technology that adds to the multiple advantages of this technology for structural, functional and molecular imaging.

Scanning optoacoustic microscopy operates in two distinct regimes optical resolution microscopy relies on a focused illumination and acoustic resolution microscopy that forms images by focusing the received acoustic field. Recently, a number of approaches have been proposed that combine those two modes of operation to create a highly scalable technique that can image at multiple penetration scales by gradually exchanging microscopic optical resolution in superficial tissues with ultrasonic resolution at diffuse (macroscopic) depths. However, scanning microscopy schemes commonly employ acquisition geometries that impede the use of synthetic aperture techniques to achieve meaningful images due to non-stationary illumination patterns and strong non-uniformity of the excitation light field.

Here we present a Weighted Synthetic Aperture Focusing Technique (W-SAFT) as a universal framework that effectively accounts for the non-uniform distribution of both the excitation light field and spatial sensitivity field of the detector. As a result, W-SAFT maintains optical resolution performance at superficial depths while improving the acoustic resolving capacity for deeper tissues. The dynamic range of the optoacoustic data is compressed using a general fluence decay term applied to the W-SAFT operator, allowing a more uniform visualization of the entire imaged volume. Our three-dimensional algorithm makes use of the sample's surface to account for the heterogeneity produced when scanning a finite-size light beam. We tested a GPU implementation of W-SAFT with numerical simulations and showcase its performance on experimental data acquired from targets embedded in tissue mimicking phantoms.

This work demonstrates the first measurements of blood flow velocity using photoacoustic flowmetry (PAF) employing a transducer array. The measurements were made in a flow phantom consisting of a tube (580 μm inner diameter) containing blood flowing steadily at physiological speeds ranging from 3 mm/s to 25 mm/s. Velocity measurements were based on the generation of two successive photoacoustic (PA) signals using two laser pulses with a wavelength of 1064 nm; the PA signals were detected using a 64-element transducer array with a -6 dB detection bandwidth of 11-17 MHz. We developed a processing pipeline to optimise a cross-correlation based velocity measurement method comprising the following processing steps: image reconstruction, filtering, displacement detection, and masking. We found no difference in flow detection accuracy when choosing different image reconstruction algorithms (time reversal, Fourier transformation, and delay-and-sum). High-pass filtering and wallfiltering were however found to be essential pre-processing steps in order to recover the correct displacement information. We masked the calculated velocity map based on the amplitude of the cross-correlation function in order to define the region of interest corresponding to highest signal amplitude. These developments enabled blood flow measurements using a transducer array, bringing PAF one step closer to clinical applicability.

Sparse recovery algorithms have shown great potential to reconstruct images with limited view datasets in optoacoustic tomography, with a disadvantage of being computational expensive. In this paper, we improve the fast convergent Split Augmented Lagrangian Shrinkage Algorithm (SALSA) method based on least square QR (LSQR) formulation for performing accelerated reconstructions. Further, coherence factor is calculated to weight the final reconstruction result, which can further reduce artifacts arising in limited-view scenarios and acoustically heterogeneous mediums. Several phantom and biological experiments indicate that the accelerated SALSA method with coherence factor (ASALSA-CF) can provide improved reconstructions and much faster convergence compared to existing sparse recovery methods.

Slit-enabled photoacoustic tomography (PAT) is a newly developed technique that improves the elevation resolution and signal to noise ratio of a linear array. The slit, placed along the transducer array focus, forms an array of virtual detectors with high receiving angle, which subsequently allows for three dimensional (3D) imaging with near-isotropic spatial resolution. Our development addressed the long-standing issue of high quality 3D imaging with a linear array and will have broad applications in preclinical and clinical imaging. This study presented the principle of slit-PAT and demonstrated its efficiency in phantom, animal, and human experiments.

For diagnosis of cervical cancer, screening by colposcope and successive biopsy are usually carried out. Colposcope, which is a mesoscope, is used to examine surface of the cervix and to find precancerous lesion grossly. However, the accuracy of colposcopy depends on the skills of the examiner and is inconsistent as a result. Additionally, colposcope lacks depth information. It is known that microvessel density and blood flow in cervical lesion increases associated with angiogenesis. Therefore, photoacoustic imaging (PAI) to detect angiogenesis in cervical lesion has been studied. PAI can diagnose cervical lesion sensitively and provide depth information. The authors have been investigating the efficacy of PAI in the diagnoses of the cervical lesion and cancer by use of the PAI and ultrasonography system with transvaginal probe developed by Fujifilm Corporation. For quantitative diagnosis by use of PAI, it is required to take the light propagation in biological medium into account. The image reconstruction of the absorption coefficient from the PA image of cervix by use of the simulation of light propagation based on finite element method has been tried in this study. Numerical simulation, phantom experiment and in vivo imaging were carried out.

A transrectral ultrasonography (TRUS) guided prostate biopsy is mandatory for histological diagnosis in patients with an elevated serum prostate-specific antigen (PSA), but its diagnostic accuracy is not satisfactory; therefore, a considerable number of patients are forced to have an unnecessary repeated biopsy.

Photoacoustic (PA) imaging has the ability to visualize the distribution of hemoglobin clearly. Thus, there is the potential to acquire different maps of small vessel networks between cancerous and normal tissue. We developed an original TRUS-type PA probe consisting of a microconvex array transducer with an optical illumination system providing coregistered PA and ultrasound images. The purpose of this study is to demonstrate the clinical possibility of a transrectral PA image.

The prostate biopsy cores obtained by transrectal systemic biopsies under TRUS guidance were stained with HE staining and anti-CD34 antibodies as a marker of the endothelium of the blood vessel in order to find a pattern in the map of a small vessel network, which allows for imaging-based identification of prostate cancer. We analyzed the association of PA signal patterns, the cancer location by a magnetic resonance imaging (MRI) study, and the pathological diagnosis with CD34 stains as a prospective intervention study.

In order to demonstrate the TRUS-merged-with-PA imaging guided targeted biopsy combined with a standard biopsy for capturing the clinically significant tumors, we developed a puncture needle guide attachment for the original TRUS-type PA probe.

Minimally invasive surgery carries the deadly risk of rupturing major blood vessels, such as the internal carotid arteries hidden by bone in endonasal transsphenoidal surgery. We propose a novel approach to surgical guidance that relies on photoacoustic-based vessel separation measurements to assess the extent of safety zones during these type of surgical procedures. This approach can be implemented with or without a robot or navigation system. To determine the accuracy of this approach, a custom phantom was designed and manufactured for modular placement of two 3.18-mm diameter vessel-mimicking targets separated by 10-20 mm. Photoacoustic images were acquired as the optical fiber was swept across the vessels in the absence and presence of teleoperation with a research da Vinci Surgical System. When the da Vinci was used, vessel positions were recorded based on the fiber position (calculated from the robot kinematics) that corresponded to an observed photoacoustic signal. In all cases, compounded photoacoustic data from a single sweep displayed the four vessel boundaries in one image. Amplitude- and coherence-based photoacoustic images were used to estimate vessel separations, resulting in 0.52-0.56 mm mean absolute errors, 0.66-0.71 mm root mean square errors, and 65-68% more accuracy compared to fiber position measurements obtained through the da Vinci robot kinematics. Results indicate that with further development, photoacoustic image-based measurements of anatomical landmarks could be a viable method for real-time path planning in multiple interventional photoacoustic applications.

This study demonstrated the performance of photoacoustic imaging at 1064 nm using phosphorus phthalocyanine (P-Pc), a contrast agent with strong absorption at 1064 nm. Due to high maximum permissible exposure of 1064 nm laser light and strong absorbance of P-Pc at 1064 nm, we demonstrated an imaging depth of 11.6 cm in chicken breast tissue. For animal imaging, we used P-Pc to target tumor and to track intestine dynamics. Thus, using a contrast medium with extreme absorption at 1064 nm readily enables high quality photoacoustic imaging at exceptional depths.

Transfontanelle ultrasound imaging (TFUSI) is a routine diagnostic brain imaging method in infants who are born prematurely, whose skull bones have not completely fused together and have openings between them, so-called fontanelles. Open fontanelles in neonates provide acoustic windows, allowing the ultrasound beam to freely pass through. TFUSI is used to rule out neurological complications of premature birth including subarachnoid hemorrhage (SAH), intraventricular (IVH), subependimal (SEPH), subdural (SDH) or intracerebral (ICH) hemorrhages, as well as hypoxic brain injuries. TFUSI is widely used in the clinic owing to its low cost, safety, accessibility, and noninvasive nature. Nevertheless, the accuracy of TFUSI is limited. To address several limitations of current clinical imaging modalities, we develop a novel transfontanelle photoacoustic imaging (TFPAI) probe, which, for the first time, should allow for non-invasive structural and functional imaging of the infant brain. In this study, we test the feasibility of TFPAI for detection of experimentally-induced intra ventricular and Intraparenchymal hemorrhage phantoms in a sheep model with a surgically-induced cranial window which will serve as a model of neonatal fontanelle. This study is towards using the probe we develop for bedside monitoring of neonates with various disease conditions and complications affecting brain perfusion and oxygenation, including apnea, asphyxia, as well as for detection of various types of intracranial hemorrhages (SAH, IVH, SEPH, SDH, ICH).

As Photoacoustic Tomography (PAT) matures and undergoes clinical translation, objective performance test methods are needed to facilitate device development, regulatory clearance and clinical quality assurance. For mature medical imaging modalities such as CT, MRI, and ultrasound, tissue-mimicking phantoms are frequently incorporated into consensus standards for performance testing. A well-validated set of phantom-based test methods is needed for evaluating performance characteristics of PAT systems. To this end, we have constructed phantoms using a custom tissue-mimicking material based on PVC plastisol with tunable, biologically-relevant optical and acoustic properties. Each phantom is designed to enable quantitative assessment of one or more image quality characteristics including 3D spatial resolution, spatial measurement accuracy, ultrasound/PAT co-registration, uniformity, penetration depth, geometric distortion, sensitivity, and linearity. Phantoms contained targets including high-intensity point source targets and dye-filled tubes. This suite of phantoms was used to measure the dependence of performance of a custom PAT system (equipped with four interchangeable linear array transducers of varying design) on design parameters (e.g., center frequency, bandwidth, element geometry). Phantoms also allowed comparison of image artifacts, including surface-generated clutter and bandlimited sensing artifacts. Results showed that transducer design parameters create strong variations in performance including a trade-off between resolution and penetration depth, which could be quantified with our method. This study demonstrates the utility of phantom-based image quality testing in device performance assessment, which may guide development of consensus standards for PAT systems.

A PEGylated quinoline-annulated porphyrin derivative was synthesized as in vivo photoacoustic tomography contrast agent. It possesses high solubility and stability in water and phosphate-buffered saline. No toxicity sign was observed in BALB/c mice. The dye demonstrates a 4-fold higher photoacoustic signal generation efficiency compared to fresh rat blood. Injection of the dye results in a significant enhancement of in vivo PAT images of murine tumors. Analysis of the mouse urine after injection revealed an unaltered renal filtration of the contrast agent.

We synthesized nitrogen-doped carbon nanodots (N-CNDs) for photoacoustic (PA) imaging and photothermal therapy (PTT) by controlling the nitrogen source and carbonizing organic acids. The N-CNDs showed strong optical absorbance in the near-infrared region, with great photostability and biodegradability. Thanks to the strong optical absorbance of NCNDs, the PA signals from N-CNDs were high enough to detect inside living animals and enabled minimally invasive PTT using N-CND. To evaluate the biodegradability and potential application of N-CNDs as a PA imaging contrast agent, we performed time-resolved PA imaging of sentinel lymph nodes (SLNs) and assessed renal clearance after hypodermic injection. SLN and vascular networks were photoacoustically visualized by an acoustic-resolution reflection-mode PA imaging system at a 680-nm optical wavelength. Furthermore, we performed whole-body PA imaging after subcutaneous injection of N-CNDs to assess their body distribution and clearance. Finally, we further investigated the use of N-CNDs for in vivo photothermal therapy in Balb/c nude xenograft HepG2-tumor model mice.

Photoacoustic (PA) imaging is advantageous in contrast agent imaging because of high spatial resolution at depth more than several millimeter inside biological tissues. To detect small tumors specifically, we are developing small organic molecule-based activatable PA probe with mechanism similar to that of the enzyme-activatable fluorescence probe that have successfully used for rapid fluorescence imaging of small tumors. The probe can be imaged also by fluorescence imaging and the fluorescence image can be merged onto the PA images. To extend the imaging depth by increasing PA signal intensity, PA probe that produce PA signals efficiently is required. To select small organic molecules suitable for PA probe, we synthesized small-organic molecule-based contrast agents with various absorption spectra and fluorescence quantum yields and then we exhaustively evaluated their PA signal generation characteristics including PA signal generation efficiencies. To analyze PA signal generation efficiencies precisely, the absolute values of PA signal pressures produced from aqueous solutions of the contrast agents were measured by P(VDF-TrFE) piezoelectric film acoustic sensor. As a result, small organic molecule with low fluorescence quantum yield produced PA signals efficiently. Thus, as opposed to fluorescence probes, PA probes should have low fluorescence quantum yields. By considering the result and other characteristics including excitation wavelengths, we could single out the small organic molecule suitable for PA probe. We synthesized the new activatable PA probe with low fluorescence quantum yield and excitation wavelength longer than 600 nm and its specificity was examined in in vitro experiment.

Photoacoustic Tomography combines the advantages of optical and acoustic imaging as it makes use of the high optical contrast of tissue and the high resolution of ultrasound. Furthermore, high penetration depths in tissue in the order of several centimeters can be achieved by the combination of these modalities. Extensive research is being done in the field of miniaturization of photoacoustic devices, as photoacoustic imaging could be of significant benefits for the physician during endoscopic interventions. All the existing miniature systems are based on contact transducers for signal detection that are placed at the distal end of an endoscopic device. This makes the manufacturing process difficult and impedance matching to the inspected surface a requirement. The requirement for contact limits the view of the physician during the intervention. Consequently, a fiber based non-contact optical sensing technique would be highly beneficial for the development of miniaturized photoacoustic endoscopic devices. This work demonstrates the feasibility of surface displacement detection using remote speckle-sensing using a high speed camera and an imaging fiber bundle that is used in commercially available video endoscopes. The feasibility of displacement sensing is demonstrated by analysis of phantom vibrations which are induced by loudspeaker membrane oscillations. Since the usability of the remote speckle-sensing for photo-acoustic signal detection was already demonstrated, the fiber bundle approach demonstrates the potential for non-contact photoacoustic detections during endoscopy.

Resting-state functional connectivity (RSFC) is a method to monitor the health of the brain and find out abnormalities in brain networks. Recently functional connectivity photoacoustic tomography (fcPAT) has been used to study RSFC in the mouse brain. The current method of RSFC data analysis is called “seed-based”. This method is not data-driven, and involves user intervention. Alternative signal processing approaches, such as singular value decomposition (SVD) and independent component analysis (ICA), will be explored to complement and cross validate the seed-based approach, possibly substituting them for the seed-based method. The methods are implemented and applied on the fcPAT data of a mouse brain.

The study of functional connectivity among different regions of the brain is tremendously valuable in the diagnosis and monitoring of various neurological disorders. While the conventional techniques fail to provide optimum results in terms of resolution, field of view and efficiency, Photo-acoustic based imaging is being considered as an alternative and complimentary modality. The cost and size are the factors that limit the further research and clinical advancements of fcPAT. Therefore we proposed an inexpensive fcPAT system. We determined its feasibility by imaging a graphite leads embedded in an agar phantom and demonstrated its application by imaging a microtubes filled with blood embedded in the chicken’s breast at a scan time of 50 seconds and number of angles as 300.

Seed-based correlation analysis is one of the most popular methods to explore the functional connectivity in the brain. Based on the time series of a seed, i.e., small regions of interest, connectivity is computed as the correlation of time series for all other pixels in the brain. Similarity metric to measure the similarity between time courses of different seeds plays an important role in the detection of functional connectivity maps. In this study, we investigate the performance of six similarity metrics including Pearson correlation, Kendall, Spearman, Goodman-Kruskal Gamma, normalized cross correlation and coherence analysis to determine their performance for the functional connectivity photoacoustic tomography (fcPAT) signals/images. The methods are implemented and applied on the fcPAT data of a mouse brain. We also add noise to the fcPAT data and explore the noise tolerance of these metrics.

Quantification of brain function is a significant milestone towards understanding of the underlying workings of the brain. Photoacoustic (PA) imaging is the emerging brain sensing modality by which the molecular light absorptive contrast can be non-invasively quantified from deep-lying tissue (~several cm). In this BRAIN initiative effort, we propose high-speed transcranial PA imaging using a novel, compact pulsed LED illumination system (Prexion Inc., Japan) with 200-uJ pulse energy for 75-ns duration, and pulse repetition frequency (PRF) up to 4kHz at near-infrared (NIR) wavelengths of 690-nm and 850-nm switchable in real-time. To validate the efficacy of the proposed system, preliminary ex vivo experiments were conducted with mice skull and human temporal bone, which included vessel-mimicking tubes filled with 10% Indian Ink solution and light absorptive rubber material, respectively. The results indicated that significant PA contrast, 150% signal-to-noise ratio (SNR), can be achieved through the mice skull only with 64 subsequent frame averaging. The minimal number of frames for averaging required was only 16 to generate signal above background noise, leading to 250 Hz frame rate in the strictest temporal frame separation. Furthermore, distinguishable PA contrast was achieved with human temporal bone with 64-frame averaging. Overall, the preliminary results indicate that the LED illumination system can be a cost-effective solution for high-speed PA brain imaging in preclinical and clinical applications, compared to expansive and bulky Nd:YAG laser systems commonly used in PA imaging.

Multispectral optoacoustic mesoscopy (MSOM) has been recently introduced for cancer imaging, it has the potential for high resolution imaging of cancer development in vivo, at depths beyond the diffusion limit. Based on spectral features, optoacoustic imaging is capable of visualizing angiogenesis and imaging cancer heterogeneity of malignant tumors through endogenous hemoglobin. However, high-resolution structural and functional imaging of whole tumor mass is limited by modest penetration and image quality, due to the insufficient capability of ultrasound detectors and the twodimensional scan geometry. In this study, we introduce a novel multi-spectral optoacoustic mesoscopy (MSOM) for imaging subcutaneous or orthotopic tumors implanted in lab mice, with the high-frequency ultrasound linear array and a conical scanning geometry. Detailed volumetric images of vasculature and oxygen saturation of tissue in the entire tumors are obtained in vivo, at depths up to 10 mm with the desirable spatial resolutions approaching 70μm. This unprecedented performance enables the visualization of vasculature morphology and hypoxia conditions has been verified with ex vivo studies. These findings demonstrate the potential of MSOM for preclinical oncological studies in deep solid tumors to facilitate the characterization of tumor’s angiogenesis and the evaluation of treatment strategies.

Although the Photo Acoustic effect was observed by Graham Bell in 1880, the first applications (gas analysis) occurred in 1970’s using the required energetic light pulses from lasers. During mid 1990’s medical imaging research begun to use Photo Acoustic effect and in vivo images were obtained in mid-2000. Since 2009, the number of patent related to Photo Acoustic Imaging (PAI) has dramatically increased. PAI machines for pre-clinical and small animal imaging have been being used in a routine way for several years. Based on its very interesting features (non-ionizing radiation, noninvasive, high depth resolution ratio, scalability, moderate price) and because it is able to deliver not only anatomical, but functional and molecular information, PAI is a very promising clinical imaging modality. It penetrates deeper into tissue than OCT (Optical Coherence Tomography) and provides a higher resolution than ultrasounds. The PAI is one of the most growing imaging modality and some innovative clinical systems are planned to be on market in 2017. Our study analyzes the different approaches such as photoacoustic computed tomography, 3D photoacoustic microscopy, multispectral photoacoustic tomography and endoscopy with the recent and tremendous technological progress over the past decade: advances in image reconstruction algorithms, laser technology, ultrasound detectors and miniaturization. We analyze which medical domains and applications are the most concerned and explain what should be the forthcoming medical system in the near future. We segment the market in four parts: Components and R&D, pre-clinical, analytics, clinical. We analyzed what should be, quantitatively and qualitatively, the PAI medical markets in each segment and its main trends. We point out the market accessibility (patents, regulations, clinical evaluations, clinical acceptance, funding). In conclusion, we explain the main market drivers and challenges to overcome and give a road map for medical approved PAI products.

Quantitative photoacoustic tomography is an emerging imaging technique aimed at estimating optical parameters inside tissue from photoacoustic images. This optical parameter estimation problem is an ill-posed inverse problem, and thus it is sensitive to measurement and modelling errors. Therefore, light propagation in quantitative photoacoustic tomography needs to be accurately modelled. A widely accepted model for light propagation in biological tissue is the radiative transfer equation. In this work, the radiative transfer equation is utilised in quantitative photoacoustic tomography. Estimating absorption and scattering distributions in quantitative photoacoustic tomography using various illuminations is investigated.

Here, we introduce a new image reconstruction algorithm that combines coherent weighting with focal-line-based three-dimensional image reconstruction. The new algorithm addresses the major limitation of a linear ultrasound transducer array, i.e., the poor elevation resolution, and does not require any modification to the imaging system or the scanning geometry. We first numerically validated our approach through simulation and then experimentally tested it in phantom and in vivo. Both simulation and experimental results proved that the method can significantly improve the elevation resolution (up to 3.4 times in our experiment) and enhance object contrast.

Linear-array-based photoacoustic computed tomography is a popular methodology for deep and high resolution imaging. However, issues such as phase aberration, side-lobe effects, and propagation limitations deteriorate the resolution. The effect of phase aberration due to acoustic attenuation and constant assumption of the speed of sound (SoS) can be reduced by applying an adaptive weighting method such as the coherence factor (CF). Utilizing an adaptive beamforming algorithm such as the minimum variance (MV) can improve the resolution at the focal point by eliminating the side-lobes. Moreover, invisibility of directional objects emitting parallel to the detection plane, such as vessels and other absorbing structures stretched in the direction perpendicular to the detection plane can degrade resolution. In this study, we propose a full-view array level weighting algorithm in which different weighs are assigned to different positions of the linear array based on an orientation algorithm which uses the histogram of oriented gradient (HOG). Simulation results obtained from a synthetic phantom show the superior performance of the proposed method over the existing reconstruction methods.

In this work, we explore different reconstruction algorithms for photoacoustic image reconstruction of spatially resolved projection data. While the commonly used back-projected reconstruction is efficient and fast to compute, it cannot deal with noise that arises during measurements. Therefore, we formulate photoacoustic image reconstruction in a variational framework where we add prior knowledge in terms of Total Generalized Variation. Using this prior knowledge, we can reduce measurement noise and improve the visibility of vessel structures.

Interventional applications of photoacoustic imaging often require visualization of point-like targets, including the circular cross sectional tips of needles and catheters or the circular cross sectional views of small cylindrical implants such as brachytherapy seeds. When these point-like targets are imaged in the presence of highly echogenic structures, the resulting photoacoustic wave creates a reflection artifact that may appear as a true signal. We propose to use machine learning principles to identify these type of noise artifacts for removal. A convolutional neural network was trained to identify the location of individual point targets from pre-beamformed data simulated with k-Wave to contain various medium sound speeds (1440-1640 m/s), target locations (5-25 mm), and absorber sizes (1-5 mm). Based on 2,412 randomly selected test images, the mean axial and lateral point location errors were 0.28 mm and 0.37 mm, respectively, which can be regarded as the average imaging system resolution for our trained network. This trained network successfully identified the location of two point targets in a single image with mean axial and lateral errors of 2.6 mm and 2.1 mm, respectively. A true signal and a corresponding reflection artifact were then simulated. The same trained network identified the location of the artifact with mean axial and lateral errors of 2.1 mm and 3.0 mm, respectively. Identified artifacts may be rejected based on wavefront shape differences. These results demonstrate strong promise to identify point targets without requiring traditional geometry-based beamforming, leading to the eventual elimination of reflection artifacts from interventional images.

Iterative image reconstruction algorithms have the potential to reduce the computational time required for photoacoustic tomography (PAT). An iterative deconvolution-based photoacoustic reconstruction with sparsity regularization (iDPARS) is presented which enables us to solve large-scale problems. The method deals with the limited angle of view and the directivity effects associated with clinically relevant photoacoustic tomography imaging with conventional ultrasound transducers. Our Graphics Processing Unit (GPU) implementation is able to reconstruct large 3-D volumes (100×100×100) in less than 10 minutes. The simulation and experimental results demonstrate iDPARS provides better images than DAS in terms of contrast-to-noise ratio and Root-Mean-Square errors.

In this study, a novel imaging modality, free space guided fluorescence diffuse optical tomography (FDOT), was perform to reconstruct the fluorophore distribution by incorporate structural priors into optical image inversion schemes. The dual modality imaging system used a rotation gantry combined with an electron-multiplying charge-coupled device (EMCCD) and a laser source in trans-illumination mode. The fluorescence data was collected from 360° range. The structural information of the object were obtained by an ultrasound linear array transducer. To validate the performance of the system, phantoms and bio tissue experiment were conducted. The results show that the imaging system achieves accurate and has the potential for further in vivo study.

Photoacoustic tomography is a hybrid imaging method that has a variety of biomedical applications. In photoacoustic tomography, the image reconstruction problem (inverse problem) is to resolve the initial pressure distribution from detected ultrasound waves generated within an object due to an illumination of a short light pulse. In this work, this problem is approached in Bayesian framework. Image reconstruction is investigated with numerical simulations in different detector geometries, including limited view setup, and utilizing different prior information. Furthermore, assessing the reliability of the estimates is investigated. The simulations show that the Bayesian approach can produce accurate estimates of the initial pressure distribution and uncertainty information even in a limited view setup if proper prior information is utilized.

Quantitative photoacoustic tomography seeks to estimate the optical parameters of a target given photoacoustic measurements as a data. Conventionally the problem is split into two steps: 1) the acoustical inverse problem of estimating the acoustic initial pressure distribution from the acoustical time series data; 2) the optical inverse problem of estimating the optical absorption and scattering from the initial pressure distributions. In this work, an approach for estimating the optical absorption and scattering directly from the acoustical time series is investigated with simulations. The work combines a homogeneous acoustical forward model, based on the Green's function solution of the wave equation, and a finite element method based diffusion approximation model of light propagation into a single forward model. This model maps the optical parameters of interest into a time domain signal. The model is used with a Bayesian approach to ill-posed inverse problems to form estimates of the posterior distributions for the parameters of interest. In addition to being able to provide point estimates of the parameters of interest, i.e. reconstruct the absorption and scattering distributions, the approach can be used to derive information on the uncertainty associated with the estimates.

Photoacoustic (PA) imaging has shown its potential for many clinical applications, but current research and usage of PA imaging are constrained by additional hardware costs to collect channel data, as the PA signals are incorrectly processed in existing clinical ultrasound systems. This problem arises from the fact that ultrasound systems beamform the PA signals as echoes from the ultrasound transducer instead of directly from illuminated sources. Consequently, conventional implementations of PA imaging rely on parallel channel acquisition from research platforms, which are not only slow and expensive, but are also mostly not approved by the FDA for clinical use. In previous studies, we have proposed the synthetic-aperture based photoacoustic re-beamformer (SPARE) that uses ultrasound beamformed radio frequency (RF) data as the input, which is readily available in clinical ultrasound scanners. The goal of this work is to implement the SPARE beamformer in a clinical ultrasound system, and to experimentally demonstrate its real-time visualization. Assuming a high pulsed repetition frequency (PRF) laser is used, a PZT-based pseudo PA source transmission was synchronized with the ultrasound line trigger. As a result, the frame-rate increases when limiting the image field-of-view (FOV), with 50 to 20 frames per second achieved for FOVs from 35 mm to 70 mm depth, respectively. Although in reality the maximum PRF of laser firing limits the PA image frame rate, this result indicates that the developed software is capable of displaying PA images with the maximum possible frame-rate for certain laser system without acquiring channel data.

Atherosclerosis, the most common cause of death, kills suddenly by arterial occlusion by thrombosis, which is caused by plaque rupture. Because a growing necrotic core is highly related to plaque rupture in atherosclerosis, distinguishing between fibrous plaque and lipid-rich plaque in real time is important, but has been challenging. Real-time photoacoustic imaging requires a pulse laser with high repetition rate, which tends to sacrifice pulse energy. Furthermore, a high repetition rate is hard to achieve at lipid-sensitive wavelengths, such as 1210 nm and 1720 nm. To address the unmet need, we have developed the algorithm for PA imaging. We successfully acquired ex vivo PA images from the lipid cores of arterial plaques in rabbit arteries, using a low-power 1064-nm laser. PA images were acquired with a custom-made catheter employing a single-element 40-MHz ultrasound transducer and a compact 1064-nm laser with the pulse energy of 5 μJ and the repetition rate of 24 kHz. Acquired raw data were processed in the time and frequency domains. In the time domain, a delay-and-sum algorithm was used for image enhancement. In the frequency domain, signals exceeding the MTF were removed. As a result, SNR was increased by about 10 dB without degrading spatial resolution. We were able to achieve high-speed and high-SNR lipid target imaging in animals in spite of the low lipid sensitivity of a 1064nm laser. These results show good promise for detecting lipid-rich plaques with a compact high-speed laser, which can be easily adapted for target clinical applications.

We propose a method to measure blood pressure of small vessels non-invasively and in-vivo: by combining PA imaging with compression US. Using this method, we have shown pressure-lumen area tracking, as well as estimation of the internal vessel pressure, located 2 mm deep in tissue. Additionally, reperfusion can be tracked by measuring the total PA signal within a region of interest (ROI) after compression has been released. The ROI is updated using cross-correlation based displacement tracking¹. The change in subcutaneous perfusion rates can be seen when the temperature of the hand of a human subject drops below the normal.

Photoacoustic imaging (PAI) has proved to be a promising non-invasive technique for diagnosis, prognosis and treatment monitoring of neurological disorders in small and large animals. Skull bone effects both light illumination and ultrasound propagation. Hence, the PA signal is largely affected. This study aims to quantify and compare the attenuation of PA signal due to the skull obstacle in the light illumination path, in the ultrasound propagation path, or in both. The effect of mouse, rat, and mesocephalic dog skull bones, ex-vivo, is quantitatively studied.

There is a need for continued research into the diagnosis, prevention and cure of neonatal brain disease and disorders. These disorders lead to fatalities and developmental disorders in infants. Non-invasive imaging techniques are being researched for this purpose. However, the availability of neonatal skull samples for this work is very low. A phantom can be used to simulate the neonatal skull and brain to improve imaging techniques. This study selects a phantom of polyurethane and titanium dioxide and proves its value as a replacement for neonatal skull in research. The methods used for this proof are validation of choice against the literature, transmissivity and acoustic experimentation compared to existing literature, and finally photoacoustic evaluation of the final choice to show its usefulness as a neonatal skull phantom.

Despite the great promising results of a recent new transcranial photoacoustic brain imaging technology, it has been shown that the presence of the skull severely affects the performance of this imaging modality. In this paper, we investigate the effect of skull on generated photoacoustic signals with a mathematical model. The developed model takes into account the frequency dependence attenuation and acoustic dispersion effects occur with the wave reflection and refraction at the skull surface. Numerical simulations based on the developed model are performed for calculating the propagation of photoacoustic waves through the skull. From the simulation results, it was found that the skull-induced distortion becomes very important and the reconstructed image would be strongly distorted without correcting these effects. In this regard, it is anticipated that an accurate quantification and modeling of the skull transmission effects would ultimately allow for skull aberration correction in transcranial photoacoustic brain imaging.

In photoacoustic/optoacoustic tomography (PAT/OAT) for a circular scanning geometry, the axial/radial resolution is not variant spatially and also do not depend on the ultrasound transducer (UST) aperture. But the tangential resolution is affected by the size of the detector aperture and is spatially variant. To counter this problem many techniques such as attaching a negative lens to the transducer surface, or using virtual detectors were proposed. However these techniques have difficulties. Therefore, a modified delay-and-sum reconstruction algorithm was proposed which can be used with the normal UST to improve the tangential resolution. In this work, we demonstrate the improvement of tangential resolution using the modified delay-and-sum reconstruction algorithm with experimental data. We have obtained more than twofold improvement of resolution in the tangential direction using non-focused and cylindrically focused USTs in a circular scanning geometry. We also observe that shape of the target object can also be preserved which is helpful for diagnosis and treatment purposes.

Photoacoustic tomography (PAT) is a non-ionizing biomedical imaging modality which finds applications in brain imaging, tumor angiogenesis, monitoring of vascularization, breast cancer imaging, monitoring of oxygen saturation levels etc. Typical PAT systems uses Q-switched Nd:YAG laser light illumination, single element large ultrasound transducer (UST) as detector. By holding the UST in horizontal plane and moving it in a circular motion around the sample in full 2π radians photoacoustic data is collected and images are reconstructed. The horizontal positioning of the UST make the scanning radius large, leading to larger water tank and also increases the load on the motor that rotates the UST. To overcome this limitation, we present a compact photoacoustic tomographic (ComPAT) system. In this ComPAT system, instead of holding the UST in horizontal plane, it is held in vertical plane and the photoacoustic waves generated at the sample are detected by the UST after it is reflected at 45° by an acoustic reflector attached to the transducer body. With this we can reduce the water tank size and load on the motor, thus overall PAT system size can be reduced. Here we show that with the ComPAT system nearly similar PA images (phantom and in vivo data) can be obtained as that of the existing PAT systems using both flat and cylindrically focused transducers.

We developed a miniaturized, simple and full field-of-view photoacoustic/ultrasonic endoscopy system, and used a flexible coil to transmit the rotational torque from the rotary stage, which enables a 360o field-of-view imaging in vivo. The developed imaging catheter was fully encapsulated by a single-use protective polyamide tube. A B-scan rate up to 5 Hz (200 A-lines/B-scan) was achieved. Three-dimensional photoacoustic and ultrasound images of the rectum from a SD rat were acquired in vivo. It suggests that this PAE system can be of great interest for clinical translation for a variety of endoscopic applications.

Red blood cell (RBC) transfusion is a critical component of the health care services. RBCs are stored in blood bags in hypothermic temperatures for a maximum of 6 weeks post donation. During this in vitro storage period, RBCs have been documented to undergo changes in structure and function due to mechanical and biochemical stress. Currently, there are no assessment methods that monitor the quality of RBCs within blood bags stored for transfusion. Conventional assessment methods require the extraction of samples, consequently voiding the sterility of the blood bags and potentially rendering them unfit for transfusions. It is hypothesized that photoacoustic (PA) technology can provide a rapid and non-invasive indication of RBC quality. In this study, a novel PA setup was developed for the acquisition of oxygen saturation (SO₂) of two blood bags in situ. These measurements were taken throughout the lifespan of the blood bags (42 days) and compared against the clinical gold standard method of the blood gas analyzer (BGA). SO₂ values of the blood bags increased monotonically throughout the storage period. A strong correlation between PA SO₂ and BGA SO₂ was found, however, PA values were on average 3.5% lower. Both techniques found the bags to increase by an SO₂ of approximately 20%, and measured very similar rates of SO₂ change. Future work will be focused on determining the cause of discrepancy between SO₂ values acquired from PA versus BGA, as well as establishing links between the measured SO₂ increase and other changes in RBC in situ.

Multi-wavelength laser sources are necessary for a functional photoacoustic (PA) spectroscopy. The use of high-power diode lasers (HPDLs) has aroused great interest for their relatively low costs and small sizes if compared to solid state lasers. However, HPDLs are only available at few wavelengths and can deliver low optical energy (normally in the order of μJ), while diode laser bars (DLBs) offer more wavelengths in the market and can deliver more optical energy. We show the simulations of optical systems for beam coupling of single high-power DLBs operating at different wavelengths (i.e. 808 nm, 880 nm, 910 nm, 940 nm, and 980 nm) into 400-μm optical fibers. Then, in a separate design, the beams of the DLBs are combined in a compact system making use of dichroic mirrors and focusing lenses for beam coupling into a 400-μm optical fiber. The use of optical fibers with small core diameter (< 1 mm) is particularly suggestive for future photoacoustic endoscopy (PAE) applications that require interior examination of the body.

We present a non-contact “frequency domain photoacoustic microscope” (Fd-PAM) in which photoacoustic signal is generated by an amplitude modulated continuous-wave (CW) laser and detected at the sample surface using two wave mixing (interferometer) in a photorefractive crystal (PRC). The optical detection eliminates the need for a coupling medium, thus making the probe contactless and mitigates loss in signal-to-noise ratio (SNR) resulting from attenuation associated with wave propagation from the sample to the sensor. The single frequency excitation enables the use of extremely narrow band detection techniques like a lock-in amplifier for noise suppression. Our approach also can image multi-layered specimen and directly produce an image that is equivalent to the maximum-intensity projection of the 3D image volume.

Quantitative analysis of glucose using conventional optical spectroscopy suffers from a lack of repeatability due to high optical scattering in skin tissue. Here we present a multi-modality analysis of glucose aqueous solution using photoacoustic spectroscopy (PAS) and broadband dielectric spectroscopy (BDS). These techniques involve the direct detection of the acoustic and electromagnetic waves propagating through or reflecting from tissue without their being scattered. They therefore have potential for better tolerance to the variation of scattering. For PAS, to differentiate signals induced by water absorption, we select another laser wavelength (1.38 μm) that exhibits the same absorbance for water at 1.61 μm. Furthermore, one of the two photoacoustic signals is used to normalize the variations of acoustic properties in differential signal. Measured results for glucose solutions (0–2 g/dL) showed that the differential signal has a sensitivity of 1.61%/g·dL⁻¹ and a detection limit of 120 mg/dL. We also tested glucose detection with BDS (500 MHz to 50 GHz) by detecting glucose hydration bonding at around 10-20 GHz. Using a partial least square analysis and first derivation on broadband spectra, we obtained an RMS error 19 mg/dL and a detection limit of 59 mg/dL. Using both the low-scattering ultrasonic and microwave detection techniques, we successfully captured the glucose footprint in the physiological range.

High sensitive detection with lock-in amplification can provide high signal noise ratio even when noise is in orders of magnitude higher than the signal. Here we report to combine lock-in amplification with a novel photoacoustic remote sensing (PARS) technology to achieve high resolution, high contrast, all optical non-contact photoacoustic imaging at depth beyond optical scattering limitation. We demonstrate phantom measurements from PARS with lock-in technology were several orders of magnitude more sensitive than those from PARS with the broadband detection techniques.

Visualization of the tip of medical devices like needles or catheters under ultrasound imaging has been a continuous topic since the early 1980’s. In this study, a needle tip visualization system utilizing photoacoustic effects is proposed. In order to visualize the needle tip, an optical fiber was inserted into a needle. The optical fiber tip is placed on the needle bevel and affixed with black glue. The pulsed laser light from laser diode was transferred to the optical fiber and converted to ultrasound due to laser light absorption of the black glue and the subsequent photoacoustic effect. The ultrasound is detected by transducer array and reconstructed into photoacoustic images in the ultrasound unit. The photoacoustic image is displayed with a superposed ultrasound B-mode image. As a system evaluation, the needle is punctured into bovine meat and the needle tip is observed with commercialized conventional linear transducers or convex transducers. The needle tip is visualized clearly at 7 and 12 cm depths with linear and convex probes, respectively, even with a steep needle puncture angle of around 90 degrees. Laser and acoustic outputs, and thermal rise at the needle tip, were measured and were well below the limits of the safety standards. Compared with existing needle tip visualization technologies, the photoacoustic needle tip visualization system has potential distinguishable features for clinical procedures related with needle puncture and injection.

Photoacoustic imaging (PAI) can be used to monitor lesion formation during high-intensity focused ultrasound (HIFU) therapy because HIFU changes the optical absorption spectrum (OAS) of the tissue. However, in traditional PAI, the change could be too subtle to be observed either because the OAS does not change very significantly at the imaging wavelength or due to low signal-to-noise ratio in general. We propose a machine-learning-based method for lesion monitoring with multi-wavelength PAI (MWPAI), where PAI is repeated at a sequence of wavelengths and a stack of multi-wavelength photoacoustic (MWPA) images is acquired. Each pixel is represented by a vector and each element in the vector reflects the optical absorption at the corresponding wavelength. Based on the MWPA images, a classifier is trained to classify pixels into two categories: ablated and non-ablated. In our experiment, we create a lesion on a block of bovine tissue with a HIFU transducer, followed by MWPAI in the 690 nm to 950 nm wavelength range, with a step size of 5 nm. In the MWPA images, some of the ablated and non-ablated pixels are cropped and fed to a neural network (NN) as training examples. The NN is then applied to several groups of MWPA images and the results show that the lesions can be identified clearly. To apply MWPAI in/near real-time, sequential feature selection is performed and the number of wavelengths is decreased from 53 to 5 while retaining adequate performance. With a fast-switching tunable laser, the method can be implemented in/near real-time.

We present a photoacoustic imaging system based on a low-cost high-power miniature light emitting diode (LED), which has the capability of in vivo mapping vasculature networks in biological tissue. Phantoms were used to demonstrate the feasibility of the system, while in vivo imaging the vasculature of mouse ear shows that LED-based photoacoustic imaging (LED-PAI) could have great potential for label-free biomedical imaging applications, overcoming the practical limitations of the use of bulky and expensive pulsed lasers.

One of the main issues of the advances in optoacoustic (OA) applications is to reduce the high costs and the big sizes of solid state lasers. High-power diode lasers (HPDLs) have been demonstrated to be a valid alternative reducing enormously the expenses, besides other advantages such as smaller sizes and higher modulation frequencies. However, in some cases it is possible to furtherly reduce their costs. We present a cost-effective OA system based on the combination of several 905-nm HPDLs with direct coupling into a fiber bundle. These HPDLs have an internal pulse driver, based on an n-channel Mosfet and two charging capacitors, which needs an external Mosfet driver circuit and a voltage supply in order to improve the optical pulse shape and energy. We compare the performances and the prices of this OA system with another similar HPDL-based OA system built with commercial elements. Results indicate good OA signal generation (~15.6 mV_pp) with pulse energy of 12.3 μJ and, especially, a cost reduction by a factor of ~15 if compared to the other HPDL-based system.

In the last few decades, high power diode lasers (HPDL) have been introduced as alternative laser sources for optoacoustic imaging (OAI), due to their high repetition rates (a few kHz) for fast OA image acquisition, lower cost and size if compared to solid state lasers. Nevertheless, their drawbacks consist in a low energy per pulse (μJ) and a relatively highly divergent beam that needs collimation optics. At this purpose, the employment of diode laser stacks significantly increases the energy per pulse up to several mJ. The diode laser stacks imply a big challenge if compared to single emitters for several reasons. Firstly, they need very demanding electronic requirements, as forward voltages and currents of several tens of volts and hundreds of amperes, respectively. Secondly, their highly divergent beam profile requires precise collimation by means of fast axis and slow axis collimation. In this work, we show an 808-nm diode laser stack driven with 17 V and ~ 200 A by a low-cost current driver for emitting pulses of 1 mJ at 1 kHz. Particular emphasis will be attributed to the design of the high current pulses driver and the optics employed to collimate and after focus the beam in a spot. The light spot will be applied to an ink inclusion hosted in turbid phantom. We demonstrate that our system is able to generate appreciable OA signals in turbid phantoms. This aspect represents a novelty in OAI systems because it is demonstrated that HPDL sources can efficiently replace solid-state lasers.

The optoacoustic effect is almost invariably produced by intensity modulated radiation, typically from a pulsed or an amplitude modulated continuous source. Given the form of the wave equation that describes the production of sound from absorption of light, it is clear that steady sources of radiation that move in space in an absorbing medium can also generate acoustic waves. Here the properties of a point source of radiation that rotates in a plane at a constant angular frequency are discussed. The source is shown to generate a spiral wave pattern that contains both compressions and rarefactions.

We have developed a low-cost, non-contact, multispectral photoacoustic microscope system to study the functional parameters of cellular choromophores. The system uses low power continuous wave lasers and a photoacoustic sensor made of a kHz microphone coupled to a resonant chamber. Methemoglobin has relatively high optical absorption at 500 nm and 630 nm. Moreover, it has an almost the same optical absorption as hemoglobin at the isosbestic point of 525 nm. Photoacoustic data collected from methemoglobin using our system at wavelengths of 473 nm, 533 nm, and 633 nm show the similar trends as the methemoglobin optical absorption spectrum. The PA amplitude at 473 nm is about 1.03 times greater than at 533 nm and about 2.4 times greater than at 633 nm. Similarly, it possesses optical absorption of about 1.08 greater than at 533 nm and 1.34 times greater than at 633 nm. The developed system can be used as a differential photoacoustic microscope.

We report photoacoustic microscopy (PAM) of arteriovenous (AV) shunts in early stage tumors in vivo, and develop a pattern recognition framework for computerized tumor detection. Here, using a high-resolution photoacoustic microscope, we implement a new blood oxygenation (sO₂)-based disease marker induced by the AV shunt effect in tumor angiogenesis. We discovered a striking biological phenomenon: There can be two dramatically different sO₂ values in bloodstreams flowing side-by-side in a single vessel. By tracing abnormal sO₂ values in the blood vessels, we can identify a tumor region at an early stage. To further automate tumor detection based on our findings, we adopt widely used pattern recognition methods and develop an efficient computerized classification framework. The test result shows over 80% averaged detection accuracy with false positive contributing 18.52% of error test samples on a 50 PAM image dataset.

Photoacoustic imaging is a fast growing in vivo imaging modality combining the optical absorption contrast and high spatial ultrasonic resolution. Using this technique deeper tissue penetration greater than optical mean free path (~1 mm in skin) is possible. Low resolution with deep penetration depth is possible utilizing acoustic focusing and high resolution with shallow imaging depth can be possible by optical focusing using different photoacoustic microscopy (PAM) systems. Here, we present a combined/switchable optical resolution and acoustic resolution photoacoustic microscopy (OR-AR-PAM) system capable of both high resolution and low resolution deep tissue imaging on the same sample. Lateral resolution of 3.9 μm using optical focusing and lateral resolution of 57 μm using acoustic focusing was successfully demonstrated using the combined system.

We present a low-cost laser scanning photoacoustic microscopy system with a pulsed laser diode as the excitation source. The system utilizes a 905 nm pulsed laser diode with 120 ns pulse width and 1 KHz repetition rate. No averaging is performed in data acquisition, resulting in a short image acquisition time. The maximum field of view is 4.6 mm × 3.7 mm and the lateral resolution is 71 μm. Images of human hairs and mouse ear are presented to demonstrate the feasibility of the system in imaging biological tissue.

Label-free photoacoustic microscopy (PAM) with nanometric resolution is important to study cellular and sub-cellular structures, microcirculation systems, micro-vascularization, and tumor angiogenesis etc. But, the lateral resolution of a conventional microscopy is limited by optical diffraction. The photonic nanojet generated by silica microspheres can break this diffraction limit. Single silica sphere can provide narrow photonic jet, however its short length and short working distance limits its applications to surface imaging. It is possible to increase the length of the photonic nanojet and its working distance by optimizing the sphere design and its optical properties. In this work, we will present various sphere designs to achieve ultra-long and long-working distance photonic nanojets for far-field imaging. The nanojets thus generated will be used to demonstrate super-resolution photo-acoustic imaging using k-wave simulations. The study will provide new opportunities for many biomedical imaging applications that require finer resolution.

Many neurological disorders are linked to abnormal activation or pathological alterations of the vasculature in the affected brain region. Obtaining simultaneous morphological and physiological information of neurovasculature is very challenging due to the acoustic distortions and intense light scattering by the skull and brain. In addition, the size of cerebral vasculature in murine brains spans an extended range from just a few microns up to about a millimeter, all to be recorded in 3D and over an area of several dozens of mm². Numerous imaging techniques exist that excel at characterizing certain aspects of this complex network but are only capable of providing information on a limited spatiotemporal scale. We present a hybrid ultrasound and dual-wavelength optoacoustic microscope, capable of rapid imaging of murine neurovasculature in-vivo, with high spatial resolution down to 12 μm over a large field of view exceeding 50mm². The dual wavelength imaging capability allows for the visualization of functional blood parameters through an intact skull while pulse-echo ultrasound biomicroscopy images are captured simultaneously by the same scan head. The flexible hybrid design in combination with fast high-resolution imaging in 3D holds promise for generating better insights into the architecture and function of the neurovascular system.

Photoacoustic (PA) field calculations using a Green’s function approach is presented. The method has been applied to predict PA spectra generated by normal (discocyte) and pathological (stomatocyte) red blood cells (RBCs). The contours of normal and pathological RBCs were generated by employing a popular parametric model and accordingly, fitted with the Legendre polynomial expansions for surface parametrization. The first frequency minimum of theoretical PA spectrum approximately appears at 607 MHz for a discocyte and 410 MHz for a stomatocyte when computed from the direction of symmetry axis. The same feature occurs nearly at 247 and 331 MHz, respectively, for those particles when measured along the perpendicular direction. The average experimental spectrum for normal RBCs is found to be flat over a bandwidth of 150-500 MHz when measured along the direction of symmetry axis. For spherical RBCs, both the theoretical and experimental spectra demonstrate negative slope over a bandwidth of 250-500 MHz. Using the Green’s function method discussed, it may be possible to rapidly characterize cellular morphology from single-particle PA spectra.

We propose an integrated platform of Aluminum Nitrate (AlN) based Piezoelectric Micromachined Ultrasonic Transducer (pMUT) phased array with Application Specific Integrated Circuit (ASIC) for medical imaging and industrial diagnosis. The ASIC provides wide driving range for frequencies between 100 kHz and 5 MHz and channelscalable, programmable application adaptive transmitting beamformer. The system supports operation in various media, including gasses, liquids and biological tissue. The scan resolution for 5 MHz operation is 68 μm in air. The beamformer covers a test volume from -30° to +30° with a step of 3° and scan depth of 10 cm. The ASIC system features low noise receiver electronics, power saving transmission circuitry, and high-voltage drive of large capacitance transducer (up to 500 pF). Integrated pMUT phased array consists of 4 channels of single-membrane ultrasonic transducer of 400 nm deflection and 20 pF feed-thru capacitance, which produce 15 Pa pressure at 500 μm distance from the surface of the transducers. The active area of the ASIC is (700×1490) μm², which includes channel scalable TX, 8-channale low noise RX, digital back end with autonomous beamformer and power management unit. The system is battery powered with 3.3V-5V standard supply, representing a truly portable solution for ultrasonic applications.

Given the CMOS-compatible fabrication process for the AlN pMUTs, dense, miniaturized arrays are possible. Furthermore the smooth surface of dielectric AlN renders optical quality MEMS surfaces for integration in miniaturized photonic + ultrasound microsystems.

Recently, various type of photoacoustic imaging (PAI) that can visualize properties and distribution of light absorber have been researched. We developed PAI system using LED light source and evaluated characteristics of photoacoustic signal intensity versus Indocyanine Green (ICG) concentration. In this experiment, a linear type PZT array transducer (128-elements, 10.0MHz center frequency) was used to be able to transmit and receive ultrasound and also detect photoacoustic signal from the target object. The transducer was connected to the PAI system, and two sets of LED light source that had 850nm wavelength chip array were set to the both side of the transducer. The transducer head was placed at a distance of 20 mm from the target in the water bath. The target object was a tube filled with ICG in it. The tubes containing ICG at concentrations from 300nanomolar to 3millimolar were made by diluting original ICG solution. We measured the photoacoustic signal strength from RF signal generated from the ICG in the tube, and the results showed that the intensity of the signal was almost linear response to the concentration in log-log scale.

Single sensor (pixel) signals require scanning of the sample in order to obtain spatial information. In this paper we show that using interference, optically induced signals can be reconstructed when recorded using interference pattern excitation, rather than a point illumination. This method reduces the need for dense scanning and requires a small number of scans, or can eliminate the need for scanning in some cases. It is shown that this method can be used in particular in photo-acoustic imaging.