This PDF file contains the front matter associated with SPIE Proceedings Volume 10494 including the Title Page, Copyright information, Table of Contents, Introduction, and Conference Committee listing.

Presenting highly sensitive functional information in subsurface tissue with spatial resolution comparable to ultrasound imaging, the emerging photoacoustic (PA) imaging may shed new lights to early diagnosis and treatment monitoring of human inflammatory arthritis. This paper will introduce our recent development of LED-based PA imaging and its application to human inflammatory arthritis. Facilitated by the high pulse repetition rate of the LED arrays, extensive averaging of PA signal can be performed, which boosts the signal-to-noise ratio of the LED-based PA imaging system to levels comparable to laser-based PA imaging systems. In the experiments on arthritis patients and normal volunteers, each target finger joint is scanned using the LED-based PA imaging system which is integrated with a B-scan ultrasound (US) facilitating dual imaging modalities simultaneously. 2D PA and US of a sagittal section in the joint can be acquired in a real-time fashion with a frame rate up to 30 Hz; while a series of 2D images acquired along the cross sections of the joint can be reconstructed into a 3D image for analyzing the volumetric biomarkers of joint inflammation. In this initial study on human subjects, we have confirmed the feasibility of LED-based PA imaging in detecting and characterizing arthritic joints by evaluating the hemodynamic changes associated with soft-tissue inflammation. PA imaging findings are compared to the results from Doppler US acquired using a commercial US unit. This study demonstrates that the LED-based PA imaging can be developed into a point-of-care diagnostic tool for rheumatology and radiology clinics.

We have conducted a pilot study to image and characterize ovarian masses using a co-registered ultrasound (US) and photoacoustic imaging (PAI) system. A total of seven patients who have ovarian masses and scheduled for surgical removal of both ovaries and Fallopian tubes, has enrolled to the study. A standard transvaginal US probe is used first to locate ovarian masses, measure ovary sizes in long and short axises . Then a second probe with four light-delivery fibers surrounded the US probe is inserted transvaginally to perform co-registered US and photoacoustic imaging in real-time with four optical wavelengths (730, 780, 800 and 830 nm). For the total of 7 patients, one patient had high-grade serous carcinoma involving both ovaries, one patient had a 2.2 cm endometrioid adenocarcinoma in right ovary, one patient had metastatic appendiceal adenocarcinoma involving both ovaries, one patient has serous borderline carcinoma involving both ovaries and the rest three patients had either abnormal or benign ovaries. For the malignant ovaries, photoacoustic images have showed significantly higher signal levels as quantitatively evaluated from maximum signal strengths from region-of-interest identified by co-registered US. Additionally, the quantitative features extracted from co-registered US and photoacoustic images, such as spectral slope, mid-band fit and zero MHz intercept, spatial heterogeneity of PAI distribution, the blood oxygen saturation, have showed significant differences between malignant and benign ovaries. Our initial results have demonstrated that photoacoustic imaging has a great potential to aid transvaginal US to quantitatively and effectively image and diagnose ovarian cancer.

Non-invasive in vivo imaging of lymphatic system is of paramount importance for analyzing the functions of lymphatic vessels, and for investigating their contribution to metastasis. Recently, we introduced a multi-wavelength real-time LED-based photoacoustic/ultrasound system (AcousticX). In this work, for the first time, we demonstrate that AcousticX is capable of real-time imaging of human lymphatic system. Results demonstrate the capability of this system to image vascular and lymphatic vessels simultaneously. This could potentially provide detailed information regarding the interconnected roles of lymphatic and vascular systems in various diseases, therefore fostering the growth of therapeutic interventions.

We have integrated photoacoustic tomography into an automated breast ultrasound scanner. This system scans the patient in the prone position, so illuminating the breast required the development of a fiber-coupled illumination system and a light-shaping diffuser to direct the light onto the tissue. This system allows simultaneous acquisition of B-mode and multi-wavelength photoacoustic tomography data in a single, operator-independent scan. We present the first characterizations of this complete scanning system, as well as phantom data used to evaluate the attainable imaging depth, resolution, and sensitivity of the hardware and reconstruction scheme.

Photoacoustic (PA) systems based on clinical linear ultrasound arrays have become increasingly popular in translational PA research. Such systems can more easily be integrated in a clinical workflow due to the simultaneous access to ultrasonic imaging and their familiarity of use to clinicians. In contrast to more complex setups, handheld linear probes can be applied to a large variety of clinical use cases. However, most translational work with such scanners is based on proprietary development and as such not accessible to the community. In this contribution, we present a custom-built, hybrid, multispectral, real-time photoacoustic and ultrasonic imaging system with a linear array probe that is controlled by software developed within the highly customisable and extendable open-source software platform Medical Imaging Interaction Toolkit (MITK). Our software offers direct control of both the laser and the ultrasonic system and may thus serve as a starting point for various translational research and development. To demonstrate the extensibility of our system, we developed an open-source software plugin for real-time in vivo blood oxygenation measurements. Blood oxygenation is estimated by spectral unmixing of hemoglobin chromophores. The performance is demonstrated on in vivo measurements of the common carotid artery as well as peripheral extremity vessels of healthy volunteers.

The aim of this study was to explore the unique imaging abilities of optoacoustic mesoscopy to visualize skin structures and microvasculature with the view of establishing a robust approach for monitoring heat-induced hyperemia in human skin in vivo. Abnormalities in the structure and function of skin vasculature are increasingly recognized as hallmarks of several systemic disorders. Purely optical techniques can sense changes in skin perfusion, but they lack reproducibility and resolution to quantify the behavior of vessels in deep skin. Using raster-scan optoacoustic mesoscopy (RSOM), we investigated whether optoacoustic mesoscopy can identify changes in skin response to local heating at microvasculature resolution in a cross-sectional fashion through skin in the human forearm. We visualized the heat-induced hyperemia for the first time with single-vessel resolution throughout the whole skin depth. We quantified changes in total blood volume in the skin and their correlation with local heating. In response to local heating, total blood volume increased 1.83- and 1.76-fold, respectively, in the volar and dorsal aspects of forearm skin. We demonstrate RSOM imaging of the dilation of individual vessels in the skin microvasculature, consistent with hyperemic response to heating at the skin surface. Our results demonstrate great potential of optoacoustic dermoscopy for elucidating the morphology, functional state and reactivity of dermal microvasculature, with implications for diagnostics and disease monitoring in dermatology.

Noninvasive measurement of cerebral venous oxygenation in neonates could provide critical information for clinicians such as cerebral hypoxia without the risks involved with invasive catheterization. Evaluation of cerebral hypoxia is important in many clinical settings such as hypoxic-ischemic encephalopathy, perfusion monitoring in cardiovascular surgery or in traumatic brain injury. By probing the superior sagittal sinus (SSS), a large central cerebral vein, we can obtain stable signals with our recently developed multi-wavelength, fiber-coupled laser diode optoacoustic system for measurement of SSS blood oxygenation. The neonatal SSS oxygenation was measured in the reflection mode through open anterior and posterior fontanelles without obscuration by the overlying calvarium. In the transmission mode it was measured through the skull in the occipital area. Our device is lightweight, easily maneuverable, and user friendly for physicians. We monitored the SSS oxygenation in neonates admitted to the Neonatal Intensive Care Unit (NICU) of UTMB with varying gestation, birth weight and clinical histories to identify normal range and difference between neonates with and without risk factors for cerebral hypoxia.

In this paper, we developed a real-time dual-modality Photoacoustic (PA) /ultrasound (US) imaging system and performed initial clinical study, including both healthy and cancerous nodules. This dual-modality imaging system was based on a commercial US device (Resona7, Mindray Inc, China) modified to be capable to acquire PA signals without scarifying US functions. We also made special mechanical components to allow this handheld probe to perform 3D scanning dual-modality operation. Optical excitation was provided by a tunable pulsed OPO (SpitLight 600 OPO, InnoLas Laser GmbH, Germany), which emits 7ns width pulses at 10Hz repetition rate. Light delivery was facilitated by a fiber bundle with bifurcated ends mounted on each axial side of a linear array transducer (L9-3U, Mindray Inc., China). The transducer consisted of 192 elements that were specially covered with a highly light scattering acoustic lens. Therefore, elements were mechanically focused 3cm beneath the surface of the lens in elevation direction and capable of detecting PA signals at frequencies of up to 7 MHz. The array was connected to the US machine to acquire and digitize all channel data in parallel. For optimal light coupling, a 7 mm-thick transparent soft gel pad was placed between the tissue surface and the probe. This pad provided a gap that allow light shining on the interested area just under the probe without sacrificing US imaging. This dual-modal US machine can show both PA and US images (including B-scan and Doppler) simultaneously, as well as functional PA imaging. Our study demonstrated that PA imaging could provide important complementary information for traditional ultrasound examination, which has a great potential for clinical diagnosis of many diseases, including breast cancer and thyroid cancer.

Dental implants are common method to replace decayed or broken tooth. As the implant treatment procedures varies according to the patients’ jawbone, bone ridge, and sinus structure, appropriate examinations are necessary for successful treatment. Currently, radiographic examinations including periapical radiology, panoramic X-ray, and computed tomography are commonly used for diagnosing and monitoring. However, these radiographic examinations have limitations in that patients and operators are exposed to radioactivity and multiple examinations are performed during the treatment. In this study, we demonstrated photoacoustic (PA) and ultrasound (US) combined imaging of dental implant that can lower the total amount of absorbed radiation dose in dental implant treatment. An acoustic resolution PA macroscopy and a clinical PA/US system was used for dental implant imaging. The acquired dual modal PA/US imaging results support that the proposed photoacoustic imaging strategy can reduce the radiation dose rate during dental implant treatment.

Photoacoustic (PA) imaging with internal light illumination through optical fiber could enable imaging of internal organs at deep penetration. We have developed a transurethral probe with a multimode fiber inserted in a rigid cystoscope sheath for illuminating the prostate. At the distal end, the fiber tip is processed to diffuse light circumferentially over 2 cm length. A parabolic cylinder mirror then reflects the light to form a rectangular-shaped parallel beam which has at least 1 cm² at the probe surface. The relatively large rectangular beam size can reduce the laser fluence rate on the urethral wall and thus reduce the potential of tissue damage. A 3 cm optical penetration in chicken tissue is achieved at a fluence rate around 7 mJ/cm² . For further validation, a prostate phantom was built with similar optical properties of the human prostate. A 1.5 cm penetration depth is achieved in the prostate mimicking phantom at 10 mJ/cm² fluence rate. PA imaging of prostate can potentially be carried out in the future by combining a transrectal ultrasound transducer and the transurethral illumination.

Photoablative laser therapy is in common use for selective destruction of malignant masses, vascular and brain abnormalities. Tissue ablation and coagulation are irreversible processes occurring shortly after crossing a certain thermal exposure threshold. As a result, accurate mapping of the temperature field is essential for optimizing the outcome of these clinical interventions. Here we demonstrate four-dimensional optoacoustic temperature mapping of the entire photoablated region. Accuracy of the method is investigated in tissue-mimicking phantom experiments. Deviations of the volumetric optoacoustic temperature readings provided at 40ms intervals remained below 10% for temperature elevations above 3°C, as validated by simultaneous thermocouple measurements. The excellent spatio-temporal resolution of the new temperature monitoring approach aims at improving safety and efficacy of laser-based photothermal procedures.

The strategy of Intensity-modulated radiotherapy (IMRT) is to deliver precise radiation doses to targeted area while minimizing the dose to surrounding healthy tissue. The intrafractional variations such as the movement of the patient or the respiratory motion, which most likely to cause misalignment during a session of radiotherapy, may compromise the outcome of the detailed dose delivery. This study examines the feasibility of real-time monitoring the alignment of the X-ray beam relative to treatment target during radiotherapy based on ultrasound (US) and X-ray acoustic (XA) dual-modality imaging. A dual-modality imaging system, which utilizes the US phase array for both US imaging and XA signal acquisition, was established based on the Verasonics US system. 2D US image achieved can be used to locate the target cancerous tissue, while 2D XA image acquired will show the shape and location of the X-ray field inside the same imaging plane quasi simultaneously. A phantom holding a large piece of veal liver, where parts of the liver tissue were removed from the middle and embedded with different types of bio-tissues (muscle, fat or kidney), was shot by the beams generated and modulated by a medical linear accelerator. The fusion images integrated with XA and US images quantitatively demonstrated whether the X-ray beam was delivered to the embedded bio-tissue with any mismatch in its shape or any shifts off the accurate position. The experiment results suggest that the US-XA dual-modality imaging is a potential tool for real-time monitoring the geometric alignment during radiotherapy.

To improve the precision in radiation therapy and optimize treatment strategies during the radiotherapy, in vivo radiation dosimetry monitoring which measures the actual dose received in and around target region during treatment becomes necessary. Given the fact that X-ray induced acoustic amplitude is proportionally correlating to X-ray absorption, we propose applying X-ray acoustic computed tomography (XACT) to monitor X-ray dosimetry during radiotherapy. A prototype X-ray acoustic (XA) detection system with single immersion ultrasound transducer, which was positioned by a motor controlled rotation stage, was synchronized with a medical linear accelerator to acquire the XA signal at each rotating position. A porcine gel phantom, which is embedded with equally spaced lard made cylindrical indicators, was shot by different X-ray beams modulated by physical wedge filters (with wedge angles of 15°, 30°, 45° and 60°). The reconstructed 2D XACT images not only showed the positions of the indicators but also displayed the different intensity profiles for each indicator, which had good correlations with the corresponding dose distributions captured in radiochromic film tests. A dose difference as small as 3.6% can be determined. Moreover, a phantom imitating complex scenario of body, which has lard made indicators covered by different materials (bone, muscle and air-gap), was shot by uniform beam. The variances of dose delivered to target regions suffering different attenuations were successfully presented in XACT images. The results of phantom experiments have proved that XACT can be a promising technique in monitoring the 2D dosimetry during radiotherapy with high sensitivity and good accuracy.

We have developed a flow cytometer based on simultaneous detection of ultrasound and photoacoustic waves from individual particles/cells flowing in a microfluidic channel. Our polydimethylsiloxane (PDMS) based hydrodynamic 3-dimensional (3D) flow-focusing microfluidic device contains a cross-junction channel, a micro-needle (ID 100 μm and OD 200 μm) insert, and a 3D printed frame to hold and align a high frequency (center frequency 375 MHz) ultrasound transducer. The focused flow passes through a narrow focal zone with lateral and axial focal lengths of 6-8 μm and 15-20 μm, respectively. Both the lateral and axial alignments are achieved by screwing the transducer to the frame onto the PDMS device. Individual particles pass through an interrogation zone in the microfluidic channel with a collinearly aligned ultrasound transducer and a focused 532 nm wavelength laser beam. The particles are simultaneously insonified by high-frequency ultrasound and irradiated by a laser beam. The ultrasound backscatter and laser generated photoacoustic waves are detected for each passing particle. The backscattered ultrasound and photoacoustic signal are strongly dependent on the size, morphology, mechanical properties, and material properties of the flowing particles; these parameters can be extracted by analyzing unique features in the power spectrum of the signals. Frequencies less than 100 MHz do not have these unique spectral signatures. We show that we can reliably distinguish between different particles in a sample using the acoustic-based flow cytometer. This technique, when extended to biomedical applications, allows us to rapidly analyze the spectral signatures from individual single cells of a large cell population, with applications towards label-free detection and characterization of healthy and diseased cells.

Radiation-induced brain damage could arise as a side effect in radiotherapy of brain tumors. We present evidence of radiation-induced damage to the skull and brain vasculature of mice as revealed by transcranial optoacoustic and ultrasound bio-microscopy in-vivo. The three-dimensional nature of the acquired optoacoustic images combined with a clear anatomical reference of the pulse-echo ultrasound data allowed clear differentiation of the skull vasculature from the superficial brain vasculature. The irradiation was selectively applied to one brain hemisphere and the effects of the ionizing radiation were evident without introduction of extrinsic labeling, owing to the strong haemoglobin contrast of optoacoustics.

Fluoroscopy is currently the standard approach for image guidance of surgical drilling procedures. In addition to the harmful radiation dose to the patient and surgeon, fluoroscopy fails to visualize critical structures such as blood vessels and nerves within the drill path. Photoacoustic imaging is a well-suited imaging method to visualize these structures and it does not require harmful ionizing radiation. However, there is currently no clinical system available to deliver light to occluded drill bit tips. To address this challenge, a prototype drill was designed, built, and tested using an internal light delivery system that allows laser energy to be transferred from a stationary laser source to the tip of a spinning drill bit. Photoacoustic images were successfully obtained with the drill bit submerged in water and with the drill tip inserted into a thoracic vertebra from a human cadaver.

Our current research explores the applications of sound, light, and nanoparticles in ophthalmology. Specifically, we consider glaucoma, a common blinding disease 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. To expedite clinical translation, non-invasive longitudinal monitoring of stem cell delivery in vivo is desired. We thus investigated ultrasound (US) and photoacoustic (PA) imaging of nanotracer-labeled mesenchymal stem cells (MSCs) and magnetic guidance of cells to the TM in the eye. Adipose-derived MSCs were incubated with photoacoustic nanoparticles to label cells (PA-MSCs), and 1000-4000 cells/µl were delivered to porcine eyes ex vivo while US/PA imaging was performed. Eyes were dissected for histology and additional spectroscopic PA imaging. Results show proof-of-concept for longitudinal detection of cell delivery using gold nanospheres and magnetically-mediated guidance using photomagnetic nanocubes. As cell number increased, the amplitude of spectroscopically unmixed signal from PA-MSCs increased, showing potential for quantitative imaging. Three-dimensional spectroscopic PA imaging and histology of the TM showed a similar ring-like morphology, with concentrations of signal from fluorescently-tagged cells matching the distribution of PA signal from the PA-MSCs. These results provide proof-of-concept for monitoring MSC ocular delivery, indicating new opportunities for development of nanoparticle-augmented imaging technologies in ophthalmic research. Tracking MSCs loaded with gold nanospheres has also provided new insights for improving delivery efficiency with photomagnetic nanoparticles, novel light delivery systems for safe, sensitive detection, and even a more physiologically relevant glaucoma model.

Synovitis is a driver of osteoarthritis. Imaging of the synovial vasculature is essential for osteoarthritis assessment, while traditional imaging techniques like contrast-enhanced MRI/CT and conventional ultrasonography have limitations such as invasive manipulation, radiation and subjective results. In this study, an emerging non-invasive ultrasound imaging technique – optoacoustic molecular imaging (OA) was applied to evaluate the synovial vasculature in a mouse model of knee joint osteoarthritis. 16 male Balb/c mice undertook destabilization of medial meniscus surgery (DMM), 8 were intact as baseline controls. Three-dimensional high-frequency ultrasonography, including B mode, Power Doppler (PD) and OA, was performed to the knees of live mice at baseline (n=8), 1 month (n=8), and 4 months (n=7) after DMM, before tissue harvest. We found that OA vascular density increased significantly at 1 month (p=0.028) and remained high at 4 months (p=0.541), indicating synovial neovascularization during osteoarthritis progression, which was consistent with uCT-based angiography and histological findings. Meanwhile, OA could also evaluate the function of the synovial vasculature by measuring blood oxygen saturation (sO2). We found OA sO2 declined significantly at 4 months compared to baseline (p=0.043) and 1 month (p=0.027), indicating vascular dysfunction at late stage osteoarthritis. Moreover, OA sO2 was found closely related with histological cartilage damage (p=0.028). In this study, we demonstrated that OA was reliable to evaluate the small vasculatures in the knee joint of DMM mouse model. Our findings provided a new technique for the non-invasive monitoring of both structure and function of the synovial vasculature during osteoarthritis progression.

Matrix metalloproteinases (MMPs) play important roles in the pathophysiology of cerebral ischemia. Here we visualized in vivo MMP activity in the transient middle cerebral artery occlusion (tMCAO) mouse model using multispectral optoacoustic imaging (MSOT) with a MMP-activatable probe. MSOT data was co-registered with structural magnetic resonance imaging (MRI) obtained at 7 T for localization of signal distribution. We demonstrated upregulated MMP signal within the focal ischemic lesion in the tMCAO mouse model using MSOT/MRI multimodal imaging. This convenient non-invasive method will allow repetitive measurement following the time course of MMP-lesion development in ischemic stroke animal model.

Transrectal ultrasound (TRUS) guided biopsy is the standard procedure for evaluating the presence and aggressiveness of prostate cancer (PCa). The microarchitecture of each biopsied tissue is assigned a Gleason score, a highly prognostic architecture-based grading system for PCa. Due to the limited sensitivity of TRUS imaging to PCa, less than 10% of the sample cores are clinically significant, yet the false negative rate could be 20% at the initial biopsies. A diagnostic modality that can assess the microarchitectures within the prostates in vivo without tissue extraction could significantly reduce the unnecessary biopsy cores and the post-procedure complications. Our previous study has shown that photoacoustic physio-chemical analysis (PAPCA) can quantify the architectural heterogeneities in the prostate. Our recently developed needle PA probe has facilitated the minimally invasive acquisition of PA signals with sufficient temporal length and narrow dynamic range in deep tissue for statistics-based PAPCA. This study investigates the PCa diagnosis by PAPCA of the signals acquired by the needle PA probe. A total of 45 interstitial measurements were acquired (21 in normal and 24 in cancerous regions) in 10 ex vivo human prostates. A significant difference was found in the architectural heterogeneities between the normal and cancerous regions (p<0.005). Areas-under-the-curve of 0.8 has been observed for identifying PCa using the quantitative features. Quantification of the architectural changes in vivo in a transgenic mouse model of PCa is under investigation. The preliminary test has shown a significant difference between the normal and cancerous mouse prostates ex vivo (p<0.005).

Optical microscopy remains a major workhorse in biological discovery despite the fact that light scattering limits its applicability to depths of ∼ 1 mm in scattering tissues. Optoacoustic imaging has been shown to overcome this barrier by resolving optical absorption with microscopic resolution in significantly deeper regions. Yet, the time domain is paramount for the observation of biological dynamics in living systems that exhibit fast motion. Commonly, acquisition of microscopy data involves raster scanning across the imaged volume, which significantly limits temporal resolution in 3D. To overcome these limitations, we have devised a fast optoacoustic micro-tomography (OMT) approach based on simultaneous acquisition of 3D image data with a high-density hemispherical ultrasound array having effective detection bandwidth around 25 MHz. We performed experiments by imaging tissue-mimicking phantoms and zebrafish larvae, demonstrating that OMT can provide nearly cellular resolution and imaging speed of 100 volumetric frames per second. As opposed to other optical microscopy techniques, OMT is a hybrid method that resolves optical absorption contrast acoustically using unfocused light excitation. Thus, no penetration barriers are imposed by light scattering in deep tissues, suggesting it as a powerful approach for multi-scale functional and molecular imaging applications.

Ischemic reperfusion injuries (IRIs) are caused by return of blood to a tissue or organ after a period without oxygen or nutrients. Damage in the microvasculature causes an inflammatory response and heterogeneous scarring, which is associated with an increase in collagen in the extracellular matrix. Although most often associated with heart attacks and strokes, IRI also occurs when blood reperfuses a transplanted organ. Currently, monitoring for IRI is limited to biopsies, which are invasive and sample a limited area. In this work, we explored photoacoustic (PA) biomarkers of scarring. IRI events were induced in mice (n=2) by clamping the left renal artery, then re-establishing flow. At 53 days post-surgery, kidneys were saline perfused and cut in half laterally. One half was immediately imaged with a VevoX system (Fujifilm-VisualSonics, Toronto) in two near infrared ranges - 680 to 970 nm (NIR), and 1200 to 1350 nm (NIR II). The other half was decellularized and then imaged at NIR and NIR II. Regions of interest were manually identified and analyzed for each kidney. For both cellularized and decellularized samples, the PA signal ratio based on irradiation wavelengths of 715:930 nm was higher in damaged kidneys than for undamaged kidneys (p < 0.0001 for both). Damaged kidneys had ROIs with spectra indicating the presence of collagen in the NIR II range, while healthy kidneys did not. Collagen rich spectra were more apparent in decellularized kidneys, suggesting that in the cellularized samples, other components may be contributing to the signal. PA imaging using spectral ratios associated with collagen signatures may provide a non-invasive tool to determine areas of tissue damage due to IRIs.

Non-invasive treatment of tumor is beneficial for the favorable prognosis of the patients. High Intensity Focused Ultrasound (HIFU) is an emerging non-invasive treatment tool that ablates tumor lesions by increasing local temperature without damaging surrounding tissues. In HIFU therapy, accurate focusing of the HIFU energy into the target lesion and real-time assessment of thermal distribution are critical for successful and safe treatment. Photoacoustic (PA) imaging is a novel biomedical imaging technique that can visualize functional information of biological tissues based on optical absorption and thermoelastic expansion. One unique feature of PA imaging is that the amplitude of the PA signal reflects the local temperature. Here, we demonstrate a real-time temperature monitoring system that can evaluate thermal distribution during HIFU therapy. We have integrated a HIFU treatment system, a clinical ultrasound (US) machine, and a tunable laser system and have acquired real-time PA/US images of in vitro phantoms and in vivo animals during HIFU therapy without interference from the therapeutic US waves. We have also evaluated the temperature monitoring capability of the system by comparing the amplitude of PA signals with the measured temperature in melanoma tumor bearing mice. Although much more updates are required for clinical applications, the results show the promising potential of the system to ensure accurate and safe HIFU therapy by monitoring the thermal distribution of the treatment area.

Minimally invasive fetal interventions, such as those used for therapy of twin-to-twin transfusion syndrome (TTTS), require accurate image guidance to optimise patient outcomes. Currently, TTTS can be treated fetoscopically by identifying anastomosing vessels on the chorionic (fetal) placental surface, and then performing photocoagulation. Incomplete photocoagulation increases the risk of procedure failure. Photoacoustic imaging can provide contrast for both haemoglobin concentration and oxygenation, and in this study, it was hypothesised that it can resolve chorionic placental vessels. We imaged a term human placenta that was collected after caesarean section delivery using a photoacoustic/ultrasound system (AcousticX) that included light emitting diode (LED) arrays for excitation light and a linear-array ultrasound imaging probe. Two-dimensional (2D) co-registered photoacoustic and B-mode pulse-echo ultrasound images were acquired and displayed in real-time. Translation of the imaging probe enabled 3D imaging. This feasibility study demonstrated that photoacoustic imaging can be used to visualise chorionic placental vasculature, and that it has strong potential to guide minimally invasive fetal interventions.

Various optical imaging modalities with different optical contrast mechanisms have been developed over the past years. Although most of these imaging techniques are being used in many biomedical applications and researches, integration of these techniques will allow researchers to reach the full potential of these technologies. Nevertheless, combining different imaging techniques is always challenging due to the difference in optical and hardware requirements for different imaging systems. Here, we developed a multimodal optical imaging system with the capability of providing comprehensive structural, functional and molecular information of living tissue in micrometer scale. This imaging system integrates photoacoustic microscopy (PAM), optical coherence tomography (OCT), optical Doppler tomography (ODT) and fluorescence microscopy in one platform. Optical-resolution PAM (OR-PAM) provides absorption-based imaging of biological tissues. Spectral domain OCT is able to provide structural information based on the scattering property of biological sample with no need for exogenous contrast agents. In addition, ODT is a functional extension of OCT with the capability of measurement and visualization of blood flow based on the Doppler effect. Fluorescence microscopy allows to reveal molecular information of biological tissue using autofluoresce or exogenous fluorophores. In-vivo as well as ex-vivo imaging studies demonstrated the capability of our multimodal imaging system to provide comprehensive microscopic information on biological tissues. Integrating all the aforementioned imaging modalities for simultaneous multimodal imaging has promising potential for preclinical research and clinical practice in the near future.

Photoacoustic imaging (PAI) has traditionally relied on slow, fragile and expensive lasers as excitation sources. Advances in solid-state device technology have recently resulted in the development of a new class of high power light emitting diodes (LEDs) that can be used as fast, robust and cheap excitation sources for PAI. Here, we report the characterization and technical validation of a dual-mode multi-wavelength LED-based PAI/ultrasound imaging system (AcousticX) that has the potential to perform real-time in vivo imaging. LEDs operating with a pulse length of 70 ns and a repetition rate of up to 4 KHz at wavelengths of 690, 750, 810, 850 and 980 nm were tested. Ultrasound detection was made using a linear-array transducer with a center frequency of 10.05 MHz and a fractional bandwidth of 77%. We performed several systematic studies to evaluate the precision, penetration depth, spatial resolution, and sensitivity of the system. Measurements were made in tissue-mimicking phantoms to independently assess the impact of system variables on precision, including sample positioning and frame averaging. Temporal variation was assessed by repeated measurements over minutes, hours and days in the phantoms. Sensitivity to spectral differences was established by imaging the phantoms using all available multi-wavelength LEDs. The LED-based PAI system was able to detect small molecule dyes at 500 nM concentration at depth and to differentiate oxy- and deoxy-hemoglobin in mouse blood. Our studies indicate that LED-based PAI would be capable of providing real-time structural, functional and molecular imaging information up to depths of 2.5 cm in tissue.

We have developed a preclinical 3D imaging instrument integrating photoacoustic tomography and fluorescence (PAFT) addressing known deficiencies in sensitivity and spatial resolution of the individual imaging components. PAFT is designed for simultaneous acquisition of photoacoustic and fluorescence orthogonal projections at each rotational position of a biological object, enabling direct registration of the two imaging modalities. Orthogonal photoacoustic projections are utilized to reconstruct large (21 cm³ ) volumes showing vascularized anatomical structures and regions of induced optical contrast with spatial resolution exceeding 100 µm. The major advantage of orthogonal fluorescence projections is significant reduction of background noise associated with transmitted or backscattered photons. The fluorescence imaging component of PAFT is used to boost detection sensitivity by providing low-resolution spatial constraint for the fluorescent biomarkers. PAFT performance characteristics were assessed by imaging optical and fluorescent contrast agents in tissue mimicking phantoms and in vivo. The proposed PAFT technology will enable functional and molecular volumetric imaging using fluorescent biomarkers, nanoparticles, and other photosensitive constructs mapped with high fidelity over robust anatomical structures, such as skin, central and peripheral vasculature, and internal organs.

We introduce a dual imaging contrast agent for ultrasound and photoacoustic (US/PA) imaging. Glycol-chitosan-coated gold nanoparticles (GC-AuNPs) were previously applied to photoacoustic imaging of lymph node mapping and cancer cell visualization. Based on the enhanced cellular uptake, strong photoacoustic signal was observed. Using this nanoparticle platform, we developed a gas-generating nanoconstruct consisting of GC-AuNP and azide compounds that produce gas upon laser irradiation. The gas-generating nanoparticles have unique properties desired for ultrasound imaging. Compared with conventional ultrasound contrast agents (i.e., microbubbles), the novel contrast agent is superior in terms of nanometer-scale size and controlled gas generation. We showed that the developed nanoparticles were feasible as an ultrasound imaging contrast agent with signal enhancement caused by laser-triggered nitrogen gas generation. More importantly, the signal enhancement was controlled through the intensity and duration of excitation laser pulses. Overall, the discovery of the photocatalytic function of gold nanoparticles in the photolysis of azide enabled synthesis of the gas-generating contrast agent consisting of less than 50 nm diameter particles. Our results strongly suggested that gas-generating nanoparticles will allow ultrasound imaging of various diseases that conventional ultrasound contrast agents cannot reach and, therefore, detect, diagnose, and characterize. In addition, the plasmonic core of the developed nanoparticles serves as contrast agent for photoacoustic imaging and allow for further increase of diagnostic sensitivity of combined US/PA imaging. Broadly, the developed gas-generating nanoparticles may play critical role in various applications of light and sound ranging from diagnostic imaging to image-guided therapy.

We present a ultra-thin endoscopy system for optical resolution photoacoustic microscopy. The system is based on a silica capillary waveguide of two hundred microns of diameter. The silica tube acts as a multi-mode optical waveguide for the illumination, while the hollow core of the capillary carries a fiber-based optical hydrophone to detect the photoacoustic waves. Embedding the ultrasound detection within the device avoids the absorption of high-frequency ultrasound by the tissue and therefore removes any limitation on the insertion depth. To control the illumination at the distal tip of the capillary, a digital micromirror device modulates the amplitude of the optical wavefront which is coupled into the capillary. The DMD allows for fast calibration approaches to reach calibration and measurement times of a few seconds, as compared with current approaches limited to hours. We obtain optical-diffraction-limited images with full field illumination recording the intensity of a series of various speckle patterns produced by different configurations of the DMD at the input, with no wavefront shaping. The intensity fluctuations from shot to shot codes for the position at which it is measured. Computational methods based on correlation, pseudo-inverse and compressed sensing approaches are investigated and compared with raster-scanning an optimized focus for image reconstruction. To best of our knowledge, our approach provides the thinest endoscope head capable of obtaining optical resolution photoacoustic images.

Endoscopic ultrasonography (EUS) is an important clinical tool for the assessment of tumours in the abdominal cavity and guiding minimally invasive surgical procedures. However, one of the drawbacks of EUS is the lack of soft tissue contrast which can compromise its ability to visualise the microvasculature and thus delineate tu mour margins. Photoacoustic endoscopy (PAE) could potentially overcome this drawback as it can visualise the microvasculature with high contrast due to the strong optical absorption of haemoglobin. Previously, we have demonstrated an all-optical laboratory demonstrator PAE probe using standard bulk optical components. We now present a miniaturized flexible PAE probe with a 6 mm outer diameter and improved acoustic sensitivity capable of high resolution volumetric imaging in the forward-viewing configuration. The probe comprises a 1.5 mm diameter flexible coherent fibre bundle consisting of 18,000 fibre-optic cores with a transparent 80MHz Fabry-Pérot ultrasound sensor at its distal end. The FP sensor thus acts as a high density 2D array composed of 18,000 individually addressable ultrasound detectors, each with a bandwidth of 80MHz and an element size and centre-to-centre spacing of 40 µm and 44 µm, respectively. The pulsed excitation light is coupled through all of the fibre-optic cores from the proximal end, and the generated photoacoustic waves are detected in backward mode by sequentially interrogating the FP sensor through individual fibre optic cores. This configuration allows for very fine sampling of the photoacoustic waves and thus high lateral spatial resolution and image fidelity. This new approach to photoacoustic endoscopy offers significant advantages over previous piezoelectric based distal-end scanning probes. These include a forward viewing capability and a wider detection bandwidth and finer spatial sampling than achievable with piezoelectric receivers, a high degree of miniaturisation, no moving parts at the distal end and relatively simple and inexpensive fabrication. We have characterized the PAE probe in terms of its field of view, noise-equivalent pressure (NEP), and lateral and axial PSF. The lateral field of view is 5 mm and the NEP is 500 Pa over 20MHz bandwidth. The lateral spatial resolution is 50 µm at a depth of 1 mm decreasing to 150µm at a depth of 3 mm. The axial resolution is 33 µm over this depth range. The probe have also been evaluated using a variety of tissue phantoms and ex vivo tissues and shown to provide excellent high resolution 3D images of the vasculature. It is anticipated that this novel forward-viewing probe will provide new opportunities for the photoacoustic assessment of tumours in the liver and other abdominal organs, cancer in the GI tract and guiding minimally invasive procedures in abdominal surgery and foetal medicine.

Optical-resolution photoacoustic microscopy offers a specific contrast to optical absorption. The limiting penetration depth of current techniques due to scattering produced by tissues makes endoscopic approaches attractive for photoacoustic imaging deep inside biological structures. Conventional approaches generally involves mechanically raster scanning a focused spot over the sample and acquiring an acoustic signal for each spot. Here, we demonstrate that a full-field illumination approach with multiple known speckle patterns generated by a multimode fiber can also provide diffraction-limited optical-resolution photoacoustic images. As a proof of principle we experimentally image micro-structured test samples illuminated with reference speckle patterns measured during a calibration step. A digital micromirror device modulating the incident light coupled into a multimode fiber provides the different speckle patterns at the distal tip of the fiber where the sample is placed. We study and compare the performance in simulations and experiments of three different approaches; the first method is based on cross-correlation between the photoacoustic signal under multiple speckle illumination with the calibrated known speckle patterns, following approaches from ghost imaging. The second method is based on computing the pseudo-inverse of the reference matrix obtained from the calibration step. A third method based on compressed sensing exploits the sparsity of the sample achieving reconstructed images with a number of speckle realizations smaller than the number of speckle grains. Additionally, speckle-illumination-based photoacoustic microscopy provides a powerful framework for the development of novel reconstruction approaches, that can demand less computation time in case of compressed sensing approaches.

Intravascular (IV) imaging in percutaneous coronary interventions can be invaluable to treat coronary artery disease, to facilitate decision making and to guide stent placement. Intravascular ultrasound (IVUS) and optical coherence tomography (OCT) are both established IV imaging modalities. However, achieving contrast for specific structures such as lipid plaques can be challenging; with OCT, visualisation is typically limited to tissue depths less than 2 mm. Photoacoustic (PA) imaging provides contrast that is complementary to those of IVUS and OCT, and with previous demonstrations, visualisation of lipid plaques at depths greater than 4 mm has been achieved. In this study, we developed an intravascular PA probe that comprises a commercial OCT catheter and a high sensitivity miniature fibre optic ultrasound sensor with a Fabry-Pérot cavity. This probe, which can provide both PA imaging and OCT, had a maximum width of 1.2 mm. The PA excitation sources included both pulsed and modulated lasers at different wavelengths. The omni-directionality of the US sensor allowed for three-dimensional PA images. The PA-OCT probe was characterised using a series of resolution phantoms, including fine carbon fibres. It was found that with PA imaging, the probe can provide a lateral resolution better than 25 µm and an axial resolution better than 100 µm at the optical focus. Co-registered PA and OCT images of blood vessels ex-vivo with stents and lipid injections were acquired. We conclude that PA imaging with OCT catheters is viable and that it has strong potential to guide clinical interventions.

Forward-looking photoacoustic imaging (PAI), with the potential to capture three-dimensional (3D) images of tissue structures and distinguish their characteristics, is desired in order to perform more efficient catheter interventions for complicated diseases, such as chronic total occlusion. However, few studies have reported forward-looking 3D PAI. In this study, we experimentally demonstrated the forward-looking 3D PAI in vitro with our optical-resolution PAI system. To construct a 3D image, an optical-resolution PAI with a piezoelectric fiber actuator is used. The actuator is attached to the fiber and oscillates it to spirally control the direction of a laser. For each laser emission, a photoacoustic signal that is generated at each laser line (or volume) is received by a capacitive micromachined ultrasound transducer (CMUT). As the laser directions and ultrasound time of flight are known, the positions of the laser-induced ultrasound sources are determined and therefore, the 3D image can be constructed. In our in vitro experiments, a laser pulse was emitted from multi-mode fiber illuminated carbon rod samples with diameters of 0.5 mm. The rod samples formed grid structures with a spacing of 1.0 mm. The distance between the CMUT receiver and the samples was about 10 mm. The structures in the resultant images, created with a rendering technique, were visualized with a signal-to-noise ratio of over 10. In the presentation, detailed results taken by the forward-looking 3D PAI system will be described.

Colorectal cancer is the second leading cause of cancer death in the United States. According to American Cancer society, the overall lifetime risk of developing colorectal cancer is about 4.7% for men and 4.4% for women. We have developed a rigid, endoscopic photoacoustic microscopy (PAM) probe for imaging of in vivo human colorectal cancers. In order to accommodate colon sections with different size (typically from 50 to 70mm), our 10mm diameter rigid probe uses an off-optical-axis, external mechanical scanning mechanism with a speed of 35deg/s instead of an on-optical-axis, internal mechanical scan mechanism. 532-nm pulsed laser light enters the ridged probe through a photonic crystal single mode fiber before it is collimated and refocused by a water-immersed objective lens onto the colon surface. A focused ultrasound ring transducer (40.5 MHz, 6.5mm focal length) receives photoacoustic signal from chromophores excited by laser beam. Imaging system performance specifications including resolution (6μm) and signal-to-noise ratio are quantified and verified from phantom imaging tests. Ex vivo human colon samples are studied to reveal microscopic features of normal colon, benign polyps, adenocarcinoma and cancer.

Photoacoustic imaging of oxygen saturation (sO2) in deep tissue has broad preclinical and clinical applications. Because the magnitude of photoacoustic signal is proportional to the product of optical absorption coefficient and local fluence, quantitative imaging of oxygen saturation usually requires knowledge of the local optical fluence. Especially in deep biological tissue, wavelength dependent optical attenuation of biological tissue presents a challenge to measure the absolute oxygen saturation. Here, we present a new method to measure the sO2 without knowing the local fluence. We measure photoacoustic signals at different wavelengths and different sO2 values. Because the optical fluence at each optical wavelength does not change with a certain sO2, the unknown optical fluence at one wavelength can be cancelled via taking the ratio between two photoacoustic amplitudes at the same optical wavelength but different sO2 values. Three wavelengths, i.e. 760,798,820nm, have been utilized to quantify the absolute sO2. Compared with conventional two-wavelength method, the proposed three wavelength dynamic sO2 method has a better performance on the estimation of absolute sO2. Preliminary phantom experiments have validated the feasibility of this method. This new method enables calibration-free quantitative imaging of absolute sO2 in deep biological tissue.

Quantification of photoacoustic (PA) images is one of the major challenges currently being addressed in PA research. Tissue properties can be quantified by correcting the recorded PA signal with an estimation of the corresponding fluence. Fluence estimation itself, however, is an ill-posed inverse problem which usually needs simplifying assumptions to be solved with state-of-the-art methods. These simplifications, as well as noise and artifacts in PA images reduce the accuracy of quantitative PA imaging (PAI). This reduction in accuracy is often localized to image regions where the assumptions do not hold true. This impedes the reconstruction of functional parameters when averaging over entire regions of interest (ROI). Averaging over a subset of voxels with a high accuracy would lead to an improved estimation of such parameters. To achieve this, we propose a novel approach to the local estimation of confidence in quantitative reconstructions of PA images. It makes use of conditional probability densities to estimate confidence intervals alongside the actual quantification. It encapsulates an estimation of the errors introduced by fluence estimation as well as signal noise. We validate the approach using Monte Carlo generated data in combination with a recently introduced machine learning-based approach to quantitative PAI. Our experiments show at least a two-fold improvement in quantification accuracy when evaluating on voxels with high confidence instead of thresholding signal intensity.

Photoacoustic (PA) signals carry information of the absorbing chromophores and the light distribution in imaged samples. The dependence of light distribution with optical wavelength affects the accuracy in PA chromophore quantification. Oxygen saturation (sO2) estimations maybe inaccurate in-depth due to the lack of proper fluence compensation. We propose the use of the PA radiofrequency spectral slope (SS) to generate a frequency filter to match the fluence across optical wavelengths. The SS is calculated from the ratio of the radiofrequency power spectra at the selected optical wavelengths. The SS relays information about the absorbers’ size and the light distribution. At the imaged optical wavelengths of the same sample, the SS-estimated size should in principle remain unchanged. This suggests that any changes in the measured SS as a function of optical wavelength can be attributed to the light distribution. A frequency filter can be designed from the computed SS and applied to compensate the PA images. A 5mm phantom consisting of fresh blood, intralipid and gelatin was imaged using the VevoLAZR system at 750 and 850nm. A square sliding window sized 1.6mm with 80% overlap is applied to segment the generated radiofrequency signals. The designed ultrasound filter was applied to each segmented signal. As a result, the fluence-induced depth fluctuations in the sO2 estimations dropped from 9.49%/mm to 1.83%/mm. This will allow for more accurate sO2 estimates that are less depth dependent. The approach provides a new perspective for fluence compensation which can aid in improving chromophore quantification using PA imaging.

Optoacoustic diagnostics is based on detection and analysis of optoacoustic waves induced in tissues. It may find a number of important clinical applications in large populations of patients such as diagnostics of cerebral hypoxia, circulatory shock, etc. Recently, we proposed Nano-Pulse Laser Therapy (NPLT) which utilizes short optical pulses (typically, shorter than hundreds of nanoseconds) to generate optoacoustic waves in tissues upon stress-confined irradiation. It is well known that continuous wave low-level near-infrared light can be used for therapy/photobiomodulation to stimulate, repair, regenerate, and protect injured tissue. In the past few years, new works emerged on therapeutic effects of low-intensity ultrasound waves. The NPLT consists of irradiating tissue by both lowlevel light and optoacoustic waves/ultrasound that combines merits of low-level light and ultrasound therapies. In this work we propose optoacoustic theranostics that can be used for diagnostics, optoacoustic therapy/NPLT, and monitoring of therapeutic response during and after therapy. We developed and built pulsed, tunable, near infrared (680-1064 nm), fiber-coupled systems for optoacoustic theranostics and tested them in rats with traumatic brain injury (TBI). Low energy pulses were used for optoacoustic monitoring of cerebral blood oxygenation, while higher energy pulses were used for the NPLT. Our studies show that TBI results in cerebral hypoxia, while a 5-minute transcranial application of NPLT significantly reduces negative effects of TBI as assessed by vestibulomotor, cognitive, and immunofluorescence tests. The obtained results suggest that the optoacoustic theranostics may be used for diagnostics and management of TBI and other disorders.

Beta-amyloid (Aβ) deposition and vascular dysfunction are important contributors to the pathogenesis in Alzheimer’s disease (AD). However, the spatio-temporal relationship between an altered oxygen metabolism and Aβ deposition in the brain remains elusive. Here we provide novel in-vivo estimates of brain Aβ load with Aβ-binding probe CRANAD-2 and measures of brain oxygen saturation by using multi-spectral optoacoustic imaging (MSOT) and perfusion imaging with magnetic resonance imaging (MRI) in arcAβ mouse models of AD. We demonstrated a decreased cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRO₂) in the cortical region of the arcAβ mice compared to wildtype littermates at 24 months. In addition, we showed proof-of-concept for the detection of cerebral Aβ deposits in brain from arcAβ mice compared to wild-type littermates.

Photoacoustic computed tomography (PACT) is a non-invasive imaging technique offering high contrast, high resolution, and deep penetration in biological tissues. We report a photoacoustic computed tomography (PACT) system equipped with a high frequency linear array for anatomical and functional imaging of the mouse whole brain. The linear array was rotationally scanned in the coronal plane to achieve the full-view coverage. We investigated spontaneous neural activities in the deep brain by monitoring the hemodynamics and observed strong interhemispherical correlations between contralateral regions, both in the cortical layer and in the deep regions.

Understanding brain functionality remains an arduous task despite decades of research employing a wide array of neural recording and imaging techniques. Genetically encoded calcium indicators (GECIs) are powerful and versatile tools for tagging fast neural activity in a large number of neurons with excellent spatial localization. Here we demonstrate that functional optoacoustic neuro-tomography (FONT) enables non-invasive imaging of sensory-evoked activity in GCaMP6-expressing mouse brain in vivo, thus holding promise for large-scale neural recording at penetration depths and spatio-temporal resolution scales not covered with the existing neuroimaging techniques. The FONT imaging system has volumetric temporal resolution of 10msec and spatial resolution of 150µm across an effective field of view of 2cm3 covering an entire mouse brain. However, the effective depth in the current study was restricted to the cortical areas due to the limited penetration of light at the visible wavelengths used for the GCaMP6 excitation. The stimulation protocol involving somatosensory electrical hindpaw stimulation was specifically designed to minimize the influence of hemodynamic changes indirectly associated with neuronal activity, which are much slower than the GCaMP-related calcium transients. Rapid optoacoustic signal transients were observed in the activated brain regions but not inside major blood vessels, thus allowing for a clear differentiation of the calcium-related activity from the underlying hemodynamic responses. Our study is the first to examine fast volumetric optoacoustic signatures of GECIs non-invasively in living mice, further showing that the corresponding changes of their fluorescence are directly related to the optoacoustic responses.

Crohn’s disease (CD) is a chronic autoimmune disease of the intestinal tract affecting 700,000 people in the United States. The pathology of CD is characterized by obstructing intestinal strictures due to inflammation (with high levels of hemoglobin), fibrosis (with high levels of collagen), or a combination of both. The accurate characterization of the intestinal strictures is critical, as the fibrotic intestinal strictures have to be removed surgically. Currently, there is no imaging modality that can differentiate the fibrotic and inflammatory strictures. Standard diagnosis by endoscopic biopsy suffers from the post-procedure complications, and limited sampling locations and depth. Combining the optical spectroscopy and ultrasound (US) imaging, photoacoustic (PA) imaging is an ideal tool for resolving the molecular components of the intestinal strictures. This study investigates the feasibility of differentiating the fibrotic and inflammatory intestinal strictures using PA-US parallel imaging in a rat model in vivo. A linear US array was used to acquire US and PA imaging transcutaneously. PA imaging with endoscopic and transcutaneous illumination was attempted in 12 and 10 animals, respectively. The PA images were acquired at 750, 850 and 1310 nm. The PA pixel intensities within the intestinal stricture regions were quantified. Blood oxygenation, as well as the relative ratio between the total hemoglobin and collagen contents, were derived. Significant differences were observed between the fibrotic and inflammatory strictures (p<0.05). The penetration of the noninvasive transcutaneous PA imaging was also tested in human subjects using a low-frequency probe. Penetration as deep as 6 cm was achieved.

In the current form of multi-parametric photoacoustic microscopy (PAM), imaging hemoglobin concentration and blood flow speed requires dense sampling. Moreover, large-scale recording beyond the focal zone of ultrasonic transducer requires time-consuming mechanical scan of the optical-acoustic dual foci. Thus, the image acquisition time of multi-parametric PAM has been severely limited by the laser repetition rate and the focal diameter of the transducer. Here, we report an ultrahigh-speed multi-parametric PAM with 1.2-MHz A-line rate for simultaneous real-time imaging of hemoglobin concentration, blood oxygenation, and blood flow in the mouse brain. Capitalizing on the pronounced stimulated Raman scattering in pure silica-core polarization-maintaining single-mode optical fibers, a dual-wavelength (532 and 558 nm) nanosecond laser with 1.2-MHz pulse repetition rate has been developed. Using a weakly focused ultrasonic transducer, we have achieved real-time acquisition of multi-parametric PAM images at a frame rate of 2.2 Hz over the 250-μm-diameter acoustic focal zone. By employing optical-mechanical hybrid scan, 25 dual-wavelength B-scans can be acquired simultaneously within one mechanical-scan trip, leading to a 25-fold improvement of imaging speed. As a result, the imaging frame rate is improved from 0.08 Hz in the conventional multi-parametric PAM to 2.2 Hz. The utility of this new PAM technology has been demonstrated in a mouse model of epilepsy by studying the dynamic neurovascular uncoupling during status epilepticus.

Ischemic reperfusion injuries (IRIs) occur after blood returns to a tissue or organ after a period without oxygen or nutrients, which causes an inflammatory response leading to heterogeneous scarring of the nearby tissue and vasculature. This is associated with long-term decreases blood flow, and necrosis. Although most commonly associated with heart attacks and strokes, IRIs are also a side effect of organ transplants, when the organ is reperfused in the recipient’s body after being transported from the donor to the transplant hospital. Currently, the optimal method of monitoring for IRI is limited to biopsies, which are invasive and poorly monitor the spatial heterogeneity of the damage. To non-invasively identify changes in kidneys, the left renal artery in mice (n=3) was clamped for 45 minutes to create an IRI event. Both kidneys of each animal were monitored using photoacoustics (PA) with the VevoLAZR system (Fujifilm-VisualSonics, Toronto) three, four and eight weeks after surgery. IRI-treated kidneys show increased picosirius red staining, indicative of collagen (0.601 vs 0.042, p < 0.0001), decreased size as assessed by cross-sectional area (7.8 mm² vs 35.9 mm² , p < 0.0001), and decreased oxygen saturation (sO2; 62% vs 77%, p = 0.02). Analysis of the photoacoustic data shows that a two-point metric, the 715:930 nm ratio of the whole kidney (1.05 vs 0.57, p = 0.049) and the optical spectral slope (OSS) (0.8 * 10^-3 vs 3.0 * 10^-3, p = 0.013) are both able to differentiate between IRI-treated and healthy kidneys. These data suggest that photoacoustics can be used as a non-invasive method to observe in vivo changes in the kidney due to IRI.

Acousto-optic imaging is a multi-modal imaging technique where coherent light diffusing in a complex medium is ‘tagged’ over time by a ballistic ultrasound pulse of frequency ωus. The photons which paths cross with the ultrasound pulse undergo the acousto-optic effect, resulting in the frequency shift of ωus that can be selectively detected using heterodyne interferometry. Since the ultrasounds propagate at a known velocity, a time-to-space map of the tagged photons results in an image I(x, z), where x is the lateral direction and z the depth direction of the diffuse medium. Images at propagation depths much greater than the average mean free path, typically ~1mm in biological tissue, can be obtained. In most images obtained so far, the ultrasounds are focused line after line to recover an image, and therefore limited by the probe emission rate which is ~1-10 KHz depending on the probe size and the acoustic pulse power. Therefore, in order to acquire acoustic images at frame rates greater than 1 Hz for ‘direct visualization’ of the system under study, it is crucial to minimize the number of individual acquisitions necessary to reconstruct an image. Here, we present an alternative probe configuration where plane waves emitted at various angles are used rather than focused waves to tag the diffuse light. This approach, first proposed by P.Kuchment and L.Kunyansky (2010), is similar to X-ray tomography since the image information is contained in the various angular scans performed for one acquisition. Because the piezo-elements on the acoustic probe are non-isotropic emitters, the angular scan is typically limited to +/20 degrees, which is sufficient to recover information and can be improved using more than one probe. An inversion algorithm based on inverse Radon-transform is than used to reconstruct the image

Vulnerable plaque is the one of the leading causes of cardiovascular disease occurrence. However, conventional intravascular imaging techniques suffer from difficulty in finding vulnerable plaque due to limitation such as lack of physiological information, imaging depth, and depth sensitivity. Therefore, new techniques are needed to help determine the vulnerability of plaque, Thermal strain imaging (TSI) is an imaging technique based on ultrasound (US) wave propagation speed that varies with temperature of medium. During temperature increase, strain occurs in the medium and its variation tendency is depending on the type of tissue, which makes it possible to use for tissue differentiation. Here, we demonstrate laser-induced photo-thermal strain imaging (pTSI) to differentiate tissue using an intravascular ultrasound (IVUS) catheter and a 1210-nm continuous-wave laser for heating lipids intensively. During heating, consecutive US images were obtained from a custom-made phantom made of porcine fat and gelatin. A cross correlation-based speckle-tracking algorithm was then applied to calculate the strain of US images. In the strain images, the positive strain produced in lipids (porcine fat) was clearly differentiated from water-bearing tissue (gelatin). This result shows that laser-induced pTSI could be a new method to distinguish lipids in the plaque and can help to differentiate vulnerability of plaque.

Imaging and identifying early metastases is, to this day, not an easy task: using MRI is expensive and ultrasound is not able to discriminate healthy and diseased tissues. Coupling ultrasound imaging to acousto-optic imaging could be a solution: the additional optical contrast would suppress the indetermination on the origin of the biological tissue. Acousto-optic imaging is a multi-wave technique which localizes light in highly scattering medium thanks to an acoustic wave: the acousto-optic effect creates frequency-shifted light, carrying local information about the insonified volume. The central challenge of acousto-optic imaging is the detection of the frequency-shifted light, because there are only very few modulated photons and they create a speckle pattern. We choose to explore the detection by spectral filtering using the spectral hole burning phenomenon in a rare earth doped crystal [1]. This filtering technique is intrinsically immune to speckle decorrelation and therefore well adapted to in vivo imaging. We use a YAG crystal doped with thulium ions under a magnetic field which increases the lifetime of the spectral hole from 10ms to more than a minute. We have undertaken a spectroscopic study to optimize the hole preparation sequence. We will present the first acousto-optic images achieved with a long-lived spectral filter in Tm:YAG, in a scattering medium. [1] Li, Y., Zhang, H., Kim, C., Wagner, K. H., Hemmer, P., & Wang, L. V. (2008). Applied Physics Letters, 93(1), 011111.

The resolution of photoacoustic imaging of blood vasculature is limited at depth by the acoustic diffraction limit. In this work, we propose to exploit the fluctuations caused by flowing absorbers (such as red blood cells in blood vessels) to perform photoacoustic imaging beyond the acoustic diffraction limit: following the super-resolution optical fluctuation imaging (SOFI) method, we analyze the n-th order statistics from the temporal photoacoustic fluctuations induced by flowing particles. We performed a proof-of-concept experiment in a 5-channel microfluidic silicon-based circuit flown with a suspension of RBC-mimicking 10 µm red-tainted polymer spheres (Microparticles, GmbH, Berlin, Germany). The sample was illuminated with a 5 ns pulsed ND-YAG laser (532 nm, Innolas, Krailling, Germany) with a fluence of 3 mJ/cm^2 and imaged at a 20 Hz rate using a L22-8v probe (128 elements, Verasonics, Redmond, WA, USA) coupled to a Verasonics Vantage 256 ultrasound scanner. Whereas the resolution of conventional photoacoustic imaging was too low to resolve individual channels, the nth order statistical analysis of the photoacoustic fluctuations provided images with a resolution enhancement scaling as n^{1/2}, in agreement with the SOFI theory and with numerical simulations. As opposed to our previous work which exploited speckle-based photoacoustic fluctuations to increase the resolution, the approach proposed here based on sample fluctuations do not require coherent light and can be readily applied to conventional photoacoustic imaging setup. Furthermore, in order to discard the oscillatory behavior of the photoacoustic point-spread-function, we extended in this work the SOFI theory to complex-valued photoacoustic images.

Conventional photoacoustic computed tomography (PACT) images the spatial distribution of optical absorption, which is approximated as an isotropic optical property. The optical absorption of many biological tissues, however, is anisotropic. This anisotropy, known as dichroism or diattenuation, encodes rich information about molecular conformation and structural alignment. Here we report a novel imaging method called dichroism-sensitive PACT (DS-PACT). Using a lock-detection strategy, our method can measure the amplitude of tissue’s dichroism and the orientation of the optic axis of uniaxial dichroic tissue, even at a depth of 3.25 transport mean free paths. We experimentally demonstrated DS-PACT by imaging plastic polarizers and ex vivo bovine tendons deep inside scattering media. Our method extends the functionality of PACT to include a new capability, imaging tissue absorption anisotropy.

In photoacoustic imaging, optically generated acoustic waves transport the information about embedded structures to the sample surface. Usually, short laser pulses are used for the acoustic excitation. Acoustic attenuation increases for higher frequencies, which reduces the bandwidth and limits the spatial resolution. One could think of more efficient waveforms than single short pulses, such as pseudo noise codes, chirped, or harmonic excitation, which could enable a higher information-transfer from the samples interior to its surface by acoustic waves. We used a linear state space model to discretize the wave equation, such as the Stoke’s equation, but this method could be used for any other linear wave equation. Linear estimators and a non-linear function inversion were applied to the measured surface data, for onedimensional image reconstruction. The proposed estimation method allows optimizing the temporal modulation of the excitation laser such that the accuracy and spatial resolution of the reconstructed image is maximized. We have restricted ourselves to one-dimensional models, as for higher dimensions the one-dimensional reconstruction, which corresponds to the acoustic wave without attenuation, can be used as input for any ultrasound imaging method, such as back-projection or time-reversal method.

The STORM and PALM techniques developed in the past decade in optics allow resolving sub-wavelength structures based on localization. Here, we demonstrate that localization can be used to go beyond the diffraction limit in acoustic resolution photoacoustic imaging. To this end, a proof-of-concept photoacoustic localization experiment was conducted. A silicone sample containing five parallel microchannels (channel’s width is 40μm; center-to center distance equals 180μm), fed with a water suspension of 10μm red coated microbeads at a constant flow rate, was exposed to 5ns laser pulses (wavelength=532nm, fluence=3.0 mJ/cm2). At each laser pulse the microbeads produced a photoacoustic response that was then detected by a linear US array (128 elements, L22-8v, Verasonics, USA) connected to an acquisition device (High Frequency Vantage 256, Verasonics, USA). The design of the microfluidic circuit and the concentration of microbeads ensured sparse but random distribution of microbeads at any moment. The photoacoustic data was processed by a delay-and-sum algorithm, whose output was correlated with the system’s PSF function to obtain the position of microbeads at each laser shot. These positions were accumulated onto the localization grid providing a super-resolved image of the micro-fluidic circuit. Although being indistinguishable in a conventional US image, the microchannels dimensions and position were accurately reconstructed on the localization grid with 34.8±1.3μm for the channel’s width and 179±2.5μm for the center-to-center distance. As the first demonstration of super-localization in photoacoustics, these results constitute the first step towards imaging of red blood cells at depth beyond the acoustic diffraction limit.

Diffraction causes blurring of high-resolution features in images and has been traditionally associated to the resolution limit in light microscopy and other imaging modalities. The resolution of an imaging system can be generally assessed via its point spread function, corresponding to the image acquired from a point source. However, the precision in determining the position of an isolated source can greatly exceed the diffraction limit. By combining the estimated positions of multiple sources, localization-based imaging has resulted in groundbreaking methods such as super-resolution fluorescence optical microscopy and has also enabled ultrasound imaging of microvascular structures with unprecedented spatial resolution in deep tissues. Herein, we introduce localization optoacoustic tomography (LOT) and discuss on the prospects of using localization imaging principles in optoacoustic imaging. LOT was experimentally implemented by real-time imaging of flowing particles in 3D with a recently-developed volumetric optoacoustic tomography system. Provided the particles were separated by a distance larger than the diffraction-limited resolution, their individual locations could be accurately determined in each frame of the acquired image sequence and the localization image was formed by superimposing a set of points corresponding to the localized positions of the absorbers. The presented results demonstrate that LOT can significantly enhance the well-established advantages of optoacoustic imaging by breaking the acoustic diffraction barrier in deep tissues and mitigating artifacts due to limited-view tomographic acquisitions.

Photoacoustic signals are typically generated using Q-switched Nd:YAG pumped OPO systems, as they can provide the necessary nanosecond pulse durations with mJ pulse energies required for photoacoustic tomography. However, these sources are often bulky, require external water cooling and regular maintenance and provide low pulse repetition frequencies (PRF<100Hz) thus limiting image frame rate. Fibre lasers can overcome these limitations and additionally offer much greater flexibility in their temporal output characteristics (e.g. pulse shaping and duration). Although fibre lasers have been used in optical-resolution photoacoustic microscopy, they have found limited application in widefield photoacoustic tomography (PAT) due to the relatively low pulse energy (<1mJ) provided by commercial systems. These low pulse energies are a consequence of small core diameter (<25m) fibres required to achieve a high beam quality. However, for widefield PAT, high beam quality is not a requirement and therefore fibre lasers with larger core diameters (>100m) can be used, enabling significantly higher pulse energies (>10mJ) to be achieved. A novel compact fibre laser which uses a custom drawn large core diameter fibre (100m) to provide high pulse energies (15mJ) and variable PRFs (100Hz-1kHz) and pulse durations (10-400ns) has been developed and evaluated. The fibre laser was combined with a fast Fabry Perot (FP) scanner in order to evaluate its suitability for PAT of biological tissue. The high PRF (>400Hz) of the laser has allowed tomographic images of the microvasculature of the palm of a hand to be obtained in less than one second, significantly quicker than previously achieved with a FP scanner. In addition, the ability to arbitrarily vary the temporal shape of the laser pulse offers new opportunities for controlling the acoustic frequency content of the photoacoustic signal in order to optimise penetration depth and image resolution. For example, the laser pulse duration can be increased in order to shift the acoustic frequency components to lower frequencies which are less attenuated by tissue acoustic absorption and thus improve SNR. To investigate these concepts, a tissue mimicking phantom was imaged for a range of tailored excitation pulses (e.g. different pulse durations, trains of pulses) and their effect on the contrast to noise ratio (CNR) and image resolution observed. A novel compact fibre laser, able to provide higher pulse energies (>10mJ) than previously reported and with enhanced functionality is presented. It is demonstrated that fibre lasers are a viable alternative to standard Q-switched lasers for photoacoustic tomographic applications in medicine and biology.

We introduce novel bias-sensitive piezoelectric transducer (relaxor) arrays for 3D photoacoustic imaging. A 64×64 element relaxor array using a crossed electrode or Top-Orthogonal to Bottom-Electrode (TOBE) wiring configuration is used to receive photoacoustic data from two crossed wires with 17.8 um diameters in an intralipid medium. A 3D image was then reconstructed. By biasing a column and receiving along a row, individual elements can be isolated for readout of signals from all elements using bias-switching-based multiplexing. We demonstrate a reconstruction technique called Hadamard-bias encoding with dynamic receive beamforming in which, rather than using a single column to index an array element, multiple columns are biased simultaneously allowing for more receiving elements and substantially improved SNR. Ongoing work will investigate in vivo imaging. The proposed arrays represent a new paradigm for 3D photoacoustic imaging.

Fabry-Pérot (FP) polymer film sensors exhibit small element sizes, high acoustic sensitivity, transparency and flat frequency response to enable high resolution 3D photoacoustic (PA) imaging in backward mode. However, conventional raster scan interrogation can result in slow data acquisition (several min for 3D images) compared to parallelized piezoelectric detector arrays. To address this limitation, parallelization using a camera-based readout of FP sensors is investigated. This approach requires the optical thickness of the polymer spacer to be sufficiently uniform over the scan area to obtain high acoustic sensitivity for all active elements. Since the deposition of passive polymer layers with sufficient homogeneity of thickness is challenging, the use of electro-optically (EO) or piezoelectric (PE) tunable polymer film spacers is investigated. The spacers are sandwiched between two dielectric mirrors and transparent electrodes to form an FP sensor. In this work, spin coated guest-host systems consisting of EO chromophores (2-methyl-4-nitroaniline) embedded in a PMMA matrix, and thermally evaporated PE film spacers (PVDF) were examined. Both systems were electrically poled using a corona discharge. The optical transfer function, the transmission spectrum of the excitation passband from 600 nm to 1100 nm and the tuning range of the FP sensors were determined. Furthermore, the detection of PA waves was demonstrated. Tunable FP sensors in conjunction with camera-based interrogation techniques have the potential to provide 3D image acquisition times on the order of seconds.

Photoacoustic remote sensing (PARS) microscopy is a novel photoacoustic modality which provides non-contact reflection-mode operation within optical penetration regimes. It has thus far demonstrated exceptional in vivo imaging capabilities with high signal-to-noise (greater than 70dB) and sub-cellular lateral resolution (on the order of 600 nm). Moreover, being non-contact opens a wide range of previously inaccessible imaging targets where acoustic coupling to the sample is impractical. One disadvantage of the technique however is the lack of time-gated depth discrimination which has long been a staple of more conventional photoacoustic methods. Rather, depth-resolving ability has been solely defined by the optical section provided by the primary objective lens. Here a pulsed short-wave infrared low-coherence detection beam in a spectral-domain OCT system is used to probe depth-resolved reflectivity before and immediately after visible pulsed excitation. A difference image between these A-scans reveals signals with optical absorption contrast. Simulations based on recently-developed time-domain modeling of low-coherence PARS reflectivity changes is used to generate software-phantom images. We used a 1310-nm ns-pulsed interrogation source with 45nm linewidth, along with a 532-nm ns-pulsed excitation beam. The effects of various material and apparatus parameters are discussed along with extensive analytical and simulation results. These showcase the potential capabilities of the approach, such as depth resolved spectral unmixing (with oxygen saturation) and discrimination of blood vessels in highly scattering media, along with foreseeable limitations and potential implementation issues.

Optical imaging modalities are commonly characterized by rapid acquisition rates, enabling real-time feedback. Photoacoustic Remote Sensing (PARS) microscopy takes advantage of intensity reflectivity modulations induced through large photoacoustic initial pressures to provide optical absorption imaging contrast. The PARS signals are characterized by short time-domain behavior independent of time-gated effects such as acoustic propagation to a detector. Here, improved imaging rates are demonstrated. This is accomplished by introducing an analog peak detection circuit, which reduces data bandwidth requirements, and by employing a high repetition rate fiber laser. These additions enable voxel scan rates in the megahertz range. High quality real-time captures, orders of magnitude faster than previous PARS systems, are presented.

Medical phantoms with accurate tissue-mimicking properties and anatomical structures are vital for evaluation of imaging system performance, calibration of medical devices, and training medical staff in techniques such as ultrasonography. Tissue-mimicking phantoms based on Agar/gelatin and polyvinyl alcohol (PVA) materials have been developed, however, they are fragile, exhibit dehydration problems and cannot reproduce complex structures. There is an ongoing need for novel tissue-mimicking materials and phantom fabrication methods. Three-dimensional (3D) printing additive technology allows direct formation of the object layer by layer and provides freedom in object design. Various 3D printing materials have been employed from metal and ceramics to resins and polymers. Unfortunately, commercially available 3D printing materials don’t have suitable physical properties to mimick tissue. In this work, we describe the development of a novel 3D printing technology based on an original soft tissue-mimicking material, Gel Wax, a mixture of polymer and mineral oil. This material is soft, optically and acoustically clear and does not dehydrate. The optical, acoustic and mechanical properties of the material can be tailored to mimic biological tissues by embedding titanium dioxide, dyes, glass microspheres, or paraffin wax. Gel Wax cannot be made into a conventional filament and we designed a novel 3D printing techniques. We demonstrate hippocampus models directly printed using our proposed Gel Wax 3D printer. This technology holds a great promise for fabricating patient-specific medical phantoms. This opens the door for 3D printing to provide new affordable medical phantoms to enable widespread application in biomedical field.

Phantoms are crucial for developing photoacoustic imaging systems and for training practitioners. Advances in 3D printing technology have allowed for the generation of detailed moulds for tissue-mimicking materials that represent anatomically realistic tissue structures such as blood vessels. Here, we present methods to generate phantoms for photoacoustic and ultrasound imaging based on patient-specific anatomy and mineral oil based compounds as tissue-mimicking materials. Moulds were created using a 3D printer with fused deposition modelling. Optical and acoustic properties were independently tuned to match different soft tissue types using additives: inorganic dyes for optical absorption, TiO2 particles for optical scattering, paraffin wax for acoustic attenuation, and solid glass spheres for acoustic backscattering. Melted mineral oil compounds with additives were poured into the 3D printed moulds to fabricate different anatomical structures. Optical absorption and reduced scattering coefficients across the wavelength range of 400 to 1600 nm were measured using a spectrophotometer with an integrating sphere, and inverse adding-doubling. The acoustic attenuation and speed-of-sound were measured in reflection mode using a 10 MHz transducer. Three phantoms were created to represent nerves and adjacent blood vessels, a human placenta obtained after caesarean section, and a human heart based on an MRI image volume. Co-registered multi-wavelength photoacoustic and ultrasound images were acquired with a system that comprised a clinical ultrasound imaging scanner, an optical parametric oscillator, and linear-array ultrasound imaging probes. We conclude that mineral oil based compounds can be well suited to create anatomically-realistic phantoms for photoacoustic and ultrasound imaging using 3D printed moulds.

Photoacoustic Imaging (PAI) is an emerging technology with strong potential for broad clinical applications from breast cancer detection to cerebral monitoring due to its ability to compute maps of blood oxygen saturation (SO₂) distribution in deep tissues using multispectral imaging. However, no well-validated consensus test methods currently exist for evaluating oximetry-specific performance characteristics of PAI devices. We have developed a phantombased flow system capable of rapid SO₂ adjustment to serve as a test bed for elucidation of factors impacting SO₂ measurement and quantitative characterization of device performance. The flow system is comprised of a peristaltic pump, membrane oxygenator, oxygen and nitrogen gas, and in-line oxygen, pH, and temperature sensors that enable real-time estimation of SO₂ reference values. Bovine blood was delivered through breast-relevant tissue phantoms containing vessel-mimicking fluid channels, which were imaged using a custom multispectral PAI system. Blood was periodically drawn for SO₂ measurement in a clinical-grade CO-oximeter. We used this flow phantom system to evaluate the impact of device parameters (e.g.,wavelength-dependent fluence corrections) and tissue parameters (e.g. fluid channel depth, blood SO₂, spectral coloring artifacts) on oximetry measurement accuracy. Results elucidated key challenges in PAI oximetry and device design trade-offs, which subsequently allowed for optimization of system performance. This approach provides a robust benchtop test platform that can support PAI oximetry device optimization, performance validation, and clinical translation, and may inform future development of consensus test methods for performance assessment of photoacoustic oximetry imaging systems.

We recently described a technique to monitor heparin anticoagulation therapy in real-time using methylene blue and photoacoustic imaging. The photoacoustic signal of methylene blue was significantly amplified in the presence of heparin, but the exact mechanism underlying this novel photoacoustic behavior remains unclear. Here, we showed that the signal amplification was due to the aggregation of methylene blue. Methylene blue formed different aggregates in water and phosphate buffered saline (PBS). In water, the absorbance maximum of methylene blue with heparin from 0 to 3 U/mL blue shifted from 660 to 570 nm and the corresponding fluorescence intensity decreased 6-fold, which indicated the methylene blue aggregated from monomer to dimer and eventually to high order aggregates. Furthermore, the corresponding 0.04 ppm chemical shift of the proton in the phenothiazine ring of methylene blue from the nuclear magnetic resonance spectrum suggested the electron delocalization and self-aggregation of methylene blue. The coupling of methylene blue molecules results in extra vibrational relaxations within the split exciton states, and this causes enhanced photoacoustic signal. In PBS, we observed the aggregation of methylene blue/heparin complex using transmission electron microscopy (size=150.5 nm), but the absorbance maximum reversed back to 660 nm. This suggested the methylene blue formed monomer bound to heparin—the heparin could not self-aggregate due to electrostatic repulsive forces. The methylene blue bound monomers experienced less degree of freedom than free monomers and therefore caused excess photoacoustic signal.

Periodontal probing is a useful diagnostic tool to estimate the periodontal pocket depth and assess the status of periodontal disease, but is limited by systematic and random errors. Here, we used photoacoustic imaging in tandem with a food grade cuttlefish ink contrast agent to specifically measure pocket depths in swine models (n=27 teeth) and then compared this to Williams probe. Photoacoustic imaging used a Vevo LAZR imaging system (Visualsonics) at 40 MHz. Spectral data was collected at both 680 and 800 nm to discriminate between the photoacoustic signal from stains and contrast agent. The pocket depths were measured on the sagittal view of the 3D images as well as with a Williams probe before photoacoustic imaging. The Bland-Altman plots show that 97% of our samples fell within ± 1.96 standard deviations of the differences between the depths measured by photoacoustic imaging and the probe (95% confidence interval) at mesial, lingual and buccal, and distal locations. Small bias values of -0.04, +0.17, and -0.2 mm were identified at mesial, lingual and buccal, and distal locations, respectively; the 95% confidence intervals are plotted as well and all are < 1.0 mm. The photoacoustic imaging approach also offered 0.01 mm precision and could cover the entire pocket versus the probe-based approach that is limited to only a few sites.

We characterized a commercially available LED-based photoacoustic system (Prexion Inc. Japan) that offered a tunable LED pulse repetition rate (1K Hz, 2K Hz, 3K Hz, and 4K Hz) and found that the temporal resolution of the scanner is dependent on the choice of repetition rate. The LED system have lower power, and averaging is used to minimize the noise. The power from the LED arrays at 690 nm and 850 nm with 70 ns pulse width was measured to be 9.85 mW/cm2 and 31.55 mW/cm2. Beam profiling showed that the average intensity at the center of the transducer was ~ 18% higher than the power on the edges of the transducer. The system had axial and lateral resolution of 268 μm and 590 μm, respectively. This system has frame rates of 30 Hz and 0.15 Hz. Pencil lead inside chicken breast could be detected up to 3.2 cm deep with a frame rate of 15 Hz. Indocyanine green (ICG), methylene blue (MB), and DiR were used as exogenous contrasts to measure the limit of detection using PLED-PAI. The limit of detection values for ICG, MB, and DiR are 9 μM, 0.75 mM, and 68 μM, respectively. For in vivo experiments, DiR (positive control), 400,000 stem cells labeled with DiR, and bare stem cells (negative control) were subcutaneously injected on the spinal cord of male mice. Results shows difference between labeled and unlabeled cells in photoacoustic intensity using PLED-PAI. This experiment shows the capability of this LED-based system to perform molecular imaging at a price point and device footprint nearly a log order smaller than systems based on an optical parametric oscillator laser.

The increasing interest around imaging and microsurgery techniques based on the photoacoustic effect has boosted active research into the development of exogenous contrast agents that may enhance the potential of this innovative approach.

In this context, plasmonic particles as gold nanorods are achieving resounding interest, owing to their efficiency of photothermal conversion, intense optical absorbance in the near infrared region, inertness in the body and convenience for conjugation with ligands of molecular targets.

On the other hand, the photoinstability of plasmonic particles remains a remarkable obstacle. In particular, gold nanorods easily reshape into nanospheres and so lose their optical absorbance in the near infrared region, under exposure to few-ns-long laser pulses. This issue is attracting much attention and stimulating ad-hoc solutions, such as the addition of rigid shells and the optimization of multiple parameters.

In this contribution, we focus on the influence of the shape of gold nanorods on their photothermal behavior and photostability. We describe the photothermal process in the gold nanorods by modeling their optical absorption and consequent temperature dynamics as a function of their aspect ratio (length / diameter).

Our results suggest that increasing the aspect ratio does probably not limit the photostability of gold nanorods, while shifting the plasmonic peak towards wavelengths around 1100 nm, which hold more technological interest.

Intraoperative tumor margin assessment during breast-conserving surgical resection is critically needed as positive margins are found in up to 34% of patients. Spectroscopic photoacoustic (sPA) molecular imaging combined with translatable antibody (Ab)-indocyanine green (ICG) contrast agent targeted to B7-H3, a molecular marker differentially expressed in breast cancer, may aid in margin assessment in murine breast cancers. ICG was conjugated to anti-B7-H3 antibodies through standard NHS chemistry. FVB/N Tg(MMTV/PyMT634Mul mice, a transgenic mouse model for breast cancer development, 5 weeks of age with ductal carcinoma in situ (DCIS) or small invasive carcinomas were given 33 μg of B7-H3-ICG 3-5 days before surgical resection. During excision of sequential sections of the lower mammary glands, fluorescence, multi-wavelength (680-900 nm, 10 nm increments) sPA, and B-mode ultrasound imaging were performed. Section specific molecular B7-H3 signal was compared to fluorescence imaging and histological (H&E) analysis. B-mode US and sPA imaging signal were able to relay accurate anatomical and molecular tissue information. sPA molecular imaging was able to detect B7-H3-ICG uptake in DCIS and early invasive carcinomas less than 1 mm in diameter. Furthermore, histological analysis of the excised tissues was able to show strong correlation between sPA imaging signal and disease state and location. While fluorescence imaging was able to detect ICG signal, there was insufficient resolution and specificity to identify and aid in resection of small cancerous foci. Adding molecular sPA imaging to help guide intraoperative margin assessment may increase the occurrence of negative margins which will decrease local disease recurrence.

Photoswitchable chromoproteins allow for molecular photoacoustic images with reduced hemoglobin background signal. We have previously introduced GAF2 a far-red photoswitchable chromoprotein similar to BphP1 [Yao et al., Nat. Meth. 13, 67–73 (2016)], but one-tenth the size. We introduce a new strategy for differentiating between spectrally similar photoswitchable chromoproteins. This strategy is based on exploiting relative spectral differences between two photoswitchable absorption states. We present in vivo photoacoustic images of GAF2 and BphP1 with differentiation based on their relative intensity change. We imaged using a custom photoswitchable photoacoustic imaging system that allows simultaneous background-free photoacoustic signal acquisition and difference spectra differentiation. E.coli expressing BphP1 and GAF2 are injected at various depths in hairless SCID mice. Background-free photoacoustic images are obtained and the chromoproteins are differentiated based on their relative intensity change. We photoconvert GAF2 and BphP1 using 607.5nm (5.2mJ/cm2) and 710nm (8.3mJ/cm2) light. The cycle of imaging and photoconversion is 20s. We are able to obtain background-free photoacoustic images 1.2cm deep in vivo and clearly differentiate between GAF2 and BphP1 via relative intensity changes despite similar imaging spectra. Photoswtichable chromoproteins and difference spectra differentiation could prove promising for deep multiplexed molecular background-free photoacoustic imaging.

Molecular photoacoustic imaging of targeted agents in vivo can be a valuable tool for biopsy guidance, tumor detection and delineation. Recently, our group has developed a prototype clinical, side looking, photoacoustic and ultrasonic system based on capacitive micro-machined ultrasound transducers. The system was used for imaging both fresh ex vivo pancreatic cancer samples labeled with functionalized IRDye-800 as well as in vivo trans-rectal imaging of prostate cancer patients with and without Indocyanine green contrast agent. Beamforming algorithms were used to provide real-time imaging albeit their low contrast to background ratio. To improve the quality images, presented here is a model-based acoustic reconstruction technique. The model is adapted to a sector scanning convention common in ultrasonography, compensates for the effects of non-ideal element directivity, impulse response and uneven responsivity as well as the ultrasound’s time dependent gain and the medium’s acoustic attenuation. Despite the low element count and the very limited viewing angle of the transducer, this technique was capable of reconstructing high-quality photoacoustic images both ex vivo and in vivo with a significant increase in contrast compared to the commonly used Universal back-projection algorithm. For ex vivo imaging, the results are also compared with fluorescence imaging showing a high degree of correlation. This study demonstrates the feasibility and the potential of the model based reconstruction approach for real-time visualization of contrast agents in vivo deep within the tissue, either intraoperatively or in routine imaging. Thus, such approach opens avenues for better cancer detection, diagnosis, treatment and monitoring.

We present a water-proof Microelectromechanical systems (MEMS) based scanning optical resolution Photoacoustic Microscopy (OR-PAM) system and its application in glioma tumor mouse model study. The presented OR-PAM system has high optical resolution (~3 μm) and high scanning speed (up to 50 kHz A-scan rate), which is ideal for cerebral vascular imaging. In this study, the mice with glioma tumor are treated with vascular disrupting agent (VDA). OR-PAM system is utilized to image the cerebral with the whole skull intact before and after the injection of VDA. By image registration, the response of every single blood vessel can be traced. This will provide us deeper understanding of the drug effect.

Conventional one-photon photoacoustic microscopy (PAM) utilizes high-frequency components of generated photoacoustic waves to improve the depth resolution. However, to obtain optically-high resolution in PAM in the depth direction, the use of high-frequency ultrasonic waves is to be avoided. It is because that the propagation distance is shortened as the frequency of ultrasonic waves becomes high. To overcome this drawback, we have proposed and developed two-photon photoacoustic microscopy (TP-PAM). Two-photon absorption occurs only at the focus point. TPPAM does not need to use the high-frequency components of photoacoustic waves. Thus, TP-PAM can improve the penetration depth while preserving the spatial resolution. However, the image acquisition time of TP-PAM is longer than that of conventional PAM, because TP-PAM needs to scan the laser spot both in the depth and transverse directions to obtain cross-sectional images. In this paper, we have introduced a focus-tunable electrically-controlled liquid lens in TP-PAM. Instead of a mechanical stepping-motor stage, we employed electrically-controlled liquid lens so that the depth of the focus spot can be quickly changed. In our system, the imaging speed of TP-PAM using the liquid lens and one-axis stepping-motor stage was 10 times faster than that using a two-axis stepping-motor stage only. TP-PAM with focus-scanning head consisting of the liquid lens and stepping-motor stage will be a promising method to investigate the inside of living tissues.

In fast functional photoacoustic microscopy (FPAM), the detection and monitoring of the oxygen saturation are important to monitor tissue functionality and disease progress. FPAM needs multi-wavelength pulsed laser sources with high pulse repetition rates, sufficient pulse energies and short wavelength switching time. Here, we develop a multi-wavelength pulsed laser source based on the stimulated Raman-scattering effect. The new laser is based on a 532-nm 1-MHz pulsed laser. The 532-nm laser pulse is split into two beams: one pumps a 5-m optical fiber to excite a 558-nm wavelength via stimulated Raman scattering; the other one propagates through a 50-m optical fiber to delay the pulse by 220 nano second so that the excitation wavelengths can be separated in time for fast functional photoacoustic imaging. The two beams are spatially combined and coupled into an optical fiber for photoacoustic excitation. Consequently, the new laser source can generate 2 million pulses per second, switch wavelengths in 220 ns, and provide hundreds of nano-Joules pulse energy for each wavelength. Using this laser source, we demonstrate optical-resolution photoacoustic imaging of microvascular structure and oxygen saturation in the mouse ear. The ultrashort wavelength switching time enables oxygen saturation imaging of flowing single red blood cells.

In this work, we employ frequency-domain photoacoustic microscopy to obtain photoacoustic images of labeled and unlabeled cells. The photoacoustic microscope is based on an intensity-modulated diode laser in combination with a focused piezo-composite transducer and allows imaging of labeled cells without severe photo-bleaching. We demonstrate that frequency-domain photoacoustic microscopy realized with a diode laser is capable of recording photoacoustic images of single cells with sub-µm resolution. As examples, we present images of undyed human red blood cells, stained human epithelial cells, and stained yeast cells.

Optoacoustic microscopy (OAM) has enabled high-resolution, label-free imaging of tissues at depths not achievable with purely optical microscopy. However, widespread implementation of OAM into existing epi-illumination microscopy setups is often constrained by the performance and size of the commonly used piezoelectric ultrasound detectors. In this work, we introduce a novel acoustic detector based on a π-phase-shifted fiber Bragg grating (π-FBG) interferometer embedded inside an ellipsoidal acoustic cavity. The cavity enables seamless integration of epi-illumination OAM into existing microscopy setups by decoupling the acoustic and optical paths between the microscope objective and the sample. The cavity also acts as an acoustic condenser, boosting the sensitivity of the π-FBG and enabling cost effective CW-laser interrogation technique. We characterize the sensor’s sensitivity and bandwidth and demonstrate hybrid OAM and second-harmonic imaging of phantoms and mouse tissue in vivo.

In biomedical imaging, all optical techniques face a fundamental trade-off between spatial resolution and tissue penetration. Therefore, obtaining an organelle-level resolution image of a whole organ has remained a challenging and yet appealing scientific pursuit. Over the past decade, optical microscopy assisted by mechanical sectioning or chemical clearing of tissue has been demonstrated as a powerful technique to overcome this dilemma, one of particular use in imaging the neural network. However, this type of techniques needs lengthy special preparation of the tissue specimen, which hinders broad application in life sciences. Here, we propose a new label-free three-dimensional imaging technique, named microtomy-assisted photoacoustic microscopy (mPAM), for potentially imaging all biomolecules with 100% endogenous natural staining in whole organs with high fidelity. We demonstrate the first label-free mPAM, using UV light for label-free histology-like imaging, in whole organs (e.g., mouse brains), most of them formalin-fixed and paraffin- or agarose-embedded for minimal morphological deformation. Furthermore, mPAM with dual wavelength illuminations is also employed to image a mouse brain slice, demonstrating the potential for imaging of multiple biomolecules without staining. With visible light illumination, mPAM also shows its deep tissue imaging capability, which enables less slicing and hence reduces sectioning artifacts. mPAM could potentially provide a new insight for understanding complex biological organs.

Zebrafish are an attractive animal model for the study of disease due to their low cost, ease of care, and ability to rapidly produce large colonies. Some mutant zebrafish, for example those from the casper line, exhibit optical properties which make them ideal specimens for in vivo optical imaging. In this study we use an ultra-high frequency photoacoustic (PA) microscope to image the head and trunk of live casper fish, and generate 3D renderings of the local functional vasculature with single-cell resolution. Five day-post-fertilization larvae were anesthetized using tricane and embedded in 1.5% (w/v) agarose to prevent movement during image acquisition. A transmission mode PA microscope equipped with a single element transducer (either 200 or 400 MHz central frequency) and a 532 nm laser focused through a 4X optical objective was used to raster scan the larvae. Reconstructions of the vasculature were created from the recorded RF data. Images of the trunk showed the caudal artery (CA) and vein (CV), as well as intersegmental vessels (ISV). The ISVs were observed to originate from either the CA or CV, and curve around the notochord to join at the dorsal longitudinal anastomotic vessel. Furthermore, individual erythrocytes were resolved within the caudal hematopoietic tissue. Reconstructions of the head revealed a tortuous organization of developing vessels in the brain. This work is the first demonstration of in vivo PAM at frequencies greater than 200 MHz, and paves the way for future studies which will explore label-free imaging of nanoparticles and cancer progression in vivo.

In cancer research, regions of increasingly lowered oxygenation in tissue (hypoxia), which are due to tumor development, are considered to play an important role in activating various signaling pathways that facilitate further development of the cancer. However, devising a minimally invasive method to monitor tissue oxygenation has remained a challenge. Photoacoustic microscopy has been posed as a solution in a variety of preclinical research studies. Here, using optical-resolution photoacoustic microscopy (OR-PAM), for the first time, we non-invasively measured oxygenation and vascularization in vivo caused by multiple myeloma (MM) progression. Mice injected with MM cells tagged with green fluorescent protein were monitored with a fluorescence microscope for tumor progression over the course of 28 days. OR-PAM evaluated the oxygen saturation (sO2) and the blood vessel density in the cerebral bone marrow, where MM cells home. At 28 days after the injection of MM cells, the total sO2 had dropped by 50% in the developing tumor regions, while in the non-tumor developing regions it had dropped by 20% compared with the value at one day after MM injection. The blood vessel density had dropped by 35% in the tumor developing regions, while in the non-tumor developing regions it had dropped by 8% compared with the value at one day after MM injection. In summary, non-invasive measurement by OR-PAM correlated the development of hypoxia with to MM progression. It revealed decreased vascularization surrounding the tumor areas, which we feel can be ascribed to the rapid tumor progression.

Reconstruction of photoacoustic images acquired with clinical ultrasound transducers is traditionally performed using the delay and sum (DAS) beamforming algorithm. Recently, the delay multiply and sum (DMAS) beamforming algorithm has been shown to provide increased contrast, signal to noise ratio (SNR) and resolution in PA imaging. The main reason for the continued use of DAS beamforming in photoacoustics is its linearity in reconstructing the PA signal to the initial pressure generated by the absorbed laser pulse. This is crucial for the identification of different chromophores in multispectral PA applications and DMAS has not yet been demonstrated to provide this property. Furthermore, due to its increased computational complexity, DMAS has not yet been shown to work in real time. We present an open-source real-time variant of the DMAS algorithm which ensures linearity of the reconstruction while still providing increased SNR and therefore enables use of DMAS for multispectral PA applications. This is demonstrated in vitro and in vivo. The DMAS and reference DAS algorithms were integrated in the open-source Medical Imaging Interaction Toolkit (MITK) and are available to the community as real-time capable GPU implementations.

Delay and sum beamformed acoustic-resolution photoacoustic images are limited in resolution by the wavelength of the received acoustic signal. We seek to improve on this resolution in the case when the received signal is known to be generated by only a few absorbers. When the absorbers to be imaged are known a priori to be sparse then the reconstruction problem can be stated as an optimization problem aiming to minimize the residual between model predictions and measured channel data and also an L1-norm-based metric of sparsity. In brief, the strategy aims to express experimentally observed curved wavefronts in channel data as a super-position of simulated point-spread functions with a constraint on sparsity. The approach is similar in spirit to recent super-resolution contrast ultrasound approaches but uses an L1-norm minimization strategy. We have applied this optimization strategy to photoacoustic beamforming in both simulation and experiment. Simulation was conducted using Field II, and an experimental measure of resolving power was obtained by imaging the cross section of two wires at successively smaller separations. Experimental channel data was acquired using a 21-MHz Visualsonics array transducers with a Verasonics Vantage ultrasound platform for data acquisition. Simulations indicate potential to beat the ultrasound diffraction limit by a factor of four or more while current experiments achieve a factor of two resolution improvement. A possible application of this approach is for providing increased resolution images of the microvasculature surrounding cancerous tumors. Ongoing work aims to investigate in vivo performance of the proposed sparsity-constrained super-resolution approach.

Quantification of tissue properties with photoacoustic (PA) imaging typically requires a highly accurate representation of the initial pressure distribution in tissue. Almost all PA scanners reconstruct the PA image only from a partial scan of the emitted sound waves. Especially handheld devices, which have become increasingly popular due to their versatility and ease of use, only provide limited view data because of their geometry. Owing to such limitations in hardware as well as to the acoustic attenuation in tissue, state-of-the-art reconstruction methods deliver only approximations of the initial pressure distribution. To overcome the limited view problem, we present a machine learning-based approach to the reconstruction of initial pressure from limited view PA data. Our method involves a fully convolutional deep neural network based on a U-Net-like architecture with pixel-wise regression loss on the acquired PA images. It is trained and validated on in silico data generated with Monte Carlo simulations. In an initial study we found an increase in accuracy over the state-of-the-art when reconstructing simulated linear-array scans of blood vessels.

To achieve high-resolution photoacoustic tomography in 3D requires either a high-density 2D detection array or multiple sequential acquisitions (scanning). Due to the cost and fabrication challenges of suitable 2D arrays, scanning systems are often used. The drawback is that scanning leads to long acquisition times and a trade-off must be made between image resolution and frame rate. This paper describes how techniques from compressed sensing can ameliorate this problem. The main idea of compressed sensing is that a fully sampled data set usually contains redundant information, and a high-quality image can be recovered from much less data by exploiting the low spatial, temporal and/or spectral complexity of the chromophore distributions. Scanners that are able to suitably sub-sample the acoustic field can acquire sufficient data much faster. To reconstruct high-quality images from sub-sampled data, iterative, model-based approaches incorporating explicit constraints on the characteristics of the chromophore distributions are used. Such algorithms can be computationally demanding, but also highly versatile. They are applicable to all scanning geometries, can be extended to incorporate complex acoustic models accounting for heterogeneous media, and could even include optical models for quantitative reconstructions. Experimental demonstrations of both static in-vivo and dynamic imaging will be described, using data obtained with a sequential scanner. The static images can be obtained 4x-8x times faster using this approach. A further increase 2x increase can be achieved when imaging dynamic processes and using motion estimation models. Finally, dynamic, high-resolution 3D PAT imaging with frame rates exceeding 1Hz will be demonstrated experimentally.

The development of photoacoustic computed tomography (PACT) for neuroimaging in humans will fill an important void left by available imaging techniques. However, due to the presence of the skull, accurate image reconstruction in transcranial PACT remains challenging. Variations in the shear and longitudinal wave speed distributions due to the skull can induce strong aberrations in the measured photoacoustic wavefields. To mitigate these artifacts, image reconstruction methods in transcranial PACT require knowledge of these acoustic properties. However, such information may be difficult to obtain in practice. To circumvent this, we developed a joint reconstruction (JR) method for transcranial PACT where the longitudinal and shear speed distributions are reconstructed concurrently with the sought-after initial pressure distribution. The joint estimation of the initial pressure, longitudinal speed, and shear speed distributions from PACT data alone is unstable. To overcome this instability, we propose to incorporate prior information about the acoustic properties of the skull. Specifically, a low-dimensional parameterized acoustic representation of the skull is established with the aid of adjunct CT data. The use of a low-dimensional representation of the acoustic skull parameters effectively overcomes the instability of the JR problem and allows stable reconstruction of the acoustic skull parameters and the initial pressure distribution concurrently. To validate the proposed method, we conducted 3D numerical studies based on realistic human skull models derived from adjunct CT data. The efficacy of the proposed JR method was demonstrated through accurate reconstruction of the initial pressure, longitudinal speed, and shear speed distributions from PACT measurement data alone.

Accurate estimation of the initial pressure distribution in photoacoustic computed tomography (PACT) requires some knowledge of the sound speed distribution. However, the sound speed distribution is typically unknown. Further, the initial pressure and sound speed distributions cannot both, in general, be stably recovered from PACT measurements alone. In this work, a joint reconstruction method for the initial pressure distribution and a low-dimensional parameterized model of the sound speed distribution is proposed. By employing a priori information about the structure of the sound speed distribution, both the initial pressure and sound speed can be accurately recovered. The joint reconstruction problem is solved by use of a proximal optimization method that allows constraints and non-smooth regularization functions for initial pressure distribution. The gradients of the cost function with respect to the initial pressure and sound speed distributions are calculated by use of an adjoint state method that has the same per-iteration computational cost as calculating the gradient with respect to the initial pressure distribution alone. This approach is quantitatively evaluated through 2D computer-simulation studies for a small animal imaging model. The impact of the choice of the parameterized sound speed model is investigated. Even when the assumed parameterized sound speed model is inconsistent with the true sound speed distribution, the estimated initial pressure distribution is more accurate than that obtained by assuming a constant sound speed. The utility of the proposed approach is also demonstrated through application to experimental in vivo measurements of a mouse.

We introduce a preclinical imaging platform – a 3D photoacoustic/fluorescence tomography (PAFT) instrument augmented with an environmentally responsive dual-contrast biocompatible nanoprobe. The PAFT instrument was designed for simultaneous acquisition of photoacoustic and fluorescence orthogonal projections at each rotational position of a biological object, enabling direct co-registration of the two imaging modalities. The nanoprobe was based on liposomes loaded with J-aggregates of indocyanine green (PAtrace). Once PAtrace interacts with the environment, a transition from J-aggregate to monomeric ICG is induced. The subsequent recovery of monomeric ICG is characterized by dramatic changes in the optical absorption spectrum and reinstated fluorescence. In the activated state, PAtrace can be simultaneously detected by both imaging modes of the PAFT instrument using 780 nm excitation and fluorescence detection at 810 nm. The fluorescence imaging component is used to boost detection sensitivity by providing lowresolution map of activated nanoprobes, which are then more precisely mapped in 3D by the photoacoustic imaging component. Activated vs non-activated particles can be distinguished based on their different optical absorption peaks, removing the requirements for complex image registration between reference and detection scans. Preliminary phantom and in vivo animal imaging results showed successful activation and visualization of PAtrace with high sensitivity and resolution. The proposed PAFT-PAtrace imaging platform could be used in various functional and molecular imaging applications including multi-point in vivo assessment of early metastasis.

We have developed a single-breath-hold photoacoustic computed tomography (SBH-PACT) system to detect tumors and reveal detailed angiographic information about human breasts. SBH-PACT provides high spatial and temporal resolutions with a deep in vivo penetration depth of over 4 cm. A volumetric breast image can be acquired by scanning the breast within a single breath hold (~15 sec). We imaged a healthy female volunteer and seven breast cancer patients. SBH-PACT clearly identified all tumors by revealing higher blood vessel densities and lower compliance associated with the tumors

Laser Optoacoustic Ultrasonic Imaging System Assembly (LOUISA-3D) was developed in response to demand of diagnostic radiologists for an advanced screening system for the breast to improve on low sensitivity of x-ray based modalities of mammography and tomosynthesis in the dense and heterogeneous breast and low specificity magnetic resonance imaging. It is our working hypothesis that co-registration of quantitatively accurate functional images of the breast vasculature and microvasculature, and anatomical images of breast morphological structures will provide a clinically viable solution for the breast cancer care. Functional imaging is LOUISA-3D is enabled by the full view 3D optoacoustic images acquired at two rapidly toggling laser wavelengths in the near-infrared spectral range. 3D images of the breast anatomical background is enabled in LOUISA-3D by a sequence of B-mode ultrasound slices acquired with a transducer array rotating around the breast. This creates the possibility to visualize distributions of the total hemoglobin and blood oxygen saturation within specific morphological structures such as tumor angiogenesis microvasculature and larger vasculature in proximity of the tumor. The system has four major components: (i) a pulsed dual wavelength laser with fiberoptic light delivery system, (ii) an imaging module with two arc shaped probes (optoacoustic and ultrasonic) placed in a transparent bowl that rotates around the breast, (iii) a multichannel electronic system with analog preamplifiers and digital data acquisition boards, and (iv) computer for the system control, data processing and image reconstruction. The most important advancement of this latest system design compared with previously reported systems is the full breast illumination accomplished for each rotational step of the optoacoustic transducer array using fiberoptic illuminator rotating around the breast independently from rotation of the detector probe. We report here a pilot case studies on one healthy volunteer and on patient with a suspicious small lesion in the breast. LOUISA3D visualized deoxygenated veins and oxygenated arteries of a healthy volunteer, indicative of its capability to visualize hypoxic microvasculature in cancerous tumors. A small lesion detected on optoacoustic image of a patient was not visible on ultrasound, potentially indicating high system sensitivity of the optoacoustic subsystem to small but aggressively growing cancerous lesions with high density angiogenesis microvasculature. The main breast vasculature (0.5-1 mm) was visible at depth of up to 40-mm with 0.3-mm resolution. The results of LOUISA-3D pilot clinical validation demonstrated the system readiness for statistically significant clinical feasibility study.

Tomographic photoacoustic (PA) images acquired using a Fabry-Perot (FP) based scanner offer high resolution and image fidelity but can result in long acquisition times due to the need for raster scanning. To reduce the acquisition times, a parallelised camera-based PA signal detection scheme is developed. The scheme is based on using a sCMOScamera and FPI sensors with high homogeneity of optical thickness. PA signals were acquired using the camera-based setup and the signal to noise ratio (SNR) was measured. A comparison of the SNR of PA signal detected using 1) a photodiode in a conventional raster scanning detection scheme and 2) a sCMOS camera in parallelised detection scheme is made. The results show that the parallelised interrogation scheme has the potential to provide high speed PA imaging.

Compressional wave detection is useful means for health monitoring of building, detection of abnormal vibration of moving objects, defect evaluation, and biomedical imaging such as echography and photoacoustic imaging. The frequency of the compressional wave is varied from quasi-static to a few tens of megahertz depending on applications. Since the dynamic range of general compressional wave detectors is limited, we need to choose a proper compressional wave detector depending on applications. For the compressional wave detection with wide dynamic range, two or more detectors with different detection ranges is required. However, these detectors with different detection ranges generally has different accuracy and precision, disabling the seamless detection over these detection ranges. In this study, we proposed a compressional wave detector employing optical frequency comb (OFC). The compressional wave was sensed with a part of an OFC cavity, being encoded into OFC. The spectrally encoded OFC was converted to radio-frequency by the frequency link nature of OFC. The compressional wave-encoded radio-frequency can therefore be directly measured with a high-speed photodetector. To enhance the dynamic range of the compressional wave detection, we developed a cavityfeedback-based system and a phase-sensitive detection system, both of which the accuracy and precision are coherently linked to these of the OFC. We provided a proof-of-principle demonstration of the detection of compressional wave from quasi-static to ultrasound wave by using the OFC-based compressional wave sensor. Our proposed approach will serve as a unique and powerful tool for detecting compressional wave versatile applications in the future.

Visualization methods are very important in biomedical imaging. As a technology that understands life, biomedical imaging has the unique advantage of providing the most intuitive information in the image. This advantage of biomedical imaging can be greatly improved by choosing a special visualization method. This is more complicated in volumetric data. Volume data has the advantage of containing 3D spatial information. Unfortunately, the data itself cannot directly represent the potential value. Because images are always displayed in 2D space, visualization is the key and creates the real value of volume data. However, image processing of 3D data requires complicated algorithms for visualization and high computational burden. Therefore, specialized algorithms and computing optimization are important issues in volume data. Photoacoustic-imaging is a unique imaging modality that can visualize the optical properties of deep tissue. Because the color of the organism is mainly determined by its light absorbing component, photoacoustic data can provide color information of tissue, which is closer to real tissue color. In this research, we developed realistic tissue visualization using acoustic-resolution photoacoustic volume data. To achieve realistic visualization, we designed specialized color transfer function, which depends on the depth of the tissue from the skin. We used direct ray casting method and processed color during computing shader parameter. In the rendering results, we succeeded in obtaining similar texture results from photoacoustic data. The surface reflected rays were visualized in white, and the reflected color from the deep tissue was visualized red like skin tissue. We also implemented the CUDA algorithm in an OpenGL environment for real-time interactive imaging.

The goal of this work was to develop and validate a spectrally resolved photoacoustic imaging method, namely multi-spectral photoacoustic elasticity tomography (PAET) for quantifying the physiological parameters and elastic modulus of biological tissues. We theoretically and experimentally examined the PAET imaging method using simulations and in vitro experimental tests. Our simulation and in vitro experimental results indicated that the reconstructions were quantitatively accurate in terms of sizes, the physiological and elastic properties of the targets.

Quantitative photoacoustic tomography (q-PAT) is a nontrivial technique can be used to reconstruct the absorption image with a high spatial resolution. Several attempts have been investigated by setting point sources or fixed-angle illuminations. However, in practical applications, these schemes normally suffer from low signal-to-noise ratio (SNR) or poor quantification especially for large-size domains, due to the limitation of the ANSI-safety incidence and incompleteness in the data acquisition. We herein present a q-PAT implementation that uses multi-angle light-sheet illuminations and a calibrated iterative multi-angle reconstruction. The approach can acquire more complete information on the intrinsic absorption and SNR-boosted photoacoustic signals at selected planes from the multi-angle wide-field excitations of light-sheet. Therefore, the sliced absorption maps over whole body can be recovered in a measurementflexible, noise-robust and computation-economic way. The proposed approach is validated by the phantom experiment, exhibiting promising performances in image fidelity and quantitative accuracy.

Photoacoustic (PA) imaging is a promising imaging modality for both preclinical research and clinical practices. Laser wavelengths in the first near infrared window (NIR-I, 650-950 nm) have been widely used for photoacoustic imaging. As compared with NIR-I window, scattering of photons by biological tissues is largely reduced in the second NIR (NIR-II) window, leading to enhanced imaging fidelity. However, the lack of biocompatible NIR-II absorbing exogenous agents prevented the use of this window for in vivo imaging. In recent years, few studies have been reported on photoacoustic imaging in NIR-II window using exogenous contrast agents. In this work, we discuss the recent work on PA imaging using 1064 nm wavelength, the fundamental of Nd:YAG laser, as an excitation wavelength. The PA imaging at 1064 nm is advantageous because of the low and homogeneous signal from tissue background, enabling high contrast in PA imaging when NIR-II absorbing contrast agents are employed.

Photoacoustic tomography is an emerging imaging modality which has paved its way in preclinical and clinical trials owing to the multiple advantages it offers. A typical PAT system consists of a laser beam which homogeneously illuminates the sample giving rise to photoacoustic (PA) waves, which are collected using an ultrasound transducer (UST) rotating around the sample. Low cost, high sensitivity and easy availability have made single-element transducers (SETs) a preferred choice for acquiring these A-lines PA signal. Two methods have been reported for collection of these A-lines by SETs- (1) Stop-and-go scan and (2) Continuous scan. In stop-and-go scan, the stepper motor moves the SET to a predefined position where the SET collects multiple A-lines. Once the desired number of A-lines at that point have been collected and saved, the stepper motor moves to the next position and the process continues. A continuous scan is one in which the stepper motor rotates the SET continuously at a predefined speed. The A-lines are thus collected by a moving SET and are saved once the motor has stopped. In this work, we have compared the two types of scanning methods in terms of image quality, signal-to-noise ratio and time of scan by performing experiments on phantoms.

Visualization of small tumors inside biological tissue is important in cancer treatment because that promotes accurate surgical resection and enables therapeutic effect monitoring. For sensitive detection of tumor, we have been developing photoacoustic (PA) imaging technique to visualize tumor-specific contrast agents, and have already succeeded to image a subcutaneous tumor of a mouse using the contrast agents. To image tumors inside biological tissues, extension of imaging depth and improvement of sensitivity were required. In this study, to extend imaging depth, we developed a PA tomography (PAT) system that can image entire cross section of mice. To improve sensitivity, we discussed the use of the P(VDF-TrFE) linear array acoustic sensor that can detect PA signals with wide ranges of frequencies. Because PA signals produced from low absorbance optical absorbers shifts to low frequency, we hypothesized that the detection of low frequency PA signals improves sensitivity to low absorbance optical absorbers. We developed a PAT system with both a PZT linear array acoustic sensor and the P(VDF-TrFE) sensor, and performed experiment using tissue-mimicking phantoms to evaluate lower detection limits of absorbance. As a result, PAT images calculated from low frequency components of PA signals detected by the P(VDF-TrFE) sensor could visualize optical absorbers with lower absorbance.

Recently we developed a multispectral LED-based photoacoustic/ultrasound imaging system (AcousticX) and have been continuously working on its technical/functional improvements. AcousticX is a linear array ultrasound transducer (128 elements, 10 MHz)-based system in which LED arrays (selectable wavelengths, pulse repetition frequency: 4 kHz, pulse width: tunable from 40 – 100 ns) are fixed on both sides of the transducer to illuminate the tissue for photoacoustic imaging. The ultrasound/photoacoustic data from all 128 elements can be simultaneously acquired, processed and displayed. We already demonstrated our system’s capability to perform photoacoustic/ultrasound imaging for dynamic imaging of the tissue at a frame rate of 10 Hz (for example to visualize the pulsation of arteries in vivo in human subjects). In this work, we present the development of a new high-speed imaging mode in AcousticX. In this mode, instead of toggling between ultrasound and photoacoustic measurements, it is possible to continuously acquire only photoacoustic data for 1.5 seconds with a time interval of 1 ms. With this improvement, we can record photoacoustic signals from the whole aperture (38 mm) at fast rate and can be reviewed later at different speeds for analyzing dynamic changes in the photoacoustic signals. We believe that AcousticX with this new high-speed mode opens up a feasible technical path for multiple dynamic studies, for example one which focus on imaging the response of voltage sensitive dyes. We envisage to improve the acquisition speed further in future for exploring ultra-high-speed applications.

Photoacoustic imaging is a hybrid biomedical imaging modality that has emerged over the last decade. In photoacoustic imaging, pulsed-light absorbed by the target emits ultrasound that can be detected using a conventional ultrasound array. This ultrasound data can be used to reconstruct the location and spatial details of the intrinsic/extrinsic light absorbers in the tissue. Recently we reported on the development of a multi-wavelength high frame-rate LED-based photoacoustic/ultrasound imaging system (AcousticX). In this work, we photoacoustically characterize the absorption spectrum of ICG and porcine blood using LED arrays with multiple wavelengths (405, 420, 470, 520, 620, 660, 690, 750, 810, 850, 925, 980 nm). Measurements were performed in a simple reflection mode configuration in which LED arrays where fixed on both sides of the linear array ultrasound probe. Phantom used consisted of micro-test tubes filled with ICG and porcine blood, which were placed in a tank filled with water. The photoacoustic spectrum obtained from our measurements matches well with the reference absorption spectrum. These results demonstrate the potential capability of our system in performing clinical/pre-clinical multispectral photoacoustic imaging.

Several annular detector arrays are compared for scanning photoacoustic imaging. Compared to single, spherically focused detectors, the arrays offer similar sensitivity, but have an extended depth of field due to their dynamic focusing capability. The investigated arrays consist either of piezoelectric polymer film (PVDF) with a large sensing area for optimized sensitivity or of fiber optic rings, where the small width of elements gives rise to high bandwidth and resolution. Simulations demonstrate the superior resolution of the fiber-optic rings over a flat piezo array. However, with inclined sensing elements also the piezo-detector reaches a similar resolution as the optical array. In phantom experiments with the PVDF array the extended depth of field and the capability of imaging complex objects are demonstrated.

Photoacoustic mesoscopy (PAMe), offering high-resolution (sub-100-μm) and high optical contrast imaging at the depth of 1-10 mm, generally obtains massive collection data using a high-frequency focused ultrasonic transducer. The spatial impulse response (SIR) of this focused transducer causes the distortion of measured signals in both duration and amplitude. Thus, the reconstruction method considering the SIR needs to be investigated in the computation-economic way for PAMe. Here, we present a modified back-projection algorithm, by introducing a SIR-dependent calibration process using a non-satationary convolution method. The proposed method is performed on numerical simulations and phantom experiments of microspheres with diameter of both 50 μm and 100 μm, and the improvement of image fidelity of this method is proved to be evident by methodology parameters. The results demonstrate that, the images reconstructed when the SIR of transducer is accounted for have higher contrast-to-noise ratio and more reasonable spatial resolution, compared to the common back-projection algorithm.

Photoacoustic (PA) imaging forms an image based on optical absorption contrasts with ultrasound (US) resolution. In contrast, US imaging is based on acoustic backscattering to provide structural information. In this study, we develop a miniature all-optical probe for high-resolution PA-US dual-modality imaging over a large imaging depth range. The probe employs three individual optical fibers (F1-F3) to achieve optical generation and detection of acoustic waves for both PA and US modalities. To offer wide-angle laser illumination, fiber F1 with a large numerical aperture (NA) is used for PA excitation. On the other hand, wide-angle US waves are generated by laser illumination on an optically absorbing composite film which is coated on the end face of fiber F2. Both the excited PA and backscattered US waves are detected by a Fabry-Pérot cavity on the tip of fiber F3 for wide-angle acoustic detection. The wide angular features of the three optical fibers make large-NA synthetic aperture focusing technique possible and thus high-resolution PA and US imaging. The probe diameter is less than 2 mm. Over a depth range of 4 mm, lateral resolutions of PA and US imaging are 104−154 μm and 64−112 μm, respectively, and axial resolutions of PA and US imaging are 72−117 μm and 31−67 μm, respectively. To show the imaging capability of the probe, phantom imaging with both PA and US contrasts is demonstrated. The results show that the probe has potential for endoscopic and intravascular imaging applications that require PA and US contrast with high resolution.

In this paper, a new imaging modality, named photoacoustic resonance imaging (PARI), is proposed and experimentally demonstrated. Being distinct from conventional single nanosecond laser pulse induced wideband PA signal, the proposed PARI method utilizes multi-burst modulated laser source to induce PA resonant signal with enhanced signal strength and narrower bandwidth. Moreover, imaging contrast could be clearly improved than conventional single-pulse laser based PA imaging by selecting optimum modulation frequency of the laser source, which originates from physical properties of different materials beyond the optical absorption coefficient. Specifically, the imaging steps is as follows: 1: Perform conventional PA imaging by modulating the laser source as a short pulse to identify the location of the target and the background. 2: Shine modulated laser beam on the background and target respectively to characterize their individual resonance frequency by sweeping the modulation frequency of the CW laser source. 3: Select the resonance frequency of the target as the modulation frequency of the laser source, perform imaging and get the first PARI image. Then choose the resonance frequency of the background as the modulation frequency of the laser source, perform imaging and get the second PARI image. 4: subtract the first PARI image from the second PARI image, then we get the contrast-enhanced PARI results over the conventional PA imaging in step 1. Experimental validation on phantoms have been performed to show the merits of the proposed PARI method with much improved image contrast.

Photoacoustic imaging (PAI) is a novel medical imaging modality that uses the advantages of the spatial resolution of ultrasound imaging and the high contrast of pure optical imaging. Analytical algorithms are usually employed to reconstruct the photoacoustic (PA) images as a results of their simple implementation. However, they provide a low accurate image. Model-based (MB) algorithms are used to improve the image quality and accuracy while a large number of transducers and data acquisition are needed. In this paper, we have combined the theory of compressed sensing (CS) with MB algorithms to reduce the number of transducer. Smoothed version of ℓ₀-norm (Sℓ₀) was proposed as the reconstruction method, and it was compared with simple iterative reconstruction (IR) and basis pursuit. The results show that Sℓ₀ provides a higher image quality in comparison with other methods while a low number of transducers were. Quantitative comparison demonstrates that, at the same condition, the Sℓ₀ leads to a peak-signal-to-noise ratio for about two times of the basis pursuit.

Photoacoustic imaging (PAI), is a promising medical imaging technique that provides the high contrast of the optical imaging and the resolution of ultrasound (US) imaging. Among all the methods, Three-dimensional (3D) PAI provides a high resolution and accuracy. One of the most common algorithms for 3D PA image reconstruction is delay-and-sum (DAS). However, the quality of the reconstructed image obtained from this algorithm is not satisfying, having high level of sidelobes and a wide mainlobe. In this paper, delay-multiply-andsum (DMAS) algorithm is suggested to overcome these limitations in 3D PAI. It is shown that DMAS algorithm is an appropriate reconstruction technique for 3D PAI and the reconstructed images using this algorithm are improved in the terms of the width of mainlobe and sidelobes, compared to DAS. Also, the quantitative results show that DMAS improves signal-to-noise ratio (SNR) and full-width-half-maximum (FW HM) for about 25 dB and 0.2 mm, respectively, compared to DAS.

Wavefront shaping using spatial light modulator (SLM) is a popular method for focusing light through a turbid media, such as biological tissues. Usually, in iterative optimization methods, due to the very large number of pixels in SLM, larger pixels are formed, bins, and the phase value of the bins are changed to obtain an optimum phase map, hence a focus. In this study an efficient optimization algorithm is proposed to obtain an arbitrary map of focus utilizing all the SLM pixels or small bin sizes. The application of such methodology in dermatology, hair removal in particular, is explored and discussed

Vesicoureteral reflux is the abnormal flow of urine from your bladder back up the tubes (ureters) that connect your kidneys to your bladder. Normally, urine flows only down from your kidneys to your bladder. Vesicoureteral reflux is usually diagnosed in infants and children. The disorder increases the risk of urinary tract infections, which, if left untreated, can lead to kidney damage. X-Ray cystography is used currently to diagnose this condition which uses ionising radiation, making it harmful for patients. In this work we demonstrate the feasibility of imaging the urinary bladder using a handheld clinical ultrasound and photoacoustic dual modal imaging system in small animals (rats). Additionally, we demonstrate imaging vesicoureteral reflux using bladder mimicking phantoms. Urinary bladder imaging is done with the help of contrast agents like black ink and gold nanoparticles which have high optical absorption at 1064 nm. Imaging up to 2 cm was demonstrated with this system. Imaging was done at a framerate of 5 frames per second.

In 2016, an estimated ~250,000 new cases of invasive and non-invasive breast cancer were diagnosed in US women. About 60–75% of these cases were treated with breast conserving surgery (BCS) as the initial therapy. To reduce the local recurrence rate, the goal of BCS is to excise the tumor with a rim of normal surrounding tissue, so that no cancer cells remain at the cut margin, while preserving as much normal breast tissue as possible. Therefore, patients with remaining cancer cells at the cut margin commonly require a second surgical procedure to obtain clear margins. Different approaches have been used to decrease the positive margin rate to avoid re-excision. However, these techniques are variously ineffective in reducing the re-operative rate, difficult to master by surgeons, or time-consuming for large specimens. Thus, 20-60% of patients undergoing BCS still require second surgeries due to positive surgical margins. The ideal tool for margin assessment would provide the same information as histological analysis, without the need for processing specimens. To achieve this goal, we have developed and refined label-free photoacoustic microscopy (PAM) for breast specimens. Exploiting the intrinsic optical contrast of tissue, ultraviolet (UV) laser illumination can highlight cell nuclei, thus providing the same contrast as hematoxylin labeling used in conventional histology and measuring features related to the histological landscape without the need for labels. We demonstrate that our UV-PAM system can provide label-free, high-resolution, and histology-like imaging of fixed, unprocessed breast tissue.

Photoacoustic imaging is anticipated for use in portraying blood vessel structures (e.g. neovascularization in inflamed regions). To reduce invasiveness and enhance ease handling, we developed a handheld photoacoustic imaging system using multiple wavelengths. The usefulness of the proposed system was investigated in phantom experiments and in vivo measurements. A silicon tube was embedded into chicken breast meat to simulate the blood vessel. The tube was filled with ovine blood. Then laser light was guided to the phantom surface by an optical fiber bundle close to the linear ultrasound probe. Photoacoustic images were obtained at 750-950 nm wavelengths. Strong photoacoustic signals from the boundary between blood and silicon tube are observed in these images. The shape of photoacoustic spectrum at the boundary resembles that of the HbO₂ absorption spectrum at 750-920 nm. In photoacoustic images, similarity between photoacoustic spectrum and HbO₂ absorption spectrum was evaluated by calculating the normalized correlation coefficient. Results show high correlation in regions of strong photoacoustic signals in photoacoustic images. These analyses demonstrate the feasibility of portraying blood vessel structures under practical conditions. To evaluate the feasibility of three-dimensional vascular imaging, in vivo experiments were conducted using three wavelengths. A right hand and ultrasound probe were set in degassed water. By scanning a probe, cross-sectional ultrasound and photoacoustic images were obtained at each location. Then, all ultrasound or photoacoustic images were piled up respectively. Then three-dimensional images were constructed. Resultant images portrayed blood vessel-like structures three-dimensionally. Furthermore, to distinguish blood vessels from other tissues (e.g. skin), distinguishing images of them were constructed by comparing photoacoustic signal intensity among three wavelengths. The resultant image portrayed blood vessels as distinguished from surrounding tissues. These results demonstrated the usefulness of the proposed imaging device.

For early diagnosis of rheumatoid arthritis (RA), it is important to visualize its potential marker, vascularization in the synovial membrane of the finger joints. Photoacoustic (PA) imaging, which can image blood vessels at high contrast and resolution is expected to be a potential modality for earlier diagnosis of RA. In previous studies of PA finger imaging, different acoustic schemes such as linear or arc-shaped arrays have been utilized, but these have limited detection views, rendering inaccurate reconstruction, and most of them require rotational detection. We are developing a photoacoustic system for finger vascular imaging using a ring-shaped array ultrasound transducer. By designing the ring-array based on simulations and phantom experiments, we have created a system that can image multiple objects of different diameters and has the potential to image small objects 0.1-0.5mm in diameter at accurate positions by providing PA and ultrasound echo images simultaneously. In addition, we determined that full width at half maximum (FWHM) of the slice direction corresponded to that of the simulation. In the future, this system may visualize the 3-D vascularization of RA patients’ fingers.

Traditional back-projection(BP) method suffers from low resolution and high peak sidelobe level (PSL). A new method named eigenspace based adaptive beamforming (EAB) is proposed for photoacoustic computed tomography (PACT). Adaptive beamforming (AB) suppresses the interference and noise signal to improve the lateral resolution, eigenspace can further restrain the off-axis signal to eliminate the artifacts. The simulated experiment shows that the lateral resolution after BP, AB and EAB are 0.99, 0.31, and 0.31mm, respectively. The actual point targets experimental results show that the new method improves the lateral resolution by 67.9% compared with BP, which is agreed with simulated results. New method also improves the PSL by 39.6dB and 42.6dB compared with BP and AB, eliminates the artifacts and improvs imaging quality of the PACT.

We present a prototype for all-optical photoacoustic projection imaging. By generating projection images, photoacoustic information of large volumes can be retrieved with less effort compared to common photoacoustic computed tomography where many detectors and/or multiple measurements are required. In our approach, an array of 60 integrating line detectors is used to acquire photoacoustic waves. The line detector array consists of fiber-optic MachZehnder interferometers, distributed on a cylindrical surface. From the measured variation of the optical path lengths of the interferometers, induced by photoacoustic waves, a photoacoustic projection image can be reconstructed. The resulting images represent the projection of the three-dimensional spatial light absorbance within the imaged object onto a two-dimensional plane, perpendicular to the line detector array. The fiber-optic detectors achieve a noise-equivalent pressure of 24 Pascal at a 10 MHz bandwidth. We present the operational principle, the structure of the array, and resulting images. The system can acquire high-resolution projection images of large volumes within a short period of time. Imaging large volumes at high frame rates facilitates monitoring of dynamic processes.

Light absorption by the chromophores (hemoglobin, melanin, water etc.) present in any biological tissue results in local temperature rise. This rise in temperature results in generation of pressure waves due to the thermoelastic expansion of the tissue. In a circular scanning photoacoustic computed tomography (PACT) system, these pressure waves can be detected using a single-element ultrasound transducer (SUST) (while rotating in full 360° around the sample) or using a circular array transducer. SUST takes several minutes to acquire the PA data around the sample whereas the circular array transducer takes only a fraction of seconds. Hence, for real time imaging circular array transducers are preferred. However, these circular array transducers are custom made, expensive and not easily available in the market whereas SUSTs are cheap and readily available in the market. Using SUST for PACT systems is still cost effective. In order to reduce the scanning time to few seconds instead of using single SUST (rotating 360° ), multiple SUSTs can be used at the same time to acquire the PA data. This will reduce the scanning time by two-fold in case of two SUSTs (rotating 180° ) or by four-fold and eight-fold in case of four SUSTs (rotating 90° ) and eight SUSTs (rotating 45° ) respectively. Here we show that with multiple SUSTs, similar PA images (numerical and experimental phantom data) can be obtained as that of PA images obtained using single SUST.

In this study, we developed a novel PIID-DTBT based semiconducting polymer dots (Pdots) that have broad and strong optical absorption in the visible-light region (500 nm - 700 nm). Gold nanoparticles (GNPs) and gold nanorods (GNRs) that have been verified as an excellent photoacoustic contrast agent were compared with Pdots based on photoacoustic imaging method. Both ex vivo and in vivo experiment demonstrated Pdots have a better photoacoustic conversion efficiency at 532 nm than GNPs and similar photoacoustic performance with GNRs at 700 nm at the same mass concentration. Our work demonstrates the great potential of Pdots as a highly effective contrast agent for precise localization of lesions relative to the blood vessels based on photoacoustic tomography imaging.

Contrast agents which can be used for more than one bio-imaging technique has gained a lot of attention from researchers in recent years. In this work, a microfluidic device employing a flow-focusing junction, is used for the continuous generation of monodisperse nitrogen microbubbles in methylene blue, an optically absorbing organic dye, for dual-modal photoacoustic and ultrasound imaging. Using an external phase of polyoxyethylene glycol 40 stearate (PEG 40), a non-ionic surfactant, and 50% glycerol solution at a flow rate of 1 ml/hr and gas pressure at 1.75 bar, monodisperse nitrogen microbubbles of diameter 7 microns were obtained. The external phase also contained methylene blue hydrate at a concentration of 1 gm/litre. The monodisperse microbubbles produced a strong ultrasound signal as expected. It was observed that the signal-to-noise (SNR) ratio of the photoacoustic signal for the methylene blue solution in the presence of the monodisperse microbubbles was 68.6% lower than that of methylene blue solution in the absence of microbubbles. This work is of significance because using microfluidics, we can precisely control the bubbles’ production rate and bubble size which increases ultrasound imaging efficiency. A uniform size distribution of the bubbles will have narrower resonance frequency bandwidth which will respond well to specific ultrasound frequencies.

Photoacoustic (PA) imaging is a biomedical imaging method that can provide both structural and functional information of living tissues beyond the optical diffusion limit by combining the concepts of conventional optical and ultrasound imaging methods. Although endogenous chromophores can be utilized to acquire PA images of biological tissues, exogenous contrast agents that absorb near-infrared (NIR) lights have been extensively explored to improve the contrast and penetration depth of PA images. Here, we demonstrate Bi2Se3 nanoplates, that strongly absorbs NIR lights, as a contrast agent for PA imaging. In particularly, the Bi₂Se₃ nanoplates produce relatively strong PA signals with an optical wavelength of 1064 nm, which has several advantages for deep tissue imaging including: (1) relatively low absorption by other intrinsic chromophores, (2) cost-effective light source using Nd:YAG laser, and (3) higher available energy than other NIR lights according to American National Standards Institute (ANSI) safety limit. We have investigated deep tissue imaging capability of the Bi2Se3 nanoplates by acquiring in vitro PA images of microtubes under chicken breast tissues. We have also acquired in vivo PA images of bladders, gastrointestinal tracts, and sentinel lymph nodes in mice after injection of the Bi₂Se₃ nanoplates to verify their applicability to a variety of biomedical research. The results show the promising potential of the Bi₂Se₃ nanoplates as a PA contrast agent for deep tissue imaging with an optical wavelength of 1064 nm.

More than 80% of the ovarian cancers are diagnosed at late stages and the survival rate is less than 50%. Currently, there is no effective screening technique available and transvaginal US can only tell if the ovaries are enlarged or not. We have developed a new real-time co-registered US and photoacoustic system for in vivo imaging and characterization of ovaries. US is used to localize ovaries and photoacoustic imaging provides functional information about ovarian tissue angiogenesis and oxygenation saturation. The system consists of a tunable laser and a commercial US system from Alpinion Inc. The Alpinion system is cable of providing channel data for both US pulse-echo and photoacoustic imaging and can be programmed as a computer terminal for display US and photoacoustic images side by side or in coregistered mode. A transvaginal ultrasound probe of 6-MHz center frequency and bandwidth of 3-10 MHz is coupled with four optical fibers surrounded the US probe to deliver the light to tissue. The light from optical fibers is homogenized to ensure the power delivered to the tissue surface is below the FDA required limit. Physicians can easily navigate the probe and use US to look for ovaries and then turn on photoacoustic mode to provide real-time tumor vasculature and So2 saturation maps. With the optimized system, we have successfully imaged first group of 7 patients of malignant, abnormal and benign ovaries. The results have shown that both photoacoustic signal strength and spatial distribution are different between malignant and abnormal and benign ovaries.

In photoacoustic (PA) imaging, the optical absorption can be acquired from the initial pressure distribution (IPD). An accurate reconstruction of the IPD will be very helpful for the reconstruction of the optical absorption. However, the image quality of PA imaging in scattering media is deteriorated by the acoustic diffraction, imaging artifacts, and weak PA signals. In this paper, we propose a sparsity-based optimization approach that improves the reconstruction of the IPD in PA imaging. A linear imaging forward model was set up based on time-and-delay method with the assumption that the point spread function (PSF) is spatial invariant. Then, an optimization equation was proposed with a regularization term to denote the sparsity of the IPD in a certain domain to solve this inverse problem. As a proof of principle, the approach was applied to reconstructing point objects and blood vessel phantoms. The resolution and signal-to-noise ratio (SNR) were compared between conventional back-projection and our proposed approach. Overall these results show that computational imaging can leverage the sparsity of PA images to improve the estimation of the IPD.

In this paper, a novel matrix used in regularization term is proposed to acquire high quality reconstructed image. The use of Central-Enhancement Laplace (CEL) matrix is essentially a improvement of the conventional L2-norm regularization algorithm. By adding this matrix into the regularization term, we can obtain the reconstructed images with higher quality. The use of CEL matrix can enhance the edge information of images while reducing the reconstruction artifacts. Combined with the results of the in-vivo reconstructed images, we can confirm the above properties of this matrix. More importantly, the reconstructed images of L2-norm regularization algorithm using CEL matrix can achieve better reconstruction quality than some of the complex L1-norm regularization algorithms. Due to the flexibility of the matrix center element settings, we can also weigh the smoothness and fineness of the reconstructed image as needed.

To fully realize the potential of photoacoustic tomography (PAT) in preclinical and clinical applications, rapid measurements and robust reconstructions are needed. Sparse-view measurements have been adopted effectively to accelerate the data acquisition. However, since the reconstruction from the sparse-view sampling data is challenging, both of the effective measurement and the appropriate reconstruction should be taken into account. In this study, we present an iterative sparse-view PAT reconstruction scheme where a virtual parallel-projection concept matching for the proposed measurement condition is introduced to help to achieve the “compressive sensing” procedure of the reconstruction, and meanwhile the spatially adaptive filtering fully considering the a priori information of the mutually similar blocks existing in natural images is introduced to effectively recover the partial unknown coefficients in the transformed domain. Therefore, the sparse-view PAT images can be reconstructed with higher quality compared with the results obtained by the universal back-projection (UBP) algorithm in the same sparse-view cases. The proposed approach has been validated by simulation experiments, which exhibits desirable performances in image fidelity even from a small number of measuring positions.

Applying standard algorithms to sparse data problems in photoacoustic tomography (PAT) yields low-quality images containing severe under-sampling artifacts. To some extent, these artifacts can be reduced by iterative image reconstruction algorithms which allow to include prior knowledge such as smoothness, total variation (TV) or sparsity constraints. These algorithms tend to be time consuming as the forward and adjoint problems have to be solved repeatedly. Further, iterative algorithms have additional drawbacks. For example, the reconstruction quality strongly depends on a-priori model assumptions about the objects to be recovered, which are often not strictly satisfied in practical applications. To overcome these issues, in this paper, we develop direct and efficient reconstruction algorithms based on deep learning. As opposed to iterative algorithms, we apply a convolutional neural network, whose parameters are trained before the reconstruction process based on a set of training data. For actual image reconstruction, a single evaluation of the trained network yields the desired result. Our presented numerical results (using two different network architectures) demonstrate that the proposed deep learning approach reconstructs images with a quality comparable to state of the art iterative reconstruction methods.

Photoacoustic microscopy (PAM) is a hybrid imaging technology using optical illumination and acoustic detection. PAM is divided into two types: optical-resolution PAM (OR-PAM) and acoustic-resolution photoacoustic microscopy (AR-PAM). Among them, AR-PAM has a great advantage in the penetration depth compared to OR-PAM because ARPAM relies on the acoustic focus, which is much less scattered in biological tissue than optical focus. However, because the acoustic focus is not as tight as the optical focus with a same numerical aperture (NA), the AR-PAM requires acoustic NA higher than optical NA. The high NA of the acoustic focus produces good image quality in the focal zone, but significantly degrades spatial resolution and signal-to-noise ratio (SNR) in the out-of-focal zone. To overcome the problem, synthetic aperture focusing technique (SAFT) has been introduced. SAFT improves the degraded image quality in terms of both SNR and spatial resolution in the out-of-focus zone by calculating the time delay of the corresponding signals and combining them. To extend the dimension of correction effect, several 2D SAFTs have been introduced, but there was a problem that the conventional 2D SAFTs cannot improve the degraded SNR and resolution as 1D SAFT can do. In this study, we proposed a new 2D SAFT that can compensate the distorted signals in x and y directions while maintaining the correction performance as the 1D SAFT.

To quantify the functional and structural information of peripheral blood vessels for diagnoses of diseases which affects peripheral blood vessels such as diabetes and peripheral vascular disease, a 3D quantitative photoacoustic tomography (QPAT) reconstructing the optical properties such as the absorption coefficient reflecting microvascular structures and hemoglobin concentration and oxygenation saturation is studied. QPAT image reconstruction algorithms based on radiative transfer equation (RTE) and photon diffusion equation (PDE) have been proposed. However, it is not easy to use RTE in the clinical practice because of the huge computational load and long calculation time. On the other hand, it is always considered problematic to use PDE, because it does not approximate RTE well near the illuminating position. In this study, we developed the 3D QPAT image reconstruction using Monte Carlo (MC) method which approximates RTE better than PDE to reconstruct the optical properties in the region near the illuminating surface. To reduce the calculation time, we applied linearization. The QPAT image reconstruction algorithm with MC method and linearization was examined in numerical simulations and phantom experiment by use of a scanning system with a single probe consisting of P(VDF-TrFE) piezo electric film and optical fiber.

The photoacoustic image reconstruction problem (inverse problem) is to estimate an initial acoustic pressure distribution from measurements of ultrasound waves generated within an object due to optical excitation with a short light pulse. In this work, the recently suggested Bayesian approach to photoacoustic tomography is extended to three dimensions and an iterative matrix-free method for the solution of the problem is described. Image reconstruction is investigated with numerical simulations and experimental data. The use of different prior information and noise models in different sensor geometries, including a limited-view setup, is investigated. The results show that the Bayesian approach can produce accurate estimates of the initial pressure distribution even in a limited-view setup provided that prior information and the noise have been properly modelled.

Photoacoustic (PA) signal of an ideal optical absorb particle is a single N-shape wave. PA signals of a complicated biological tissue can be considered as the combination of individual N-shape waves. However, the N-shape wave basis not only complicates the subsequent work, but also results in aliasing between adjacent micro-structures, which deteriorates the quality of the final PA images. In this paper, we propose a method to improve PA image quality through signal processing method directly working on raw signals, which including deconvolution and empirical mode decomposition (EMD). During the deconvolution procedure, the raw PA signals are de-convolved with a system dependent point spread function (PSF) which is measured in advance. Then, EMD is adopted to adaptively re-shape the PA signals with two constraints, positive polarity and spectrum consistence. With our proposed method, the built PA images can yield more detail structural information. Micro-structures are clearly separated and revealed. To validate the effectiveness of this method, we present numerical simulations and phantom studies consist of a densely distributed point sources model and a blood vessel model. In the future, our study might hold the potential for clinical PA imaging as it can help to distinguish micro-structures from the optimized images and even measure the size of objects from deconvolved signals.

An endoscopic probe with high resolution and multiple contrasts provides useful diagnostic information. For example, a miniature probe capable of multimodal imaging including photoacoustic imaging, optical coherence tomography (OCT), and ultrasound has been reported. However, microscale resolution is only realized in OCT modality in the probe, which may restrict the applications where high resolutions for multiple contrasts are required. Here, we present an approach to construct a miniature scanhead with 2.8 mm in diameter that achieves high-resolution multispectral photoacoustic microscopy (PAM) and OCT. The method realizes high resolution of ∼10 μm for both PAM and OCT. We experimentally demonstrate ex vivo and in vivo imaging using the scanhead.

Photoacoustic tomography (PAT) is a novel modality that can visualize blood vessels without contrast agents. It clearly shows blood vessels near the body surface. However, these vessels obstruct the observation of deep blood vessels. As the existence range of each vessel is determined by the distance from the body surface, they can be separated if the position of the skin is known. However, skin tissue, which does not contain hemoglobin, does not appear in PAT results, therefore, manual estimation is required. As this task is very labor-intensive, its automation is highly desirable. Therefore, we developed a method to estimate the body surface using the cloth-simulation technique, which is a commonly used method to create computer graphics (CG) animations; however, it has not yet been employed for medical image processing. In cloth simulations, the virtual cloth is represented by a two-dimensional array of mass nodes. The nodes are connected with each other by springs. Once the cloth is released from a position away from the body, each node begins to move downwards under the effect of gravity, spring, and other forces; some of the nodes hit the superficial vessels and stop. The cloth position in the stationary state represents the body surface. The body surface estimation, which required approximately 1 h with the manual method, is automated and it takes only approximately 10 s with the proposed method. The proposed method could facilitate the practical use of PAT.

We propose a differential photoacoustic spectroscopy (PAS), wherein two wavelengths of light with the same absorbance are selected, and differential signal is linearized by one of the two signals for a non-invasive blood glucose monitoring. PAS has the possibility to overcome the strong optical scattering in tissue, but there are still remaining issues: the water background and instability due to the variation in acoustic resonance conditions. A change in sample solution temperature is one of the causes of the variation in acoustic resonance conditions. Therefore, in this study, we investigated the sensitivity against glucose concentration under the condition where the temperature of the sample water solution ranges 30 to 40 °C. The glucose concentration change is simulated by shifting the wavelength of irradiated laser light, which can effectively change optical absorption. The temperature also affects optical absorption and the acoustic resonance condition (acoustic velocity). A distributed-feedback (DFB) laser, tunable wavelength laser (TWL) and an acoustic sensor were used to obtain the differential PAS signal. The wavelength of the DFB laser was 1.382 μm, and that of TWL was switched from 1.600 to 1.610 μm to simulate the glucose concentration change. Optical absorption by glucose occurs at around 1.600 μm. The sensitivities against temperature are almost the same: 1.9 and 1.8 %/°C for 1.600 and 1.610 μm. That is, the glucose dependence across the whole temperature range remains constant. This implies that temperature correction is available.

We previously proposed a method of removing reflection artifacts in photoacoustic images that uses deep learning. Our approach generally relies on using simulated photoacoustic channel data to train a convolutional neural network (CNN) that is capable of distinguishing sources from artifacts based on unique differences in their spatial impulse responses (manifested as depth-based differences in wavefront shapes). In this paper, we directly compare a CNN trained with our previous continuous transducer model to a CNN trained with an updated discrete acoustic receiver model that more closely matches an experimental ultrasound transducer. These two CNNs were trained with simulated data and tested on experimental data. The CNN trained using the continuous receiver model correctly classified 100% of sources and 70.3% of artifacts in the experimental data. In contrast, the CNN trained using the discrete receiver model correctly classified 100% of sources and 89.7% of artifacts in the experimental images. The 19.4% increase in artifact classification accuracy indicates that an acoustic receiver model that closely mimics the experimental transducer plays an important role in improving the classification of artifacts in experimental photoacoustic data. Results are promising for developing a method to display CNN-based images that remove artifacts in addition to only displaying network-identified sources as previously proposed.

Medium intensity focused ultrasound (MIFU) holds promise in important clinical applications. Generally, the aim in MIFU is to stimulate physiological mechanisms that reinforce healing responses, avoiding reaching temperatures that can cause permanent tissue damage. The outcome of interventions is then strongly affected by the temperature distribution in the treated region, and accurate monitoring represents a significant clinical need. In this work, we showcase the capacities of 4D optoacoustic imaging to monitor tissue heating during MIFU. The proposed method allows localizing the ultrasound focus, estimating the peak temperature and measuring the size of the heat-affected volume. Calibration experiments in a tissue-mimicking phantom demonstrate that the optoacoustically-estimated temperature accurately matches thermocouple readings. The good performance of the suggested approach in real tissues is further showcased in experiments with bovine muscle samples.

We previously derived spatial coherence theory to be implemented for studying theoretical properties of ShortLag Spatial Coherence (SLSC) beamforming applied to photoacoustic images. In this paper, our newly derived theoretical equation is evaluated to generate SLSC images of a point target and a 1.2 mm diameter target and corresponding lateral profiles. We compared SLSC images simulated solely based on our theory to SLSC images created after beamforming acoustic channel data from k-Wave simulations of 1.2 mm-diameter disc target. This process was repeated for a point target and the full width at half the maximum signal amplitudes were measured to estimate the resolution of each imaging system. Resolution as a function of lag was comparable for the first 10% of the receive aperture (i.e., the short-lag region), after which resolution measurements diverged by a maximum of 1 mm between the two types of simulated images. These results indicate the potential for both simulation methods to be utilized as independent resources to study coherence-based photoacoustic beamformers when imaging point-like targets.

Photoacoustic (PA) imaging technology is expected to be applied to clinical assessment for peripheral vascularity. We started a clinical evaluation with the prototype PA imaging system we recently developed. Prototype PA imaging system was composed with in-house Q-switched Alexandrite laser system which emits short-pulsed laser with 750 nm wavelength, handheld ultrasound transducer where illumination optics were integrated and signal processing for PA image reconstruction implemented in the clinical ultrasound (US) system. For the purpose of quantitative assessment of PA images, an image analyzing function has been developed and applied to clinical PA images. In this analyzing function, vascularity derived from PA signal intensity ranged for prescribed threshold was defined as a numerical index of vessel fulfillment and calculated for the prescribed region of interest (ROI). Skin surface was automatically detected by utilizing B-mode image acquired simultaneously with PA image. Skinsurface position is utilized to place the ROI objectively while avoiding unwanted signals such as artifacts which were imposed due to melanin pigment in the epidermal layer which absorbs laser emission and generates strong PA signals. Multiple images were available to support the scanned image set for 3D viewing. PA images for several fingers of patients with systemic sclerosis (SSc) were quantitatively assessed. Since the artifact region is trimmed off in PA images, the visibility of vessels with rather low PA signal intensity on the 3D projection image was enhanced and the reliability of the quantitative analysis was improved.

Channel data acquisition, and synchronization between laser excitation and PA signal acquisition, are two fundamental hardware requirements for photoacoustic (PA) imaging. Unfortunately, however, neither is equipped by most clinical ultrasound scanners. Therefore, less economical specialized research platforms are used in general, which hinders a smooth clinical transition of PA imaging. In previous studies, we have proposed an algorithm to achieve PA imaging using ultrasound post-beamformed (USPB) RF data instead of channel data. This work focuses on enabling clinical ultrasound scanners to implement PA imaging, without requiring synchronization between the laser excitation and PA signal acquisition. Laser synchronization is inherently consisted of two aspects: frequency and phase information. We synchronize without communicating the laser and the ultrasound scanner by investigating USPB images of a point-target phantom in two steps. First, frequency information is estimated by solving a nonlinear optimization problem, under the assumption that the segmented wave-front can only be beamformed into a single spot when synchronization is achieved. Second, after making frequencies of two systems identical, phase delay is estimated by optimizing the image quality while varying phase value. The proposed method is validated through simulation, by manually adding both frequency and phase errors, then applying the proposed algorithm to correct errors and reconstruct PA images. Compared with the ground truth, simulation results indicate that the remaining errors in frequency correction and phase correction are 0.28% and 2.34%, respectively, which affirm the potential of overcoming hardware barriers on PA imaging through software solution.

The recent development of novel transdermal drug delivery systems (TDDS) using microneedle technology allows micron-sized conduits to be formed within the outermost skin layers attracting keen interest in skin as an interface for localized and systemic delivery of therapeutics. In light of this, researchers are using microneedles as tools to deliver nanoparticle formulations to targeted sites for effective therapy. However, in such studies the use of traditional histological methods are employed for characterization and do not allow for the in vivo visualization of drug delivery mechanism. Hence, this study presents a novel imaging technology to characterize microneedle based nanoparticle delivery systems using optical resolution-photoacoustic microscopy (OR-PAM). In this study in vivo transdermal delivery of gold nanoparticles using microneedles in mice ear and the spatial distribution of the nanoparticles in the tissue was successfully illustrated. Characterization of parameters that are relevant in drug delivery studies such as penetration depth, efficiency of delivered gold nanoparticles were monitored using the system. Photoacoustic microscopy proves an ideal tool for the characterization studies of microneedle properties and the studies shows microneedles as an ideal tool for precise and controlled drug delivery.

In order to understand the pathophysiology of glaucoma, Lamina cribrosa (LC) perfusion needs to be the subject of thorough investigation. It is currently difficult to obtain high resolution images of the embedded microcapillary network of the LC using conventional imaging techniques. In this study, an optical resolution photoacoustic microscopy (OR-PAM) system was used for imaging lamina cribrosa of an ex vivo porcine eye. Extrinsic contrast agent was used to perfuse the eye via its ciliary arteries. The OR-PAM system have a lateral resolution of 4 μm and an axial resolution of 30 μm. The high resolution system could able resolve a perfused LC microcapillary network to show vascular structure within the LC thickness. OR-PAM could be a promising imaging modality to study the LC perfusion and hence could be used to elucidate the hemodynamic aspect of glaucoma.

It is always a great challenge for pure optical techniques to maintain good resolution and imaging depth at the same time. Photoacoustic imaging is an emerging technique which can overcome the limitation by pulsed light illumination and acoustic detection. Here, we report a Near Infrared Acoustic-Resolution Photoacoustic Microscopy (NIR-AR-PAM) systm with 30 MHz transducer and 1064 nm illumination which can achieve a lateral resolution of around 88 μm and imaging depth of 9.2 mm. Compared to visible light NIR beam can penetrate deeper in biological tissue due to weaker optical attenuation. In this work, we also demonstrated the in vivo imaging capabilty of NIRARPAM by near infrared detection of SLN with black ink as exogenous photoacoustic contrast agent in a rodent model.

Super-resolution microscopy has been increasingly important to delineate nanoscale biological structures or nanoparticles. With these increasing demands, several imaging modalities, including super-resolution fluorescence microscope (SRFM) and electron microscope (EM), have been developed and commercialized. These modalities achieve nanoscale resolution, however, SRFM cannot image without fluorescence, and sample preparation of EM is not suitable for biological specimens. To overcome those disadvantages, we have numerically studied the possibility of superresolution photoacoustic microscopy (SR-PAM) based on near-field localization of light. Photoacoustic (PA) signal is generally acquired based on optical absorption contrast; thus it requires no agents or pre-processing for the samples. The lateral resolution of the conventional photoacoustic microscopy is limited to ~200 nm by diffraction limit, therefore reducing the lateral resolution is a major research impetus. Our approach to breaking resolution limit is to use laser pulses of extremely small spot size as a light source. In this research, we simulated the PA signal by constructing the three dimensional SR-PAM system environment using the k-Wave toolbox. As the light source, we simulated ultrashort light pulses using geometrical nanoaperture with near-field localization of surface plasmons. Through the PA simulation, we have successfully distinguish cuboids spaced 3 nm apart. In the near future, we will develop the SR-PAM and it will contribute to biomedical and material sciences.

Current clinical available retinal imaging techniques have limitations, including limited depth of penetration or requirement for the invasive injection of exogenous contrast agents. Here, we developed a novel multimodal imaging system for high-speed, high-resolution retinal imaging of larger animals, such as rabbits. The system integrates three state-of-the-art imaging modalities, including photoacoustic microscopy (PAM), optical coherence tomography (OCT), and fluorescence microscopy (FM). In vivo experimental results of rabbit eyes show that the PAM is able to visualize laser-induced retinal burns and distinguish individual eye blood vessels using a laser exposure dose of ~80 nJ, which is well below the American National Standards Institute (ANSI) safety limit 160 nJ. The OCT can discern different retinal layers and visualize laser burns and choroidal detachments. The novel multi-modal imaging platform holds great promise in ophthalmic imaging.

We present a system for large depth-of-field photoacoustic scanning macroscopy (PASMac). Photoacoustic waves are detected optically with a fiber-optic annular detector array. Pressure changes, induced by the photoacoustic waves, modulate the refractive index in the fibers. The resulting variation of the optical path is measured by interferometric means. The ring-shape of the fibers results in a higher sensitivity to pressure waves stemming from the ring symmetry axis compared to off-axis signals. Hence, the fiber-optic ring-shaped detector array embodies a focused ultrasound transducer, however with a greatly extended depth-of-field. To reduce off-axis sensitivity, the signals from multiple rings with varying diameters are summed up using coherence weighting. By raster scanning of the sensor array, threedimensional or cross-sectional images are formed. We report on the design of an enhanced detector with 8 concentric rings for targeted imaging depths ranging from 5 mm to 50 mm.

Obesity with a body mass index is greater than 30 kg/m² is one of the rapidly growing diseases in advanced societies and can lead to stroke, type 2 diabetes, and heart failure. Common methods of removing subcutaneous adipose tissues are liposuction and laser treatment. In this study, we used photoacoustic imaging to monitor the anti-obesity photothermal degradation process. To improve the photothermal lipid degradation efficiency without any invasive methods, we synthesized hyaluronic acid hollow hold nanosphere adipocyte targeting sequence peptide (HA-HAuNS-ATS) conjugates. The conjugate enhanced the skin penetration ability and biodegradability of the nanoparticles using hyaluronate and enhanced the targeting effect on adipose tissue with adipocyte targeting sequence peptide. Thus, the conjugate can be delivered to the adipose tissue by simply spreading the conjugate on the skin without any invasive method. Then, the photothermal lipolysis and delivery of the conjugate were photoacoustically monitored in vivo. These results demonstrate the potential for photoacoustic method to be applied for photothermal lipolysis monitoring.

In this work, protein-modified hydrophilic copper sufide (CuS) nanotriangles with tunable absorption in the second near-infrared (NIR-II) region are developed, which can be served as contrast agents for enhanced in vivo photoacoustic imaging. In vitro and in vivo toxicity analysis are also performed, which show that the nanoprobes are biocompatible for most of the test cases. As a result, the nanoprones is able to pave a new avenue for improving the photoacoustic imaigng contrast and penetration depth in cancer detection. It should be pointed out that other functional blocks may also be linked on it, which makes it a general method to design multifunctional nanoprobes.

Monitoring dynamic interactions of T cells migrating toward tumor is beneficial to understand how cancer immunotherapy works. Optical-resolution photoacoustic microscope (OR-PAM) can provide not only high spatial resolution but also deeper penetration than conventional optical microscopy. With the aid of exogenous contrast agents, the dual-wavelength OR-PAM can be applied to map the distribution of CD8+ cytotoxic T lymphocytes (CTLs) with gold nanospheres (AuNS) under 523nm laser irradiation and Hepta1-6 tumor spheres with indocyanine green (ICG) under 800nm irradiation. However, at 1K laser PRF, it takes approximately 20 minutes to obtain a full sample volume of 160 × 160 × 150 μm³ . To increase the imaging rate, we propose a random non-uniform sparse sampling mechanism to achieve fast sparse photoacoustic data acquisition. The image recovery process is formulated as a low-rank matrix recovery (LRMR) based on compressed sensing (CS) theory. We show that it could be stably recovered via nuclear-norm minimization optimization problem to maintain image quality from a significantly fewer measurement. In this study, we use the dual-wavelength OR-PAM with CS to visualize T cell trafficking in a 3D culture system with higher temporal resolution. Data acquisition time is reduced by 40% in such sample volume where sampling density is 0.5. The imaging system reveals the potential to understand the dynamic cellular process for preclinical screening of anti-cancer drugs.

The formation of amyloid – aggregate of misfolded proteins – is associated with more than 50 human pathologies, including Alzheimer’s disease, Parkinson’s disease, and Type 2 diabetes mellitus. Investigating protein aggregation is a critical step in drug discovery and development of therapeutics targeted to these pathologies. However, screens to identify protein aggregates are challenging due to the stochastic character of aggregate nucleation. Here we employ photoacoustics (PA) to screen thermodynamic conditions and solution components leading to formation of protein aggregates. Particularly, we study the temperature dependence of the Gruneisen parameter in optically-contrasted, undersaturated and supersaturated solutions of glycoside hydrolase (lysozyme). As nucleation of protein aggregates proceeds in two steps, where the first is liquid-liquid separation (rearrangement of solute’s density), the PA response from complex solutions and its temperature-dependence monitor nucleation and differentiate undersaturated and supersaturated protein solutions. We demonstrate that in the temperature range from 22 to 0° C the PA response of contrasted undersaturated protein solution behaves similar to water and exhibits zero thermal expansion at 4°C or below, while the response of contrasted supersaturated protein solution is nearly temperature independent, similar to the behavior of oils. These results can be used to develop a PA assay for high-throughput screening of multi-parametric conditions (pH, ionic strength, chaperone, etc.) for protein aggregation that can become a key tool in drug discovery, targeting aggregate formation for a variety of amyloids.

In this paper we discuss the influence of the excited state lifetimes of molecules onto the photoacoustic signal generation. Usually, photoacoustic signal generation is performed either with nanosecond laser pulses or modulation frequencies in the MHz up to the GHz regime. The molecules’ singlet relaxation times typically lie in the order of nanoseconds, while triplet lifetimes can be considerably longer. As both time scales, i.e. the laser pulse duration used for excitation and the relaxation times, lie in the same order of magnitude there exists an influence of the excited state lifetimes onto the photoacoustic signal generation. Here, we theoretically discuss at which frequencies this influence becomes significant and what impact this has on the performance and optimum design of photoacoustic microscopes.

Visualizing biological markers and delivering bioactive agents to living organisms are important to biological research. In recent decades, photoacoustic imaging (PAI) has been significantly improved in the area of molecular imaging, which provides high-resolution volume imaging with high optical absorption contrast. To demonstrate the ability of nanoprobes to target tumors using PAI, we synthesize convertible nanostructured agents with strong photothermal and photoacoustic properties and linked the nanoprobe with biotin to target tumors in small animal model. Interestingly, these nanoprobes allow partial to disassemble in the presence of targeted proteins that switchable photoactivity, thus the nanoprobes provides a fluorescent-cancer imaging with high signal-to-background ratios. The proposed nanoprobe produce a much stronger PA signal compared to the same concentration of methylene blue (MB), which is widely used in clinical study and contrast agent for PAI. The biotin conjugated nanoprobe has high selectivity for biotin receptor positive cancer cells such as A549 (human lung cancer). Then we subsequently examined the PA properties of the nanoprobe that are inherently suitable for in vivo PAI. After injecting of the nanoprobe via intravenous method, we observed the mice’s whole body by PA imaging and acquired the PA signal near the cancer. The PA signal increased linearly with time after injection and the fluorescence signal near the cancer was confirmed by fluorescence imaging. The ability to target a specific cancer of the nanoprobe was well verified by PA imaging. This study provides valuable perspective on the advancement of clinical translations and in the design of tumor-targeting phototheranostic agents that could act as new nanomedicines.

Determination of the precise location and degree of condition of the Choroidal neovascularization (CNV) lesion is essential for diagnosation Neovascular age-related macular degeneration (AMD) and evaluation the efficacy of treatment. Given the complimentary contrast mechanisms of Photoacoustic microscopy (PAM) and Optical coherence tomography (OCT), the combination of PAM and OCT imaging could potentially provide much sensitive and specific detection of CNV. In this paper, we validated the opportunity to evaluate the information of laser-induced CNV and presented the in vivo time-serial evaluation of the CNV by simultaneously using PAM and OCT techniques. In vivo PAM and OCT examination was performed after laser photocoagulation applied to the rat fundus at days 1, 3, 5, 7, 14. Time-serial results showed that CNV in rats increased to its maximum at day 7 and decreased at day 14. Evolution of CNV information was given in PAM images with a high contrast and details of high axial resolution OCT images were simultaneously given to show the hyperreflective reaction progress.

Fluorescence imaging is widely employed in all fields of cell and molecular biology due to its high sensitivity, high contrast and ease of implementation. However, the low spatial resolution and lack of depth information, especially in strongly-scattering samples, restrict its applicability for deep-tissue imaging applications. On the other hand, optoacoustic imaging is known to deliver a unique set of capabilities such as high spatial and temporal resolution in three dimensions, deep penetration and spectrally-enriched imaging contrast. Since fluorescent substances can generate contrast in both modalities, simultaneous fluorescence and optoacoustic readings can provide new capabilities for functional and molecular imaging of living organisms. Optoacoustic images can further serve as valuable anatomical references based on endogenous hemoglobin contrast. Herein, we propose a hybrid system for in vivo real-time planar fluorescence and volumetric optoacoustic tomography, both operating in reflection mode, which synergistically combines the advantages of stand-alone systems. Validation of the spatial resolution and sensitivity of the system were first carried out in tissue mimicking phantoms while in vivo imaging was further demonstrated by tracking perfusion of an optical contrast agent in a mouse brain in the hybrid imaging mode. Experimental results show that the proposed system effectively exploits the contrast mechanisms of both imaging modalities, making it especially useful for accurate monitoring of fluorescence-based signal dynamics in highly scattering samples.

In this paper, we report a new photoacoustic sensing probe design consisting of two optical fibers. One optical fiber is used for delivering the excitation light pulses. The other one serves as an acoustic delay line to relay the generated PA signal from the target to an outside ultrasound transducer. With the addition of suitable time delay, the original PA signal can be easily separated from the interference signals. To demonstrate this new design, a prototype probe was designed, fabricated and tested. The PA sensing performance was characterized with different concentration of black dye solutions. The testing results show that the PA sensing probe can provide good sensitivity and can maintain high linearity over a wide range of concentrations.

Sensitive detection is always crucial to photoacoustic sensing and imaging applications owing to the extremely low conversion efficiency from light to sound. Conventional approach to enhance the signal-to-noise ratio (SNR) of the photoacoustic signal is data averaging, which is quite time-consuming due to multiple data acquisitions for each photoacoustic measurement. Especially for high power pulsed laser source with only 10-20 pulse repetition rate, multiple data averaging will severely degrade the frame rate. In this paper, we present a simple but efficient way, called adaptive coherent photoacoustic (aCPA) sensing to obviously enhance the detected signal SNR with only single laser pulse. More specifically, The proposed aCPA employs an adaptive matched filter to cross-correlate with the raw time-domain PA signal iteratively. The optimum matched filter could be found after several iterations, leading to improved signal SNR. In vivo experimental results show that the proposed aCPA method improved the signal SNR by about 60 dB with single PA measurement. In conventional data averaging, 106 times PA measurements is required to achieve same SNR improvement. In other words, sensing and imaging speed is improved by 10⁶ times in theory. It demonstrates the potential of aCPA to perform highly sensitive photoacoustic sensing and imaging with significantly accelerated speed.

Light propagation in turbid media, such as biological tissues, experience scattering due to inhomogeneous distribution of refractive indices. Control of light scattering is important for focusing the light or imaging through scattering medium. By spatially shaping the wave-front of the incident beam, using spatial light modulator (SLM), the scattered light can be focused to a point. Iterative optimization is a popular way of obtaining the most optimum phase map on the SLM. In this study, we implement six optimization algorithms including continuous sequential, partitioning algorithm, transmission matrix estimation method, genetic algorithms, particle swarm optimization, and simulated annealing to obtain the optimum phase map of the SLM. The main characteristics of the algorithms such as convergence time, improvement ratio and performance are compared and discussed.

Despite the promising results of the recent novel transcranial photoacoustic (PA) brain imaging technology, it has been demonstrated that the presence of the skull severely affects the performance of this imaging modality. We theoretically investigate the effects of acoustic heterogeneity induced by skull on the PA signals generated from single particles, with firstly developing a mathematical model for this phenomenon and then explore experimental validation of the results. The model takes into account the frequency dependent attenuation and dispersion effects occur with wave reflection, refraction and mode conversion at the skull surfaces. Numerical simulations based on the developed model are performed for calculating the propagation of photoacoustic waves through the skull. The results show a strong agreement between simulation and ex-vivo study. The findings are as follow: The thickness of the skull is the most PA signal deteriorating factor that affects both its amplitude (attenuation) and phase (distortion). Also we demonstrated that, when the depth of target region is low and it is comparable to the skull thickness, however, the skull-induced distortion becomes increasingly severe and the reconstructed image would be strongly distorted without correcting these effects. It is anticipated that an accurate quantification and modeling of the skull transmission effects would ultimately allow for aberration correction in transcranial PA brain imaging.

In this research, we present the results of applying simulated annealing (SA) which is a heuristic optimization algorithm for focusing light through a turbid media. The performance of this algorithm on both phase optimization and amplitude optimization has been demonstrated. A guideline to set up the SA parameters is also suggested. The performance of SA algorithm in different levels of measurement noise has been also explored. The results showed that the SA algorithm performs effectively in measurement noises as high as 0.3⟨I0⟩.

In recent years, the minimum variance (MV) beamforming has been widely studied due to its high resolution and contrast in B-mode Ultrasound imaging (USI). However, the performance of the MV beamformer is degraded at the presence of noise, as a result of the inaccurate covariance matrix estimation which leads to a low quality image. Second harmonic imaging (SHI) provides many advantages over the conventional pulse-echo USI, such as enhanced axial and lateral resolutions. However, the low signal-to-noise ratio (SNR) is a major problem in SHI. In this paper, Eigenspace-based minimum variance (EIBMV) beamformer has been employed for second harmonic USI. The Tissue Harmonic Imaging (THI) is achieved by Pulse Inversion (PI) technique. Using the EIBMV weights, instead of the MV ones, would lead to reduced sidelobes and improved contrast, without compromising the high resolution of the MV beamformer (even at the presence of a strong noise). In addition, we have investigated the effects of variations of the important parameters in computing EIBMV weights, i.e., K, L, and δ, on the resolution and contrast obtained in SHI. The results are evaluated using numerical data (using point target and cyst phantoms), and the proper parameters of EIBMV are indicated for THI.

Non-invasive vascular elastography is an emerging technique in vascular tissue imaging. During the past decades, several techniques have been suggested to estimate the tissue elasticity by measuring the displacement of the Carotid vessel wall. Cross correlation-based methods are the most prevalent approaches to measure the strain exerted in the wall vessel by the blood pressure. In the case of a low pressure, the displacement is too small to be apparent in ultrasound imaging, especially in the regions far from the center of the vessel, causing a high error of displacement measurement. On the other hand, increasing the compression leads to a relatively large displacement in the regions near the center, which reduces the performance of the cross correlation-based methods. In this study, a non-rigid image registration-based technique is proposed to measure the tissue displacement for a relatively large compression. The results show that the error of the displacement measurement obtained by the proposed method is reduced by increasing the amount of compression while the error of the cross correlationbased method rises for a relatively large compression. We also used the synthetic aperture imaging method, benefiting the directivity diagram, to improve the image quality, especially in the superficial regions. The best relative root-mean-square error (RMSE) of the proposed method and the adaptive cross correlation method were 4.5% and 6%, respectively. Consequently, the proposed algorithm outperforms the conventional method and reduces the relative RMSE by 25%.

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. The conventional illumination method for photoacoustic is a called top-illumination were diffused light hit the target from one side. However, this method is not suitable for all target shapes and body parts like breast. To overcome this problem we proposed a novel side-illumination scheme where light comes from a set of fibers all around the object. We showed that this method can improve the obtained images with phantom experiments and simulations. This method is particularly useful for in-plane imaging where the ring of fibers scan long objects.

Photoacoustic imaging (PAI) is an imaging modality for obtaining absorption coefficient at every location inside the tissue based on the detected photoacoustic signals. PA image reconstruction aims to determine the initial PA pressure everywhere inside the tissue. The pressure is proportional to both absorption coefficient and light fluence. Provided that fluence is homogenous, the reconstructed image will be an accurate mapping of the absorption coefficient of the tissue. Here we presented a method for obtaining uniform fluence inside the region of interest. We created a large dataset of fluence maps for different source locations, diameters and numerical apertures with Monte Carlo simulations, then used this dataset to solve an optimization problem for finding the source configuration which results in the best fluence distribution.

The accurate quantification of lesions located in deep tissue is an important challenge in diffuse optical tomography (DOT), while photoacoustic tomography (PAT) as a non-invasive optical imaging provides high-resolution imaging of optical contrast in deep tissue that can be served as a complementary modality to improve the accuracy of DOT. Here, we coupled advantages of photoacoustic tomography (PAT) to diffuse optical tomography (DOT) for enhancing reconstructed DOT images. Using a priori information provided by PAT was used to reasonably regularize the DOT inversion procedure.. The results show that hybrid DOT-PAT can provide high-resolution image in deep tissue.