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Optical microscopy is indispensable to biomedical research and clinical investigations. As all molecules absorb light, optical-resolution photoacoustic microscopy (PAM) is an important tool to image molecules at high resolution without labeling. However, due to tissue-induced optical aberration, the imaging quality degrades with increasing imaging depth. To mitigate this effect, we develop an imaging method, called acoustic-feedback wavefront-adapted PAM (AWA-PAM), to dynamically compensate for tissue-induced aberration at depths. In contrast to most existing adaptive optics assisted optical microscopy, AWA-PAM employs acoustic signals rather than optical signals to indirectly determine the optimized wavefront. To demonstrate this technique, we imaged zebrafish embryos and mouse ears in vivo. Experimental results show that compensating for tissue-induced aberration in live tissue effectively improves both signal strength and lateral resolution. With this capability, AWA-PAM reveals fine structures, such as spinal cords and microvessels, that were otherwise unidentifiable using conventional PAM. We anticipate that AWA-PAM will benefit the in vivo imaging community and become an important tool for label-free optical imaging in the quasi-ballistic regime. © 2024 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement © 2024 Optica Publishing Group.

Photoacoustic tomography (PAT) is a rapidly-evolving medical imaging modality that combines optical absorption contrast with ultrasound imaging depth. One challenge in PAT is image reconstruction with inadequate acoustic signals due to limited sensor coverage or due to the density of the transducer array. Such cases call for solving an ill-posed inverse reconstruction problem. In this work, we use score-based diffusion models to solve the inverse problem of reconstructing an image from limited PAT measurements. The proposed approach allows us to incorporate an expressive prior learned by a diffusion model on simulated vessel structures while still being robust to varying transducer sparsity conditions., Comment: 5 pages

Understanding ultrafast dynamics in the femtosecond timescale plays a pivotal role in fundamental research and technology innovation. Spatiotemporal observation of those events in real-time requires imaging speeds greater than 10¹² frames per second (fps), far beyond the fundamental speed limits of the ubiquitous semiconductor sensor technologies. In addition, a majority of femtosecond events are non-repeatable or difficult-to-repeat since they either work in a highly unstable nonlinear regime or require extreme or rare conditions to initiate. Therefore, the traditional pump-probe imaging approach fails since it heavily depends on precise event repetition. Single-shot ultrafast imaging emerges as the only solution; however, existing techniques cannot reach more than 15×10¹² fps, and they only record an insufficient number of frames. Compressed ultrafast spectral photography (CUSP) is proposed to overcome these limitations. Here, CUSP's full design space is explored by manipulating the ultrashort optical pulse in the active illumination. Via parameter optimization, an extraordinarily fast frame rate of 219 × 10¹² fps is achieved. This implementation of CUSP is also highly flexible, allowing various combinations of imaging speeds and numbers of frames (several hundred up to 1000) to be readily deployed in diverse scientific studies, such as laser-induced transient birefringence, self-focusing, and filaments in dielectric media.

Imaging hemodynamics is crucial for the diagnosis, treatment, and prevention of vascular diseases. However, current imaging techniques are limited due to the use of ionizing radiation or contrast agents, short penetration depth, or complex and expensive data acquisition systems. Photoacoustic tomography shows promise as a solution to these issues. However, existing photoacoustic tomography methods collect signals either sequentially or through numerous detector elements, leading to either low imaging speed or high system complexity and cost. To address these issues, here we introduce a method to capture a 3D photoacoustic image of vasculature using a single laser pulse and a single-element detector that functions as 6,400 virtual ones. Our method enables ultrafast volumetric imaging of hemodynamics in the human body at up to 1 kHz and requires only a single calibration for different objects and for long-term operations. We demonstrate 3D imaging of hemodynamics at depth in humans and small animals, capturing the variability in blood flow speeds. This concept can inspire other imaging technologies and find applications such as home-care monitoring, biometrics, point-of-care testing, and wearable monitoring.

In many clinical conditions, such as head trauma, stroke, and low cardiac output states, the brain is at risk for hypoxic-ischemic injury. The metabolic rate and oxygen consumption of the brain are reflected in internal jugular venous oxygen saturation (sijvO2). The current gold standard for monitoring brain oxygenation is invasive; it requires jugular vein catheterization under fluoroscopic guidance and therefore is rarely used. Photoacoustic (PA) measurement, on the other hand, can estimate the oxygen consumption of the brain non-invasively in real time. Such a convenient method can potentially aid earlier detection and prevention of impending hypoxic brain injury. A dual-wavelength photoacoustic tomography (PAT) and ultrasound imaging (US) system was used to image the internal jugular vein (IJV) and estimate the sijvO2 in seven healthy volunteers. The system captured simultaneous co-registered PAT and US images at a rate of five frames per second. For each volunteer, the internal jugular vein was identified under ultrasound guidance, then, additional PA images from two optical wavelengths were collected and used to estimate the oxygen saturation of the internal jugular vein. For each volunteer, the oxygen saturation was calculated from transverse and longitudinal views of the internal jugular vein. The average sijvO2 was 72 +/- 7 %. The preliminary results are encouraging and agree with those reported in the literature. Photoacoustic images were successfully used to calculate the blood hemoglobin oxygen saturation in the internal jugular vein. These results raise confidence that this emerging technology can be used clinically for accurate, noninvasive estimation of sijvO2. In addition, the fast co-registration with US images makes the technique suitable for studying the temporal variations of oxygen saturation in response to physiologic challenges in clinical settings.

Obtaining frozen sections of bone tissue for intraoperative examination is challenging. To identify the bony edge of resection, orthopaedic oncologists therefore rely on pre-operative X-ray computed tomography or magnetic resonance imaging. However, these techniques do not allow for accurate diagnosis or for intraoperative confirmation of the tumour margins, and in bony sarcomas, they can lead to bone margins up to 10-fold wider (1,000-fold volumetrically) than necessary. Here, we show that real-time three-dimensional contour-scanning of tissue via ultraviolet photoacoustic microscopy in reflection mode can be used to intraoperatively evaluate undecalcified and decalcified thick bone specimens, without the need for tissue sectioning. We validate the technique with gold-standard haematoxylin-and-eosin histology images acquired via a traditional optical microscope, and also show that an unsupervised generative adversarial network can virtually stain the ultraviolet-photoacoustic-microscopy images, allowing pathologists to readily identify cancerous features. Label-free and slide-free histology via ultraviolet photoacoustic microscopy may allow for rapid diagnoses of bone-tissue pathologies and aid the intraoperative determination of tumour margins.

Clinical oncology can benefit substantially from imaging technologies that reveal physiological characteristics with multiscale observations. Complementing conventional imaging modalities, photoacoustic imaging (PAI) offers rapid imaging (for example, cross-sectional imaging in real time or whole-breast scanning in 10–15 s), scalably high levels of spatial resolution, safe operation and adaptable configurations. Most importantly, this novel imaging modality provides informative optical contrast that reveals details on anatomical, functional, molecular and histological features. In this Review, we describe the current state of development of PAI and the emerging roles of this technology in cancer screening, diagnosis and therapy. We comment on the performance of cutting-edge photoacoustic platforms, and discuss their clinical applications and utility in various clinical studies. Notably, the clinical translation of PAI is accelerating in the areas of macroscopic and mesoscopic imaging for patients with breast or skin cancers, as well as in microscopic imaging for histopathology. We also highlight the potential of future developments in technological capabilities and their clinical implications, which we anticipate will lead to PAI becoming a desirable and widely used imaging modality in oncological research and practice.

Objective: Ultra-rapid insulin formulations control postprandial hyperglycemia; however, inadequate understanding of injection site absorption mechanisms is limiting further advancement. We used photoacoustic imaging to investigate the injection site dynamics of dye-labeled insulin lispro in the Humalog and Lyumjev formulations using the murine ear cutaneous model and correlated it with results from unlabeled insulin lispro in pig subcutaneous injection model. Methods: We employed dual-wavelength optical-resolution photoacoustic microscopy to study the absorption and diffusion of the near-infrared dye-labeled insulin lispro in the Humalog and Lyumjev formulations in mouse ears. We mathematically modeled the experimental data to calculate the absorption rate constants and diffusion coefficients. We studied the pharmacokinetics of the unlabeled insulin lispro in both the Humalog and Lyumjev formulations as well as a formulation lacking both the zinc and phenolic preservative in pigs. The association state of insulin lispro in each of the formulations was characterized using SV-AUC and NMR spectroscopy. Results: Through experiments using murine and swine models, we show that the hexamer dissociation rate of insulin lispro is not the absorption rate-limiting step. We demonstrated that the excipients in the Lyumjev formulation produce local tissue expansion and speed both insulin diffusion and microvascular absorption. We also show that the diffusion of insulin lispro at the injection site drives its initial absorption; however, the rate at which the insulin lispro crosses the blood vessels is its overall absorption rate-limiting step. Conclusions: This study provides insights into injection site dynamics of insulin lispro and the impact of formulation excipients. It also demonstrates photoacoustic microscopy as a promising tool for studying protein therapeutics. The results from this study address critical questions around the subcutaneous behavior of insulin lispro and

Optical techniques offer a wide variety of applications as light-matter interactions provide extremely sensitive mechanisms to probe or treat target media. Most of these implementations rely on the usage of ballistic or quasi-ballistic photons to achieve high spatial resolution. However, the inherent scattering nature of light in biological tissues or tissue-like scattering media constitutes a critical obstacle that has restricted the penetration depth of non-scattered photons and hence limited the implementation of most optical techniques for wider applications. In addition, the components of an optical system are usually designed and manufactured for a fixed function or performance. Recent advances in wavefront shaping have demonstrated that scattering- or component-induced phase distortions can be compensated by optimizing the wavefront of the input light pattern through iteration or by conjugating the transmission matrix of the scattering medium. This offers unprecedented opportunities in many applications to achieve controllable optical delivery or detection at depths, or dynamically configurable functionalities by using scattering media to substitute conventional optical components. In this article, the recent progress of wavefront shaping in multidisciplinary fields is reviewed, from optical focusing and imaging with scattering media, functionalized devices, modulation of mode coupling and nonlinearity in multimode fiber, and to multimode fiber-based applications. Apart from insights into the underlying principles and recent advances in wavefront shaping implementations, practical limitations and roadmap for future development are discussed in depth. Looking back and looking forward, it is believed that wavefront shaping holds a bright future that will open new avenues for noninvasive or minimally invasive optical interactions and arbitrary control inside deep tissues. The high degree of freedom with multiple scattering will also provide unprecedented opportu

The last decade has seen the development of a wide set of tools, such as wavefront shaping, computational or fundamental methods, that allow to understand and control light propagation in a complex medium, such as biological tissues or multimode fibers. A vibrant and diverse community is now working on this field, that has revolutionized the prospect of diffraction-limited imaging at depth in tissues. This roadmap highlights several key aspects of this fast developing field, and some of the challenges and opportunities ahead.

Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics' agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, this status report reviews an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion report, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed, and provide an outlook for the future directions.

Recent advances in functional ultrasound imaging (fUS) and photoacoustic tomography (PAT) offer powerful tools for studying brain function. Complementing each other, fUS and PAT, respectively, measure the cerebral blood flow (CBF) and hemoglobin concentrations, allowing synergistic characterization of cerebral hemodynamics. Here, cross-ray ultrasound tomography (CRUST) and its combination with PAT are presented. CRUST employs a virtual point source from a spherically focused ultrasonic transducer (SFUST) to provide widefield excitation at a 4-kHz pulse repetition frequency. A full-ring-shaped ultrasonic transducer array whose imaging plane is orthogonal to the SFUST's acoustic axis receives scattered ultrasonic waves. Superior to conventional fUS, whose sensitivity to blood flow is angle-dependent and low for perpendicular flow, the crossed transmission and panoramic detection fields of CRUST provide omnidirectional sensitivity to CBF. Using CRUST-PAT, the CBF, oxygen saturation, and hemoglobin concentration changes of the mouse brain during sensory stimulation are measured, with a field of view of ≈7 mm in diameter, spatial resolution of ≈170 µm, and temporal resolution of 200 Hz. The results demonstrate CRUST-PAT as a unique tool for studying cerebral hemodynamics.

Herein we report the first in-human transcranial imaging of brain function using photoacoustic computed tomography. Functional responses to benchmark motor tasks were imaged on both the skull-less and the skull-intact hemispheres of a hemicraniectomy patient. The observed brain responses in these preliminary results demonstrate the potential of photoacoustic computed tomography for achieving transcranial functional imaging.

Long-duration in vivo simultaneous imaging of multiple anatomical structures is useful for understanding physiological aspects of diseases, informative for molecular optimization in preclinical models, and has potential applications in surgical settings to improve clinical outcomes. Previous studies involving simultaneous imaging of multiple anatomical structures, e.g., blood and lymphatic vessels as well as peripheral nerves and sebaceous glands, have used genetically engineered mice, which require expensive and time-consuming methods. Here, an IgG4 isotype control antibody is labeled with a near-infrared dye and injected into a mouse ear to enable simultaneous visualization of blood and lymphatic vessels, peripheral nerves, and sebaceous glands for up to 3 hours using photoacoustic microscopy. For multiple anatomical structure imaging, peripheral nerves and sebaceous glands are imaged inside the injected dye-labeled antibody mass while the lymphatic vessels are visualized outside the mass. The efficacy of the contrast agent to label and localize deep medial lymphatic vessels and lymph nodes using photoacoustic computed tomography is demonstrated. The capability of a single injectable contrast agent to image multiple structures for several hours will potentially improve preclinical therapeutic optimization, shorten discovery timelines, and enable clinical treatments.

This Conference Presentation, “Functional photoacoustic tomography of the human brain,” was recorded at SPIE Photonics West held in San Francisco, California, United States.

Long-duration in vivo simultaneous imaging of multiple anatomical structures is useful for understanding physiological aspects of diseases, informative for molecular optimization in preclinical models, and has potential applications in surgical settings to improve clinical outcomes. Previous studies involving simultaneous imaging of multiple anatomical structures, for example, blood and lymphatic vessels as well as peripheral nerves and sebaceous glands, have used genetically engineered mice, which require expensive and time-consuming methods. Here, an IgG4 isotype control antibody is labeled with a near-infrared dye and injected into a mouse ear to enable simultaneous visualization of blood and lymphatic vessels, peripheral nerves, and sebaceous glands for up to 3 h using photoacoustic microscopy. For multiple anatomical structure imaging, peripheral nerves and sebaceous glands are imaged inside the injected dye-labeled antibody mass while the lymphatic vessels are visualized outside the mass. The efficacy of the contrast agent to label and localize deep medial lymphatic vessels and lymph nodes using photoacoustic computed tomography is demonstrated. The capability of a single injectable contrast agent to image multiple structures for several hours will potentially improve preclinical therapeutic optimization, shorten discovery timelines, and enable clinical treatments.

Obtaining frozen sections of bone tissue for intraoperative examination is challenging. To identify the bony edge of resection, orthopaedic oncologists therefore rely on pre-operative X-ray computed tomography or magnetic resonance imaging. However, these techniques do not allow for accurate diagnosis or for intraoperative confirmation of the tumour margins, and in bony sarcomas, they can lead to bone margins up to 10-fold wider (1,000-fold volumetrically) than necessary. Here, we show that real-time three-dimensional contour-scanning of tissue via ultraviolet photoacoustic microscopy in reflection mode can be used to intraoperatively evaluate undecalcified and decalcified thick bone specimens, without the need for tissue sectioning. We validate the technique with gold-standard haematoxylin-and-eosin histology images acquired via a traditional optical microscope, and also show that an unsupervised generative adversarial network can virtually stain the ultraviolet-photoacoustic-microscopy images, allowing pathologists to readily identify cancerous features. Label-free and slide-free histology via ultraviolet photoacoustic microscopy may allow for rapid diagnoses of bone-tissue pathologies and aid the intraoperative determination of tumour margins.

Photoacoustic tomography with genetically encoded near-infrared probes enables visualization of specific cell populations in vivo at high resolution in deep tissues. Here, we report a near-infrared Forster resonance energy transfer biosensor based on a pair of the near-infrared fluorescent proteins, miRFP670-iRFP720, which enables dynamic photoacoustic imaging of active biological processes. Specifically, we detected apoptosis in single cells at a resolution of ~3 µm in a mouse ear and mouse brain tumors (>3 mm in depth) at a spatial resolution of ~150 µm. These results open the way for high-resolution photoacoustic imaging of dynamic biological processes in deep tissues.

One of the primary challenges in breast cancer diagnosis and treatment is intratumor heterogeneity (ITH), i.e., the coexistence of different genetically and epigenetically distinct malignant cells within the same tumor. Identification of ITH is critical for designing better treatments and hence to increase patient survival rates. Here, we report a non-invasive approach that integrates photoacoustic computed tomography (PACT) with multitargeted and multiplexed patchy polymeric photoacoustic contrast agents (MTMPPPCAs). The target specificity of MTMPPPCAs to distinguish estrogen and progesterone receptor-positive in breast tumors was demonstrated through both fluorescence and photoacoustic measurements and validated by tissue pathology analysis.

Over 100 monoclonal antibodies have been approved by the FDA for clinical use; however, a paucity of knowledge exists regarding the injection site behavior of these formulated therapeutics, i.e., the effect of antibody and formulation on the tissue around the injection site and vice versa. In this report, we injected a near-infrared dye-labeled IgG4 isotope control antibody into the subcutaneous space in mouse ears to analyze the injection site dynamics, including quantifying molecular movement. Surprisingly, we discovered that the antibody reduces the local blood oxygen saturation levels in mice after prolonged anesthesia without affecting the total hemoglobin content and oxygen extraction fraction. The local oxygen saturation results open a new pathway to study the functional effects of monoclonal antibodies.

Blood-oxygen-level-dependent (BOLD) functional magnetic resonance imaging of the human brain requires bulky equipment for the generation of magnetic fields. Photoacoustic computed tomography obviates the need for magnetic fields by using light and sound to measure deoxyhaemoglobin and oxyhaemoglobin concentrations to then quantify oxygen saturation and blood volumes. Yet, the available imaging speeds, fields of view (FOV), sensitivities and penetration depths have been insufficient for functional imaging of the human brain. Here, we show that massively parallel ultrasonic transducers arranged hemispherically around the human head can produce tomographic images of the brain with a 10-cm-diameter FOV and spatial and temporal resolutions of 350 µm and 2 s, respectively. In patients who had a hemicraniectomy, a comparison of functional photoacoustic computed tomography and 7 T BOLD functional magnetic resonance imaging showed a strong spatial correspondence in the same FOV and a high temporal correlation between BOLD signals and photoacoustic signals, with the latter enabling faster detection of functional activation. Our findings establish the use of photoacoustic computed tomography for human brain imaging.

Photoacoustic tomography (PAT) with genetically encoded near-infrared probes enables visualization of specific cell populations in vivo at high resolution deeply in biological tissues. However, because of a lack of proper probes, PAT of cellular dynamics remains unexplored. Here, the authors report a near-infrared Forster resonance energy transfer (FRET) biosensor based on a miRFP670-iRFP720 pair of the near-infrared fluorescent proteins, which enables dynamic functional imaging of active biological processes in deep tissues. By photoacoustically detecting the changes in the optical absorption of the miRFP670 FRET-donor, they monitored cell apoptosis in deep tissue at high spatiotemporal resolution using PAT. Specifically, they detected apoptosis in single cells at a resolution of ≈3 µm in a mouse ear tumor, and in deep brain tumors (>3 mm beneath the scalp) of living mice at a spatial resolution of ≈150 µm with a 20 Hz frame rate. These results open the way for high-resolution photoacoustic imaging of dynamic biological processes in deep tissues using NIR biosensors and PAT.

Photoacoustic imaging (PAI) is an emerging imaging modality that shows great potential for preclinical research and clinical practice. As a hybrid technique, PAI uniquely combines the advantages of optical excitation and of acoustic detection. Optical excitation provides a rich contrast mechanism from either endogenous or exogenous chromophores, allowing PAI to perform biochemical, functional, and molecular imaging. Acoustic detection benefits from the low scattering of ultrasound in biological tissue, enabling PAI to generate high-resolution images in both the optical ballistic and diffusive regimes. Accordingly, this hybrid imaging modality features high sensitivity to optical absorption and wide scalability of spatial resolution with the desired imaging depth. Over the past two decades, the photoacoustic technique has led to a variety of exciting discoveries and applications from laboratory research to clinical patient care. In biological research, PAI has become an irreplaceable tool, providing functional optical contrast with high spatiotemporal resolution. Translational PAI also attracted growing interest in clinical applications including tumor margin examination, internal organ imaging, breast cancer screening, and sentinel lymph node mapping, among others.

Photoacoustic imaging (PAI) is an emerging imaging modality that shows great potential for preclinical research and clinical practice. As a hybrid technique, PAI uniquely combines the advantages of optical excitation and of acoustic detection. Optical excitation provides a rich contrast mechanism from either endogenous or exogenous chromophores, allowing PAI to perform biochemical, functional, and molecular imaging. Acoustic detection benefits from the low scattering of ultrasound in biological tissue, enabling PAI to generate high-resolution images in both the optical ballistic and diffusive regimes. Accordingly, this hybrid imaging modality features high sensitivity to optical absorption and wide scalability of spatial resolution with the desired imaging depth. Over the past two decades, the photoacoustic technique has led to a variety of exciting discoveries and applications from laboratory research to clinical patient care. In biological research, PAI has become an irreplaceable tool, providing functional optical contrast with high spatiotemporal resolution. Translational PAI also attracted growing interest in clinical applications including tumor margin examination, internal organ imaging, breast cancer screening, and sentinel lymph node mapping, among others.

Background Worldwide, 10% of babies are born preterm, defined as a live birth before 37 weeks of gestation. Preterm birth is the leading cause of neonatal death, and survivors face lifelong risks of adverse outcomes. New approaches with large sample sizes are needed to identify strategies to predict and prevent preterm birth. The primary aims of the Washington University Prematurity Research Cohort Study were to conduct three prospective projects addressing possible causes of preterm birth and provide data and samples for future research. Study Design Pregnant patients were recruited into the cohort between January 2017 and January 2020. Consenting patients were enrolled into the study before 20 weeks’ gestation and followed through delivery. Participants completed demographic and lifestyle surveys; provided maternal blood, placenta samples, and cord blood; and participated in up to three projects focused on underlying physiology of preterm birth: cervical imaging (Project 1), circadian rhythms (Project 2), and uterine magnetic resonance imaging and electromyometrial imaging (Project 3). Results A total of 1260 participants were enrolled and delivered during the study period. Of the participants, 706 (56%) were Black/African American, 494 (39%) were nulliparous, and 185 (15%) had a previous preterm birth. Of the 1260 participants, 1220 (97%) delivered a live infant. Of the 1220 with a live birth, 163 (14.1%) had preterm birth, of which 74 (6.1%) were spontaneous preterm birth. Of the 1220 participants with a live birth, 841 participated in cervical imaging, 1047 contributed data and/or samples on circadian rhythms, and 39 underwent uterine magnetic resonance imaging. Of the 39, 25 underwent electromyometrial imaging. Conclusion We demonstrate feasibility of recruiting and retaining a diverse cohort in a complex prospective, longitudinal study throughout pregnancy. The extensive clinical, imaging, survey, and biologic data obtained will be used to explore cervical, u

Ultrasound (US) and optoacoustic (OA) imaging provide complementary information for quantitative analysis of the tumor microenvironment. Herein, we demonstrate the unique capabilities of transmission-reflection optoacoustic ultrasound (TROPUS) for characterizing breast cancer in tumor-bearing mice. For this, 4 different mice featuring orthotopic tumor of different sizes were scanned with a full-ring ultrasound transducer array to simultaneously render pulse-echo US images, speed of sound (SoS) maps and OA images. The tumor size, vascular density and its elastic parameters were further quantified in the images. Our results pave the way toward clinical translation of the hybrid TROPUS imaging for tumor detection and characterization.

Laser ablation (LA) is gaining acceptance for the treatment of tumors as a viable alternative to surgical resection. In parallel, optoacoustic tomography (OAT) has enabled defining new regimes for diagnosis and characterization of malignant neoplastic lesions with high sensitivity and specificity. Even though pulsed nanosecond lasers are commonly used for both imaging and therapeutic purposes, real-time thermal treatment monitoring with a single laser source has not been previously attempted. Herein, we demonstrate the feasibility of combined OAT and LA by percutaneous irradiation of subcutaneous tumors with a 100 mJ short-pulsed (~5 ns) laser operating at 1064 nm and 100 Hz pulse repetition frequency. The OAT images rendered with a spherical ultrasound transducer array enabled real-time monitoring of the LA lesion progression, which is essential for determining the optimal treatment end-point. Local changes in the optoacoustic signal intensity associated with the induced temperature changes as well as structural alterations in the tumor vasculature could clearly be observed. The optoacoustic volumetric projections further correlated with crosssections extracted from the excised tumors. This newly enabled capability anticipates new theranostic approaches in cancer research and treatment with potential applicability in a clinical setting.

Visualizing whole-body dynamics across entire living organisms is crucial for understanding complex biology, disease progression as well as evaluating efficacy of new drugs and therapies. Existing small animal functional and molecular imaging modalities either suffer from low spatial and temporal resolution, limited penetration depth or poor contrast. In this work, we present flash scanning volumetric optoacoustic tomography (fSVOT) imaging system that enables the acquisition speeds required for visualizing fast kinetics and biodistribution of optical contrast agents across whole mice. fSVOT can render images of intricate vascular and organ anatomy with rich contrast by capitalizing on the large angular coverage of a spherical matrix array transducer rapidly scanned around the mouse. Volumetric (three-dimensional) images with 200 µm resolution can be acquired within 45 seconds, which corresponds to an imaging speed gain of an order of magnitude with respect to existing state-of-the-art modalities offering comparable resolution performance. We demonstrate volumetric tracking and quantification of gold nanorod kinetics and their differential uptake across the spleen, liver and kidneys. Overall, fSVOT offers unprecedented capabilities for multi-scale imaging of pharmacokinetics and bio-distribution of agents with high contrast, resolution and image acquisition speed.

Optical-resolution photoacoustic microscopy (OR-PAM) has demonstrated high-spatial-resolution imaging of optical absorption in biological tissue. To date, most OR-PAM systems rely on mechanical scanning with confocally aligned optical excitation and ultrasonic detection, limiting the wide-field imaging speed of these systems. Although several multifocal OR-PA (MFOR-PA) systems have attempted to address this limitation, they are hindered by the complex design in a constrained physical space. Here, we present a two-dimensional (2D) MFOR-PAM system that utilizes a 2D microlens array and an acoustic ergodic relay. Using a single-element ultrasonic transducer, this system can detect PA signals generated from 400 optical foci in parallel and then raster scan the optical foci patterns to form an MFOR-PAM image. This system improves the imaging resolution of an acoustic ergodic relay system from 220 to 13 μm and enables 400-folds shorter scanning time than that of a conventional OR-PAM system at the same resolution and laser repetition rate. We demonstrated the imaging ability of the system with both in vitro and in vivo experiments. © 2020, The Author(s).

Tremendous progress in synthetic micro/nanomotors has been made for potential biomedical applications. However, existing micro/nanomotor platforms are inefficient for deep tissue imaging and motion control in vivo. Here, we present a photoacoustic computed tomography (PACT) guided investigation of micromotors in intestines in vivo. The micromotors enveloped in microcapsules exhibit efficient propulsion in various biofluids once released. PACT has visualized the migration of micromotor capsules toward the targeted regions in real time in vivo. The integration of the developed microrobotic system and PACT enables deep imaging and precise control of the micromotors in vivo.

Photoacoustic tomography (PAT), in the forms of computed tomography or microscopy, provides in vivo functional, metabolic, molecular, and histologic imaging. PAT has the unique strength of high-resolution multiscale imaging across organelles, cells, tissues, organs, and small-animal organisms with consistent optical absorption contrast. The wavelength of the excitation laser can be broadly tuned to target various endogenous or exogenous molecules. PAT has the potential to empower omniscale biology and accelerate trans-scale clinical translation. Potential medical applications include imaging of the breast, brain, thyroid, muscle, joint, skin, vascular system, lymphatic system, prostate, esophagus, colon, cervix, bladder, and uterus.

The spatial resolution of photoacoustic (PA) computed tomography (PACT) is limited by acoustic diffraction. Here, we report in vivo superresolution PACT, which breaks the acoustic diffraction limit by localizing the centers of single dyed droplets. The dyed droplets generate much stronger PA signals than blood and can flow smoothly in blood vessels; thus, they are excellent tracers for localization-based superresolution imaging. The flowing droplets were first localized, and then their center positions were used to construct a superresolution image that exhibits sharper features and more finely resolved vascular details. A 6-fold improvement in spatial resolution has been realized in vivo.

Photoacoustic tomography (PAT), in the forms of computed tomography or microscopy, provides in vivo functional, metabolic, molecular, and histologic imaging. PAT has the unique strength of high-resolution multiscale imaging across organelles, cells, tissues, organs in both animals and humans as well as small-animal organisms with consistent optical absorption contrast. The wavelength of the excitation laser can be broadly tuned to target various endogenous or exogenous molecules. PAT has the potential to empower omniscale biology and accelerate trans-scale clinical translation. Potential medical applications include imaging of the breast, brain, thyroid, muscle, joint, skin, vascular system, lymphatic system, prostate, esophagus, colon, cervix, bladder, and uterus.

Neuroscience has become one of the most exciting contemporary research areas with major breakthroughs expected in the coming decades. Modern imaging techniques have enabled scientific understanding of the neural system by revealing anatomical, functional, metabolic, and molecular information about the brain. Among these techniques, photoacoustic tomography (PAT), drawing more and more attention, is playing an increasingly important role in brain studies, thanks to its rich optical absorption contrast, high spatiotemporal resolution, and deep penetration. More importantly, PAT’s unique scalability empowers neuroscientists to examine the brain at multiple spatial scales using the same contrast mechanism, bridging microscopic insights to macroscopic observations of the brain. In this chapter, we review the principles of PAT, present the major implementations, and summarize the representative neuroscience applications. We also discuss challenges in translating PAT to human brain imaging and envision its potential promise.

Understanding the mechanisms of cardiac disorders largely depends on availability of multi-dimensional and multiparametric imaging methods capable of quantitative assessment of cardiac morphology and function. The imaging modalities commonly employed in cardiac research, such as ultrasonography and magnetic resonance imaging, are lacking sufficient contrast and/or spatio-temporal resolution in 3D in order to reveal the multi-scale nature of rapid electromechanical activity in a beating heart. Our recently developed volumetric optoacoustic tomography (VOT) platform offers versatile observations of the heart function with rich optical contrast at otherwise unattainable temporal and spatial resolutions. Herein, we further advance the imaging performance by developing compressed acquisition scheme to boost the temporal resolution of VOT into the kilohertz range, thus enabling 3D mapping of electromechanical wave propagation in the heart. Experiments in isolated mouse hearts were performed by exciting the entire imaged tissue volume with nanosecond-duration laser pulses at 1 kHz repetition rate pulse operating at 532 nm and sparse tomographic signal sampling using a custom-made 512-element spherical matrix ultrasound array. By analyzing the strain maps obtained from the rapid VOT image sequence, it was possible to quantify the phase velocity of the electromechanical cardiac waves, in good agreement with previously reported values.

Particles with sizes in the order of a few micrometers can significantly enhance the capabilities of optoacoustic imaging systems by improving visualization of arbitrarily oriented vascular structures and achieving resolution beyond the acoustic diffraction barrier. Particle tracking may also be used for mapping the blood flow in two and three dimensions. However, a trade-off exists between the particle absorption properties and size, whereas large sized microparticles also tend to arrest in the capillary network. We analyzed the flow of microparticles in an intracardiac perfusion mouse model in which blood is effectively substituted by artificial cerebrospinal fluid (ACSF). This enables mitigating the strong blood absorption background in the optoacoustic images thus facilitating the visualization of microparticles. A sequence of three-dimensional optoacoustic images of the mouse brain is then acquired at a high frame rate of 100 Hz after injection of the particles in the left heart ventricle. By visualizing the flow of particles of different sizes in microvascular structures it is possible to establish optimal trade-offs between the particle size, their optoacoustic signal and perfusion properties.

Multispectral optoacoustic tomography (MSOT) offers the unique capability to map the distribution of spectrally distinctive endogenous and exogenous substances in heterogeneous biological tissues by exciting the sample at various wavelengths and detecting the optoacoustically-induced ultrasound waves. This powerful functional and molecular imaging capability can greatly benefit from hybridization with pulse-echo ultrasound (US), which provides additional information on tissue anatomy and blood flow. However, speed of sound variations and acoustic mismatches in the imaged object generally lead to errors in the coregistration of compounded images and loss of spatial resolution in both imaging modalities. The spatially- and wavelength-dependent light fluence attenuation further limits the quantitative capabilities of MSOT. Proper segmentation of different regions and assignment of corresponding acoustic and optical properties turns then essential for maximizing the performance of hybrid optoacoustic and ultrasound (OPUS) imaging. Particularly, accurate segmentation of the boundary of the sample can significantly improve the images rendered. Herein, we propose an automatic segmentation method based on a convolutional neural network (CNN) for segmenting the mouse boundary in a pre-clinical OPUS system. The experimental performance of the method, as characterized with the Dice coefficient metric between the network output and the ground truth (manually segmented) images, is shown to be superior than that of a state-of-the-art active contour segmentation method in a series of two-dimensional (cross-sectional) OPUS images of the mouse brain, liver and kidney regions.

An estimated ∼250,000 new cases of both invasive and non-invasive breast cancer were diagnosed in US women almost every year. To reduce the local recurrence rate, the breast conserving surgery (BCS) is widely used as the initial therapy, which is to excise the tumor with a rim of normal surrounding tissue such that no cancer cells remain at the cut margin, Patients with positive margin commonly require a second surgical procedure to obtain clear margins. To this end, optical-resolution photoacoustic microscopy (OR-PAM) with ultraviolet (UV) laser illumination (OR-UV-PAM) has been developed for providing label-free, high-resolution, and histology like imaging of fixed, unprocessed breast tissue. To further improve the performance of OR-UV-PAM, here, we introduce dual-view UV-PAM (DV-UV-PAM) to significantly improve the axial resolution, achieving three-dimensional (3D) resolution isotropy. We first use 0.5 μm polystyrene beads and carbon fibers to validate the resolution isotropy improvement. Imaging of mouse brain slices further demonstrates the improved resolution isotropy, revealing the 3D structure of cell nuclei in detail, which facilitates quantitative cell nuclear analysis. © COPYRIGHT SPIE. Downloading of the abstract is permitted for personal use only.

We developed photoacoustic tomography to peer deep into biological tissue. Photoacoustic tomography (PAT) provides in vivo omniscale functional, metabolic, molecular, and histologic imaging across the scales of organelles through organisms. We also developed compressed ultrafast photography (CUP) to record 10 trillion frames per second, 10 orders of magnitude faster than commercially available camera technologies. CUP can tape the fastest phenomenon in the universe, namely, light propagation, and can be slowed down for slower phenomena such as combustion. PAT physically combines optical and ultrasonic waves. Conventional high-resolution optical imaging of scattering tissue is restricted to depths within the optical diffusion limit (~1 mm in the skin). Taking advantage of the fact that ultrasonic scattering is orders of magnitude weaker than optical scattering per unit path length, PAT beats this limit and provides deep penetration at high ultrasonic resolution and high optical contrast by sensing molecules. Broad applications include early-cancer detection and brain imaging. The annual conference on PAT has become the largest in SPIE’s 20,000-attendee Photonics West since 2010. CUP can image in 2D non-repeatable time-evolving events. CUP has a prominent advantage of measuring an x, y, t (x, y, spatial coordinates; t, time) scene with a single exposure, thereby allowing observation of transient events occurring on a time scale down to 100 femtoseconds, such as propagation of a light pulse. Further, akin to traditional photography, CUP is receive-only—avoiding specialized active illumination required by other single-shot ultrafast imagers. CUP can be coupled with front optics ranging from microscopes to telescopes for widespread applications in both fundamental and applied sciences.

Normal development of the visual system in infants relies on clear images being projected onto the retina, which can be disrupted by lens opacity caused by congenital cataract. This disruption, if uncorrected in early life, results in amblyopia (permanently decreased vision even after removal of the cataract). Doctors are able to prevent amblyopia by removing the cataract during the first several weeks of life, but this surgery risks a host of complications which can be equally visually disabling. Here, we investigated the feasibility of focusing light noninvasively through highly scattering cataractous lenses to stimulate the retina, thereby preventing amblyopia. This approach would allow the cataractous lens removal surgery to be delayed and hence greatly reduce the risk of complications from early surgery. Employing a wavefront shaping technique named time-reversed ultrasonically encoded (TRUE) optical focusing in reflection mode, we focused 532 nm light through a highly scattering ex vivo adult human cataractous lens of 112 mean free path thick. This work demonstrates a potential clinical application of wavefront shaping techniques.

Label-free functional photoacoustic microscopy (fPAM) has become a popular technology in small-animal hemodynamic studies. Here we report a stimulated-Raman-scattering-based (SRS) dual-wavelength high-speed fPAM that has achieved volumetric imaging at a 1 MHz A-line rate with capillary-level resolution. Potassium gadolinium tungstate (KGd(WO4)2) crystal is used as a Raman shifter to convert the pump 532 nm picosecond-pulsed laser to the first order Stokes line at 558 nm through the SRS effect with ~40% efficiency and a much narrower line width compared with previous fiber-based SRS PAMs. We also developed a water-immersible micro-electro-mechanical system scanner for scanning a ~4-mm range at a 500 Hz B-scan rate, while maintaining the optic-acoustic confocal alignment. This scanner is assembled entirely from commercially available components, facilitating replication. The detection sensitivity of our fPAM is also improved by employing a high numerical aperture polyvinylidene fluoride ultrasonic transducer, whose acoustic impedance matches better with tissue coupling medium than traditional ceramic transducers. The high sensitivity combined with ~2.4 µm resolution enabled our fPAM to image single red blood cells with a signal-to-noise ratio of ~27 dB. Compared with our previous laser-pulse-width based fPAM, we achieved simultaneous imaging of hemoglobin concentration and oxygenation with a 5-fold increase in imaging speed. Moreover, our system works in a convenient free-space manner compared to previous SRS-based PAMs. We applied it to imaging vasculature and blood oxygen saturation on mouse brains in both resting and stimulated states.

Photoacoustic tomography has been developed for in vivo functional, metabolic, molecular, and histologic imaging by physically combining optical and ultrasonic waves. Broad applications include early-cancer detection and brain imaging. High-resolution optical imaging—such as confocal microscopy, two-photon microscopy, and optical coherence tomography—is limited to superficial imaging within the optical diffusion limit (~1 mm in the skin) of the surface of scattering tissue. By synergistically combining light and sound, photoacoustic tomography conquers the optical diffusion limit and provides deep penetration at high ultrasonic resolution and high optical contrast. Photoacoustic tomography has two major embodiments: photoacoustic computed tomography and photoacoustic microscopy. In photoacoustic computed tomography, a pulsed broad laser beam illuminates the biological tissue to generate a small but rapid temperature rise, which leads to emission of ultrasonic waves due to thermoelastic expansion. The unscattered pulsed ultrasonic waves are then detected by ultrasonic transducers. High-resolution tomographic images of optical contrast are then formed through image reconstruction. In photoacoustic microscopy, a pulsed laser beam is delivered into the biological tissue to generate ultrasonic waves, which are then detected with a focused ultrasonic transducer to form a depth resolved 1D image. Raster scanning yields 3D high-resolution tomographic images. Super-depths beyond the optical diffusion limit have been reached with high spatial resolution. The annual conference on PAT has grown exponentially since early 2000 and become the largest in SPIE’s 20,000-attendee Photonics West since 2010.

The International Photoacoustic Standardisation Consortium (IPASC) emerged from SPIE 2018, established to drive consensus on photoacoustic system testing. As photoacoustic imaging (PAI) matures from research laboratories into clinical trials, it is essential to establish best-practice guidelines for photoacoustic image acquisition, analysis and reporting, and a standardised approach for technical system validation. The primary goal of the IPASC is to create widely accepted phantoms for testing preclinical and clinical PAI systems. To achieve this, the IPASC has formed five working groups (WGs). The first and second WGs have defined optical and acoustic properties, suitable materials, and configurations of photoacoustic image quality phantoms. These phantoms consist of a bulk material embedded with targets to enable quantitative assessment of image quality characteristics including resolution and sensitivity across depth. The third WG has recorded details such as illumination and detection configurations of PAI instruments available within the consortium, leading to proposals for system-specific phantom geometries. This PAI system inventory was also used by WG4 in identifying approaches to data collection and sharing. Finally, WG5 investigated means for phantom fabrication, material characterisation and PAI of phantoms. Following a pilot multi-centre phantom imaging study within the consortium, the IPASC settled on an internationally agreed set of standardised recommendations and imaging procedures. This leads to advances in: (1) quantitative comparison of PAI data acquired with different data acquisition and analysis methods; (2) provision of a publicly available reference data set for testing new algorithms; and (3) technical validation of new and existing PAI devices across multiple centres.

In recent years, Polymer optical fibre (POF) has been receiving increasing attention for sensing applications. For applications such as endoscopic ultrasound and photo-acoustics, PMMA polymer fibres deliver ever improving performance and more types of polymer are being trialled for these applications. The fundamental properties of POF, when correctly leveraged, deliver at least an order of magnitude in improvements over silica fibres. POF delivers lower acoustic impedance, a reduced Young's Modulus and a higher acoustic sensitivity within the megahertz region. In contrast, existing piezo-electric transducers have an inherent narrow acoustic bandwidth and a proportionality to size that causes difficulties for applications such as endoscopy within the biomedical domain. With the increasing take-up of POF, improvements have been made in fibre dopant distribution, the range of polymers available and connectorisation techniques. While newer polymer fibres are under preliminary study, PMMA has shown itself to be the most sensitive fibre to date despite historically higher levels of dopant inconsistency and an implementation around 830 nm that has a lower signal-to-noise ratio than possible. The prior approach of edge filtering a Bragg grating with a high speed photodiode has been shown to function and is therefore maintained. We present a step index PMMA Bragg grating ultrasonic probe in the L-band for the first time. Detection is achieved using a Bragg grating less than 1cm in length in a fibre under 10cm long. We examine the temporal and frequency response of the sensor over a 1-15 MHz range.

Model-based reconstruction techniques have been successfully implemented in optoacoustic tomography and acoustic-resolution microscopy to retrieve improved image quality over delay-and-sum methods. In scanning optical resolution optoacoustic microscopy (OR-OAM), no reconstruction methods are employed while post- processing is usually limited to basic frequency filtering and envelope extraction with the Hilbert transform. This results in considerable deterioration of the acoustically-determined resolution in the axial (depth) direction. In addition, when OR-OAM is used for transcranial mouse brain imaging, the skull strongly attenuates high ultrasonic frequencies and induces reverberations, which need to be accounted for during the reconstruction process to avoid image distortions and further deterioration of the axial resolution. Here we show a basic implementation of a model-based reconstruction to increase the axial resolution in OR-OAM. The model matrix is calculated using Field II for free field conditions, taking into account the shape and bandwidth of the spherically focused transducer. Assuming the confinement of the optoacoustic sources within the limits of the optical focus, one may calculate the model matrix by assuming a line source of small absorbing spheres equal in size to the optical beam. In addition, a plate model used in the recently reported virtual-craniotomy deconvolution algorithm is incorporated into the model matrix to tackle the transcranial acoustic transmission problem. The free-field model-based results are compared against the plate model for transcranial brain data obtained in-vivo.

Photoacoustic computed tomography (PACT), a fast-developing modality for deep tissue imaging, images the spatial distribution of optical absorption. PACT usually treats the absorption coefficient as a scalar. However, the absorption coefficients of many biological tissues exhibit an anisotropic property, known as dichroism or diattenuation, which depends on molecular conformation and structural alignment. Here, we present a novel imaging method called dichroism- sensitive PACT (DS-PACT), which measures both the amplitude of tissue’s dichroism and the orientation of the optic axis of uniaxial dichroic tissue. By modulating the polarization of linearly polarized light and measuring the alternating signals through lock-in detection, DS-PACT can boost dichroic signals from biological tissues. To validate the proposed approach, we experimentally demonstrated the performance of DS-PACT by imaging plastic polarizers and ex vivo bovine tendons deep inside scattering media. We successfully detected the orientation of the optic axis of uniaxial dichroic materials, even at a depth of 4.5 transport mean free paths. We anticipate that the proposed method will extend the capability of PACT to imaging tissue absorption anisotropy. © 2018 Optical Society of America.

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.