Non-telecentric photoacoustic microscopy for multi-scale imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) excels in precisely imaging a biological tissue based on absorption contrast. However, existing OR-PAMs are confined by fixed compromises between spatial resolution and field of view (FOV), preventing the integration of large FOV and local high-resolution within one system. Here, we present a non-telecentric OR-PAM (nTC-PAM) that empowers efficient adaptation of FOV and spatial resolution to match the multi-scale requirement of diverse biological imaging. Our method allows for a large-scale transformation in FOV and even surpassing the nominal FOV of the objective with minimal marginal degradation of the lateral resolution. We demonstrate the advantage of nTC-PAM through multi-scale imaging of the leaf phantom, mouse ear, and cortex. The results reveal that nTC-PAM can switch the FOV and spatial resolution to meet the requirements of different biological tissues, such as large-scale imaging of the whole cerebral cortex and high-resolution imaging of microvascular structures in local brain regions.

High spatiotemporal mapping of cortical blood flow velocity with an enhanced accuracy.

Cerebral blood flow velocity is one of the most essential parameters related to brain functions and diseases. However, most existing mapping methods suffer from either inaccuracy or lengthy sampling time. In this study, we propose a particle-size-related calibration method to improve the measurement accuracy and a random-access strategy to suppress the sampling time. Based on the proposed methods, we study the long-term progress of cortical vasculopathy and abnormal blood flow caused by glioma, short-term variations of blood flow velocity under different anesthetic depths, and cortex-wide connectivity of the rapid fluctuation of blood flow velocities during seizure onset. The experimental results demonstrate that the proposed calibration method and the random-access strategy can improve both the qualitative and quantitative performance of velocimetry techniques and are also beneficial for understanding brain functions and diseases from the perspective of cerebral blood flow.

Wearable photoacoustic watch for humans.

Longitudinal detection of hemodynamic changes based on wearable devices is imperative for monitoring human healthcare. Photoacoustic effect is extremely sensitive to variations in hemoglobin. Therefore, wearable photoacoustic devices are apt to monitor human healthcare via the observation of hemodynamics. However, the bulky system and difficulties in miniaturizing and optimizing the imaging interface restrict the development of wearable photoacoustic devices for human use. In this study, we developed a wearable photoacoustic watch with a fully integrated system in a backpack that has a size of 450 mm × 300 mm × 200 mm and an affordable weight of 7 kg for an adult to wear. The watch has a size of 43 mm × 30 mm × 24 mm, weighs 40 g, and features a lateral resolution of 8.7 µm, a field of view (FOV) of 3 mm in diameter, and a motorized adjustable focus for optimizing the imaging plane for different individuals. We recruited volunteers to wear the watch and the backpack and performed in vivo imaging of the vasculatures inside human wrists under the conditions of walking and human cuff occlusion to observe hemodynamic variations during different physiological states. The results suggest that the focus shifting capability of the watch makes it suitable for different individuals, and the compact and stable design of the entire system allows free movements of humans.

Near-Infrared II Semiconducting Polymer Dots: Chain Packing Modulation and High-Contrast Vascular Imaging in Deep Tissues.

Fluorescence imaging in the second near-infrared (NIR-II) window has attracted considerable interest in investigations of vascular structure and angiogenesis, providing valuable information for the precise diagnosis of early stage diseases. However, it remains challenging to image small blood vessels in deep tissues because of the strong photon scattering and low fluorescence brightness of the fluorophores. Here, we describe our combined efforts in both fluorescent probe design and image algorithm development for high-contrast vascular imaging in deep turbid tissues such as mouse and rat brains with intact skull. First, we use a polymer blending strategy to modulate the chain packing behavior of the large, rigid, NIR-II semiconducting polymers to produce compact and bright polymer dots (Pdots), a prerequisite for in vivo fluorescence imaging of small blood vessels. We further developed a robust Hessian matrix method to enhance the image contrast of vascular structures, particularly the small and weakly fluorescent vessels. The enhanced vascular images obtained in whole-body mouse imaging exhibit more than an order of magnitude improvement in the signal-to-background ratio (SBR) as compared to the original images. Taking advantage of the bright Pdots and Hessian matrix method, we finally performed through-skull NIR-II fluorescence imaging and obtained a high-contrast cerebral vasculature in both mouse and rat models bearing brain tumors. This study in Pdot probe development and imaging algorithm enhancement provides a promising approach for NIR-II fluorescence vascular imaging of deep turbid tissues.

Split Ring Resonator Topology Based Microwave Induced Thermoacoustic Imaging (SRR-MTAI).

Microwave-induced thermoacoustic imaging (MTAI) using low-energy and long-wavelength microwave photons has great potential in detecting deep-seated diseases due to its unique ability of visualizing intrinsic electric properties of tissue in high resolution. However, the low contrast in conductivity between a target (e.g., a tumor) and the surroundings sets a fundamental limit for achieving a high imaging sensitivity, which significantly hinders its biomedical applications. To overcome this limit, we develop a split ring resonator (SRR) topology based MTAI (SRR-MTAI) approach to achieve highly sensitive detection by precise manipulation and efficient delivery of microwave energy. The in vitro experiments show that SRR-MTAI demonstrates an ultrahigh sensitivity of distinguishing a 0.4% difference in saline concentrations and a 2.5-fold enhancement of detecting a tissue target which mimicks a tumor embedded at a depth of 2 cm. The in vivo animal experiments conducted indicate that the imaging sensitivity between a tumor and the surrounding tissue is increased by 3.3-fold using SRR-MTAI. The dramatic enhancement in imaging sensitivity suggests that SRR-MTAI has the potential to open new avenues for MTAI to tackle a variety of biomedical problems that were impossible previously.

Handheld volumetric photoacoustic/ultrasound imaging using an internal scanning mechanism.

Photoacoustic/ultrasound (PA/US) dual-modality imaging has been evolving rapidly for the last two decades. Handheld PA/US probes with different implementations have attracted particular attention due to their convenience and high applicability. However, developing a volumetric dual-modality PA/US imaging probe with a compact design remains a challenge. Here, we develop a handheld volumetric PA/US imaging probe with a special light-ultrasound coupling design and an internal scanning mechanism. A coaxial design for the excitation and detection paths in a customized 3D-printed housing with a size of 110 × 90 × 64 mm3 is proposed to optimize the signal-to-noise ratio (SNR) of the handheld probe for deep tissue imaging. Two parallel and synchronously rotational acoustic reflectors allow for volumetric imaging with an effective field of view (FOV) of more than 30 mm × 20 mm × 8 mm. In addition to simulation and phantom validations, in vivo human trials are successfully carried out, demonstrating the high imaging quality and stability of the system for potential clinical translations.

Visualization of blood-brain barrier disruption with dual-wavelength high-resolution photoacoustic microscopy.

The blood-brain barrier (BBB) strictly regulates the substance exchange between the vascular network and the central nervous system, and plays a critical role in maintaining normal brain homeostasis. Impaired BBB is often accompanied with the emergence of cerebral diseases and probably further leads to severe neuroinflammation or even neurological degeneration. Hence, there is an urgent need to precisely monitor the impaired BBB to understand its pathogenesis and better guide the enactment of therapeutic strategies. However, there is a lack of high-resolution imaging techniques to visualize and evaluate the large-scale BBB disruption in pre-clinical and clinical aspects. In this study, we propose a dual-wavelength photoacoustic imaging (PAI) methodology that simultaneously reveals the abnormal microvasculature and impaired BBB within the cerebral cortex. In in vivo studies, BBB disruption in both mice and rats were induced by local hot-water stimulation and unilateral carotid arterial perfusion of hyperosmolar mannitol, respectively. Subsequently, the exogenous contrast agent (CA) was injected into the microcirculation via the tail vein, and photoacoustic (PA) images of the microvasculature and leaked CA within the cerebral cortex were obtained by dual-wavelength photoacoustic microscopy to evaluate the BBB disruption. Besides, analysis of distribution and concentration of leaked CA in lesion region was further conducted to quantitatively reveal the dynamic changes of BBB permeability. Furthermore, we exploited this approach to investigate the reversibility of BBB disruption within the two distinct models. Based on the experimental results, this new proposed approach presents excellent performance in visualizing microvasculature and leaked CA, and enabling it possesses great potential in evaluating the abnormal microvasculature and impaired BBB result from cerebrovascular diseases.

Photoacoustic microscopy visualizes glioma-induced disruptions of cortical microvascular structure and function.

Objective. Glioma growth may cause pervasive disruptions of brain vascular structure and function. Revealing both structural and functional alterations at a fine spatial scale is challenging for existing imaging techniques, which could confound the understanding of the basic mechanisms of brain diseases.Approach. In this study, we apply photoacoustic microscopy with a high spatial-temporal resolution and a wide field of view to investigate the glioma-induced alterations of cortical vascular morphology, hemodynamic response, as well as functional connectivity at resting- and stimulated- states.Main results.We find that glioma promotes the growth of microvessels and leads to the increase of vascular proportion in the cerebral cortex by deriving structural parameters. The glioma also causes the loss of response in the ipsilateral hemisphere and abnormal response in the contralateral hemisphere, and further induces brain-wide alterations of functional connectivity in resting and stimulated states.Significance.The observed results show the foundation of employing photoacoustic microscopy as a potential technique in revealing the underlying mechanisms of brain diseases.

Multicolor Photoacoustic Volumetric Imaging of Subcellular Structures.

Photoacoustic imaging (PAI) has been widely used in multiscale and multicontrast imaging of biological structures and functions. Optical resolution photoacoustic microscopy (OR-PAM), an emerging submodality of PAI, features high lateral resolution and rich optical contrast, indicating great potential in visualizing cellular and subcellular structures. However, three-dimensional (3D) imaging of subcellular structures using OR-PAM has remained a challenge due to the limited axial resolution. In this study, we propose a multicolor 3D photoacoustic microscopy with high lateral/axial resolutions of 0.42/2 and 0.5/2.5 µm at 532 and 780 nm excitation, respectively. Owing to the significantly increased axial resolution, we could visualize the volumetric subcellular structures of melanoma cells using intrinsic contrast. In addition, we carried out multicolor imaging of labeled microtubules/clathrin-coated pits (CCP) and microtubules/mitochondria, respectively, with one scanning by using two different excitation wavelengths. The internal connections between different subcellular structures are revealed by quantitatively comparing the spatial distributions of microtubules/CCP and microtubules/mitochondria in a single cell. Current results suggest that the proposed OR-PAM may serve as an efficient tool for subcellular and cytophysiological studies.

Detachable head-mounted photoacoustic microscope in freely moving mice.

Optical resolution photoacoustic microscopy (ORPAM) is a promising tool for investigating anatomical and functional dynamics in the cerebral cortex. However, observation in freely moving mice has been a longstanding challenge for ORPAM. In this Letter, we extended ORPAM from anesthetized, head-restrained to awake, freely moving mice by using a detachable head-mounted ORPAM probe. We used a micro-electro-mechanical-system scanner and a miniaturized piezoelectric ultrasonic detector to scan the excitation laser beam and detect generated photoacoustic signals, respectively. The probe weighs 1.8 g and has a large field of view of ∼3mm×3mm. We evaluated the performance of the probe by carrying out phantom experiments and the imaging of vascular networks in a mouse cerebral cortex. The results suggest that the ORPAM probe is capable of providing stable and high-quality ORPAM images in freely moving mice.

Dual-model wearable photoacoustic microscopy and electroencephalograph: study of neurovascular coupling in anesthetized and freely moving rats.

Observing microscale neurovascular dynamics under different physiological conditions is of great importance to understanding brain functions and disorders. Here, we report a dual-model wearable device and an auxiliary data processing algorithm to derive neurovascular dynamics. The device integrates high-resolution photoacoustic microscopy and electroencephalography (EEG), which allows observing capillary-level hemodynamics and neural activities in anesthesia and freely moving rats. By using the developed algorithm, multiple photoacoustic/EEG parameters extracted and correlated enables investigation of the interplay between neural and vascular activities. We employed this platform to study the neurovascular coupling during different types of seizures in rats under various physiological conditions. We observed cerebral vascular vasodilation/constriction corresponding well to the seizure on/off in rats under regular anesthesia conditions, showing a strong neurovascular coupling coefficient. In rats under weak anesthesia and freely moving conditions, more intense cerebral hemodynamics and neural activities occurred with a weaker neurovascular coupling coefficient. The comprehensively quantitative analyses suggest that anesthesia has a dominant impact on the seizure onset and affect the neurovascular coupling correlation in the current drug-induced localized seizure model. Our study reveals that the designed platform has the potential to support studies on brain functions and disorders in diseased rodent models in various physiological states.

Multi-modality photoacoustic/ultrasound imaging based on a commercial ultrasound platform.

Multimodal imaging takes advantage of each modality and has become a recent trend in the field of biomedical imaging. In this Letter, we develop and evaluate an integrated multi-modality imaging system combining photoacoustic computed tomography, optical resolution photoacoustic microscopy, brightness mode, and power Doppler ultrasound imaging on a commercial ultrasonographic platform. Using different imaging modalities enables the hybrid system to recover dense vascular networks and hemodynamic and morphological variations in both superficial and deep tissues. To evaluate the performance and illustrate the advantages of this system, we carried out both phantom and in vivo experiments. In addition to the complementary tissue information offered by different imaging modalities, the use of a commercial ultrasound platform shows the feasibility of the proposed method for future clinical translation.

Light-Controlled Precise Delivery of NIR-Responsive Semiconducting Polymer Nanoparticles with Promoted Vascular Permeability.

The endothelial barrier plays an essential role in health and disease by protecting organs from toxins while allowing nutrients to access the circulation. However, it is the major obstacle that limits the delivery of therapeutic drugs to the diseased tissue. Here, it is reported for the first time that near-infrared (NIR) laser pulses can transiently promote the delivery of semiconducting polymer nanoparticles passing the vascular barrier via photoacoustic-effect-induced accumulation, only by the aid of pulse laser irradiation. This strategy enables selective and substantial accumulation of the NIR-absorbing nanoparticles inside specific tissues, implying the discovery of an unprecedented approach for light-controlled nanoparticle delivery. Especially, the nanoparticle delivery in solid tumors by 10-min laser scanning is approximately six times higher than that of the enhanced permeability and retention (EPR) effect in 24 h under current experimental conditions. Further results confirm that this strategy facilitates substantial accumulation of nanoparticles in the mouse brain with intact skull. This approach thus opens a new door for tissue-specific delivery of nanomaterials with an unprecedented level of efficiency and precision.

High-resolution in vivo imaging of rhesus cerebral cortex with ultrafast portable photoacoustic microscopy.

Revealing the structural and functional change of microvasculature is essential to match vascular response with neuronal activities in the investigation of neurovascular coupling. The increasing use of rhesus models in fundamental and clinical studies of neurovascular coupling presents an emerging need for a new imaging modality. Here we report a structural and functional cerebral vascular study of rhesus monkeys using an ultrafast, portable, and high resolution photoacoustic microscopic system with a long working distance and a special scanning mechanism to eliminate the relative displacement between the imaging interface and samples. We derived the structural and functional response of the cerebral vasculature to the alternating normoxic and hypoxic conditions by calculating the vascular diameter and functional connectivity. Both vasodilatation and vasoconstriction were observed in hypoxia. In addition to the change of vascular diameter, the decrease of functional connectivity is also an important phenomenon induced by the reduction of oxygen ventilatory. These results suggest that photoacoustic microscopy is a promising method to study the neurovascular coupling and cerebral vascular diseases due to the advanced features of high spatiotemporal resolution, excellent sensitivity to hemoglobin, and label-free imaging capability of observing hemodynamics.

Photoacoustic Force-Guided Precise and Fast Delivery of Nanomedicine with Boosted Therapeutic Efficacy.

Precise and efficient delivery of nanomedicine to the target site has remained as a major roadblock in advanced cancer treatment. Here, a novel photoacoustic force (PAF)-guided nanotherapeutic system is reported based on a near-infrared (NIR)-absorbing semiconducting polymer (SP), showing significantly improved tumor accumulation and deep tissue penetration for enhanced phototherapeutic efficacy. The accumulation of nanoparticles in 4T1 tumor-bearing mice induced by the PAF strategy displays a fivefold enhancement in comparison with that of the traditional passive targeting pathway, in a significantly shortened time (45 min vs 24 h) with an enhanced penetration depth in tumors. Additionally, a tumor-bearing mouse model is rationally designed to unveil the mechanism, indicating that the nanoparticles enter solid tumors through enhanced transportation across blood vessel barriers via both inter-endothelial gaps and active trans-endothelial pathways. This process is specifically driven by PAF generated from the nanoparticles under NIR laser irradiation. The study thus demonstrates a new nanotherapeutic strategy with low dose, enhanced delivery efficiency in tumor, and boosted therapeutic efficacy, opening new doors for designing novel nanocarriers.

Progress of clinical translation of handheld and semi-handheld photoacoustic imaging.

Photoacoustic imaging (PAI), featuring rich contrast, high spatial/temporal resolution and deep penetration, is one of the fastest-growing biomedical imaging technology over the last decade. To date, numbers of handheld and semi-handheld photoacoustic imaging devices have been reported with corresponding potential clinical applications. Here, we summarize emerged handheld and semi-handheld systems in terms of photoacoustic computed tomography (PACT), optoacoustic mesoscopy (OAMes), and photoacoustic microscopy (PAM). We will discuss each modality in three aspects: laser delivery, scanning protocol, and acoustic detection. Besides new technical developments, we also review the associated clinical studies, and the advantages/disadvantages of these new techniques. In the end, we propose the challenges and perspectives of miniaturized PAI in the future.

Optical resolution photoacoustic computed microscopy.

Optical resolution photoacoustic microscopy (ORPAM) has demonstrated both high resolution and rich contrast imaging of optical chromophores in biologic tissues. To date, sensitivity remains a major challenge for ORPAM, which limits the capability of resolving biologic microvascular networks. In this study, we propose and evaluate a new ORPAM modality termed as optical resolution photoacoustic computed microscopy (ORPACM), through the combination of a two-dimensional laser-scanning system with a medical ultrasonographic platform. Apart from conventional ORPAMs, we record multiple photoacoustic (PA) signals using a 128-element ultrasonic transducer array for each pulse excitation. Then, we apply a reconstruction algorithm to recover one depth-resolved PA signal referred to as an A-line, which reveals more detailed information compared with conventional single-element transducer-based ORPAMs. In addition, we carried out both in vitro and in vivo experiments as well as quantitative analyses to show the advanced features of ORPACM.

Photoacoustic endoscopy: A progress review.

Endoscopy has been widely used in biomedical imaging and integrated with various optical and acoustic imaging modalities. Photoacoustic imaging (PAI), one of the fastest growing biomedical imaging modalities, is a noninvasive and nonionizing method that owns rich optical contrast, deep acoustic penetration depth, multiscale and multiparametric imaging capability. Hence, it is preferred to miniaturize the volume of PAI and develop an emerged endoscopic imaging modality referred to as photoacoustic endoscopy (PAE). It has been developed for more than one decade since the first report of PAE. Unfortunately, until now, there is no mature photoacoustic endoscopic technique recognized in clinic due to various technical limitations. To address this concern, recent development of new scanning mechanisms, adoption of novel optical/acoustic devices, utilization of superior computation methods and exploration of multimodality strategies have significantly promoted the progress of PAE toward clinic. In this review, we comprehensively reviewed recent progresses in single- and multimodality PAE with new physics, mechanisms and strategies to achieve practical devices for potential applicable scenarios including esophageal, gastrointestinal, urogenital and intravascular imaging. We ended this review with challenges and prospects for future development of PAE.

Fluorination Enhances NIR-II Fluorescence of Polymer Dots for Quantitative Brain Tumor Imaging.

Here, we describe a fluorination strategy for semiconducting polymers for the development of highly bright second near-infrared region (NIR-II) probes. Tetrafluorination yielded a fluorescence QY of 3.2 % for the polymer dots (Pdots), over a 3-fold enhancement compared to non-fluorinated counterparts. The fluorescence enhancement was attributable to a nanoscale fluorous effect in the Pdots that maintained the molecular planarity and minimized the structure distortion between the excited state and ground state, thus reducing the nonradiative relaxations. By performing through-skull and through-scalp imaging of the brain vasculature of live mice, we quantitatively analyzed the vascular morphology of transgenic brain tumors in terms of the vessel lengths, vessel branches, and vessel symmetry, which showed statistically significant differences from the wild type animals. The bright NIR-II Pdots obtained through fluorination chemistry provide insightful information for precise diagnosis of the malignancy of the brain tumor.

Quantitative investigation of vascular response to mesenteric venous thrombosis using large-field-of-view photoacoustic microscopy.

Mesenteric venous thrombosis (MVT) is one of major causes leading to severe mesenteric ischemia. Vascular network plays an important role during the occurrence and development of MVT. However, there lacks an appropriate imaging method, which features advanced volumetric resolving capability, superior sensitivity to hemoglobin, and ultra-large field-of-view (FOV), to investigate vascular response of MVT. In this study, we developed and applied a large-FOV optical resolution photoacoustic microscopy to quantify the vascular response during the entire course of two different MVT models in which we ligated the superior mesenteric vein and inferior mesenteric vein, respectively. Furthermore, we developed a quantitative algorithm to derive total vascular length, relative concentration of total hemoglobin and vascular density over the FOV to reveal different vascular responses in different MVT models.