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In photoacoustic computed tomography (PACT) with short-pulsed laser excitation, wideband acoustic signals are generated in biological tissues with frequencies related to the effective shapes and sizes of the optically absorbing targets. Low-frequency photoacoustic signal components correspond to slowly varying spatial features and are often omitted during imaging due to the limited detection bandwidth of the ultrasound transducer, or during image reconstruction as undesired background that degrades image contrast. Here we demonstrate that low-frequency photoacoustic signals, in fact, contain functional and molecular information, and can be used to enhance structural visibility, improve quantitative accuracy, and reduce spare-sampling artifacts. We provide an in-depth theoretical analysis of low-frequency signals in PACT, and experimentally evaluate their impact on several representative PACT applications, such as mapping temperature in photothermal treatment, measuring blood oxygenation in a hypoxia challenge, and detecting photoswitchable molecular probes in deep organs. Our results strongly suggest that low-frequency signals are important for functional and molecular PACT.

Significance: Photoacoustic microscopy (PAM) offers advantages in high-resolution and high-contrast imaging of biomedical chromophores. The speed of imaging is critical for leveraging these benefits in both preclinical and clinical settings. Ongoing technological innovations have substantially boosted PAM's imaging speed, enabling real-time monitoring of dynamic biological processes., Aim: This concise review synthesizes historical context and current advancements in high-speed PAM, with an emphasis on developments enabled by ultrafast lasers, scanning mechanisms, and advanced imaging processing methods., Approach: We examine cutting-edge innovations across multiple facets of PAM, including light sources, scanning and detection systems, and computational techniques and explore their representative applications in biomedical research., Results: This work delineates the challenges that persist in achieving optimal high-speed PAM performance and forecasts its prospective impact on biomedical imaging., Conclusions: Recognizing the current limitations, breaking through the drawbacks, and adopting the optimal combination of each technology will lead to the realization of ultimate high-speed PAM for both fundamental research and clinical translation., (© 2024 The Authors.)

Significance: Based on acoustic detection of optical absorption, photoacoustic tomography (PAT) allows functional and molecular imaging beyond the optical diffusion limit with high spatial resolution. However, multispectral functional and molecular PAT is often limited by decreased spectroscopic accuracy and reduced detection sensitivity in deep tissues, mainly due to wavelength-dependent optical attenuation and inaccurate acoustic inversion., Aim: Previous work has demonstrated that reversible color-shifting can drastically improve the detection sensitivity of PAT by suppressing nonswitching background signals. We aim to develop a new color switching-based PAT method using reversibly switchable thermochromics (ReST)., Approach: We developed a family of ReST with excellent water dispersion, biostability, and temperature-controlled color changes by surface modification of commercial thermochromic microcapsules with the hydrophilic polysaccharide alginate., Results: The optical absorbance of the ReST was switched on and off repeatedly by modulating the surrounding temperature, allowing differential photoacoustic detection that effectively suppressed the nonswitching background signal and substantially improved image contrast and detection sensitivity. We demonstrate reversible thermal-switching imaging of ReST in vitro and in vivo using three PAT modes at different length scales., Conclusions: ReST-enabled PAT is a promising technology for high-sensitivity deep tissue imaging of molecular activity in temperature-related biomedical applications, such as cancer thermotherapy., (© 2023 The Authors.)

Thromboembolism in blood vessels can lead to stroke or heart attack and even sudden death unless brought under control. Sonothrombolysis enhanced by ultrasound contrast agents has shown promising outcome on effective treatment of thromboembolism. Intravascular sonothrombolysis was also reported recently with a potential for effective and safe treatment of deep thrombosis. Despite the promising treatment results, the treatment efficiency for clinical application may not be optimized due to the lack of imaging guidance and clot characterization during the thrombolysis procedure. In this paper, a miniaturized transducer was designed to have an 8-layer PZT-5A stacked with an aperture size of 1.4 × 1.4 mm 2 and assembled in a customized two-lumen 10-Fr catheter for intravascular sonothrombolysis. The treatment process was monitored with internal-illumination photoacoustic tomography (II-PAT), a hybrid imaging modality that combines the rich contrast of optical absorption and the deep penetration of ultrasound detection. With intravascular light delivery using a thin optical fiber integrated with the intravascular catheter, II-PAT overcomes the penetration depth limited by strong optical attenuation of tissue. In-vitro PAT-guided sonothrombolysis experiments were carried out with synthetic blood clots embedded in tissue phantom. Clot position, shape, stiffness, and oxygenation level can be estimated by II-PAT at clinically relevant depth of ten centimeters. Our findings have demonstrated the feasibility of the proposed PAT-guided intravascular sonothrombolysis with real-time feedback during the treatment process.

Non-invasive small-animal imaging technologies, such as optical imaging, magnetic resonance imaging and x -ray computed tomography, have enabled researchers to study normal biological phenomena or disease progression in their native conditions. However, existing small-animal imaging technologies often lack either the penetration capability for interrogating deep tissues (e.g., optical microscopy), or the functional and molecular sensitivity for tracking specific activities (e.g., magnetic resonance imaging). To achieve functional and molecular imaging in deep tissues, we have developed an integrated photoacoustic, ultrasound and acoustic angiographic tomography (PAUSAT) system by seamlessly combining light and ultrasound. PAUSAT can perform three imaging modes simultaneously with complementary contrast: high-frequency B-mode ultrasound imaging of tissue morphology, microbubble-enabled acoustic angiography of tissue vasculature, and multi-spectral photoacoustic imaging of molecular probes. PAUSAT can provide three-dimensional (3D) multi-contrast images that are co-registered, with high spatial resolutions at large depths. Using PAUSAT, we performed proof-of-concept in vivo experiments on various small animal models: monitoring longitudinal development of placenta and embryo during mouse pregnancy, tracking biodistribution and metabolism of near-infrared organic dye on the whole-body scale, and detecting breast tumor expressing genetically-encoded photoswitchable phytochromes. These results have collectively demonstrated that PAUSAT has broad applicability in biomedical research, providing comprehensive structural, functional, and molecular imaging of small animal models.

Optogenetic manipulation and optical imaging in the near-infrared range allow non-invasive light-control and readout of cellular and organismal processes in deep tissues in vivo. Here, we exploit the advantages of Rhodopseudomonas palustris BphP1 bacterial phytochrome, which incorporates biliverdin chromophore and reversibly photoswitches between the ground (740-800 nm) and activated (620-680 nm) states, to generate a loxP-BphP1 transgenic mouse model. The mouse enables Cre-dependent temporal and spatial targeting of BphP1 expression in vivo. We validate the optogenetic performance of endogenous BphP1, which in the activated state binds its engineered protein partner QPAS1, to trigger gene transcription in primary cells and living mice. We demonstrate photoacoustic tomography of BphP1 expression in different organs, developing embryos, virus-infected tissues and regenerating livers, with the centimeter penetration depth. The transgenic mouse model provides opportunities for both near-infrared optogenetics and photoacoustic imaging in vivo and serves as a source of primary cells and tissues with genomically encoded BphP1., (© 2022. The Author(s).)

Linear-array-based photoacoustic tomography has shown broad applications in biomedical research and preclinical imaging. However, the elevational resolution of a linear array is fundamentally limited due to the weak cylindrical focus of the transducer element. While several methods have been proposed to address this issue, they have all handled the problem in a less time-efficient way. In this work, we propose to improve the elevational resolution of a linear array through Deep-E, a fully dense neural network based on U-net. Deep-E exhibits high computational efficiency by converting the three-dimensional problem into a two-dimension problem: it focused on training a model to enhance the resolution along elevational direction by only using the 2D slices in the axial and elevational plane and thereby reducing the computational burden in simulation and training. We demonstrated the efficacy of Deep-E using various datasets, including simulation, phantom, and human subject results. We found that Deep-E could improve elevational resolution by at least four times and recover the object's true size. We envision that Deep-E will have a significant impact in linear-array-based photoacoustic imaging studies by providing high-speed and high-resolution image enhancement.

Thrombosis in the circulation system can lead to major myocardial infarction and cardiovascular deaths. Understanding thrombosis formation is necessary for developing safe and effective treatments. In this work, using digital light processing (DLP)-based 3D printing, we fabricated sophisticated in vitro models of blood vessels with internal microchannels that can be used for thrombosis studies. In this regard, photoacoustic microscopy (PAM) offers a unique advantage for label-free visualization of the 3D-printed vessel models, with large penetration depth and functional sensitivity. We compared the imaging performances of two PAM implementations: optical-resolution PAM and acoustic-resolution PAM, and investigated 3D-printed vessel structures with different patterns of microchannels. Our results show that PAM can provide clear microchannel structures at depths up to 3.6 mm. We further quantified the blood oxygenation in the 3D-printed vascular models, showing that thrombi had lower oxygenation than the normal blood. We expect that PAM can find broad applications in 3D printing and bioprinting for in vitro studies of various vascular and other diseases., (© 2022 IOP Publishing Ltd.)

The in vivo hemodynamic impact of sodium nitroprusside (SNP), a widely used antihypertensive agent, has not been well studied. Here, we applied functional optical-resolution photoacoustic microscopy (OR-PAM) to study the hemodynamic responses to SNP in mice in vivo. As expected, after the application of SNP, the systemic blood pressure (BP) was reduced by 53%. The OR-PAM results show that SNP induced an arterial vasodilation of 24% and 23% in the brain and skin, respectively. A weaker venous vasodilation of 9% and 5% was also observed in the brain and skin, respectively. The results show two different types of blood oxygenation response. In mice with decreased blood oxygenation, the arterial and venous oxygenation was respectively reduced by 6% and 13% in the brain, as well as by 7% and 18% in the skin. In mice with increased blood oxygenation, arterial and venous oxygenation was raised by 4% and 22% in the brain, as well as by 1% and 9% in the skin. We observed venous change clearly lagged the arterial change in the skin, but not in the brain. Our results collectively show a correlation among SNP induced changes in systemic BP, vessel size and blood oxygenation., (© 2021 Wiley-VCH GmbH.)

The rapidly evolving field of photoacoustic tomography utilizes endogenous chromophores to extract both functional and structural information from deep within tissues. It is this power to perform precise quantitative measurements in vivo- with endogenous or exogenous contrast - that makes photoacoustic tomography highly promising for clinical translation in functional brain imaging, early cancer detection, real-time surgical guidance, and the visualization of dynamic drug responses. Considering photoacoustic tomography has benefited from numerous engineering innovations, it is of no surprise that many of photoacoustic tomography's current cutting-edge developments incorporate advances from the equally novel field of artificial intelligence. More specifically, alongside the growth and prevalence of graphical processing unit capabilities within recent years has emerged an offshoot of artificial intelligence known as deep learning. Rooted in the solid foundation of signal processing, deep learning typically utilizes a method of optimization known as gradient descent to minimize a loss function and update model parameters. There are already a number of innovative efforts in photoacoustic tomography utilizing deep learning techniques for a variety of purposes, including resolution enhancement, reconstruction artifact removal, undersampling correction, and improved quantification. Most of these efforts have proven to be highly promising in addressing long-standing technical obstacles where traditional solutions either completely fail or make only incremental progress. This concise review focuses on the history of applied artificial intelligence in photoacoustic tomography, presents recent advances at this multifaceted intersection of fields, and outlines the most exciting advances that will likely propagate into promising future innovations.

Significance: Acoustically detecting the rich optical absorption contrast in biological tissues, photoacoustic tomography (PAT) seamlessly bridges the functional and molecular sensitivity of optical excitation with the deep penetration and high scalability of ultrasound detection. As a result of continuous technological innovations and commercial development, PAT has been playing an increasingly important role in life sciences and patient care, including functional brain imaging, smart drug delivery, early cancer diagnosis, and interventional therapy guidance., Aim: Built on our 2016 tutorial article that focused on the principles and implementations of PAT, this perspective aims to provide an update on the exciting technical advances in PAT., Approach: This perspective focuses on the recent PAT innovations in volumetric deep-tissue imaging, high-speed wide-field microscopic imaging, high-sensitivity optical ultrasound detection, and machine-learning enhanced image reconstruction and data processing. Representative applications are introduced to demonstrate these enabling technical breakthroughs in biomedical research., Conclusions: We conclude the perspective by discussing the future development of PAT technologies.

Kidney stone disease is a major health problem worldwide. Shockwave lithotripsy (SWL), which uses high-energy shockwave pulses to break up kidney stones, is extensively used in clinic. However, despite its noninvasiveness, SWL can produce cavitation in vivo. The rapid expansion and violent collapse of cavitation bubbles in small blood vessels may result in renal vascular injury. To better understand the mechanism of tissue injury and improve treatment safety and efficiency, it is highly desirable to concurrently detect cavitation and vascular injury during SWL. Current imaging modalities used in SWL ( e.g. , C-arm fluoroscopy and B-mode ultrasound) are not sensitive to vascular injuries. By contrast, photoacoustic imaging is a non-invasive and non-radiative imaging modality that is sensitive to blood, by using hemoglobin as the endogenous contrast. Moreover, photoacoustic imaging is also compatible with passive cavitation detection by sharing the ultrasound detection system. Here, we have integrated shockwave treatment, photoacoustic imaging, and passive cavitation detection into a single system. Our experimental results on phantoms and in vivo small animals have collectively demonstrated that the integrated system is capable of capturing shockwave-induced cavitation and the resultant vascular injury simultaneously. We expect that the integrated system, when combined with our recently developed internal-light-illumination photoacoustic imaging, will find important applications for monitoring shockwave-induced vascular injury in deep tissues during SWL.

Photoacoustic tomography (PAT) has become increasingly popular for molecular imaging due to its unique optical absorption contrast, high spatial resolution, deep imaging depth, and high imaging speed. Yet, the strong optical attenuation of biological tissues has traditionally prevented PAT from penetrating more than a few centimeters and limited its application for studying deeply seated targets. A variety of PAT technologies have been developed to extend the imaging depth, including employing deep-penetrating microwaves and X-ray photons as excitation sources, delivering the light to the inside of the organ, reshaping the light wavefront to better focus into scattering medium, as well as improving the sensitivity of ultrasonic transducers. At the same time, novel optical fluence mapping algorithms and image reconstruction methods have been developed to improve the quantitative accuracy of PAT, which is crucial to recover weak molecular signals at larger depths. The development of highly-absorbing near-infrared PA molecular probes has also flourished to provide high sensitivity and specificity in studying cellular processes. This review aims to introduce the recent developments in deep PA molecular imaging, including novel imaging systems, image processing methods and molecular probes, as well as their representative biomedical applications. Existing challenges and future directions are also discussed.

Hypoxia, a low tissue oxygenation condition caused by insufficient oxygen supply, leads to potentially irreversible tissue damage, such as brain infarction during stroke. Intravascular oxygenation has long been used by photoacoustic imaging, among other imaging modalities, to study hypoxia. However, intravascular oxygenation describes only the oxygen supply via microcirculation, which does not directly reflect the amount of free oxygen available for metabolism in the interstitial fluid. Therefore, to fully understand hypoxia, it is highly desirable to monitor blood oxygenation as well as tissue oxygenation during the same biological process. In this work, by combining high-resolution photoacoustic microscopy (PAM) and a novel bioreducible N-oxide-based hypoxia-sensitive probe HyP-650, we have demonstrated simultaneous imaging of intravascular oxygenation and tissue hypoxia. We have established detailed chemical, optical, and photoacoustic properties of HyP-650 for hypoxic activation in vitro and in living cells. We have also performed PAM on hindlimb ischemia models and tumor-bearing mice to study the correlation between intravascular oxygenation and tissue oxygenation at various hypoxic levels. We expect that Hyp-650 enhanced photoacoustic imaging will find a variety of applications in brain and cancer research.

Non-invasive photoacoustic tomography (PAT) of mouse brains with intact skulls has been a challenge due to the skull's strong acoustic attenuation, aberration, and reverberation, especially in the high-frequency range (>15 MHz). In this paper, we systematically investigated the impacts of the murine skull on the photoacoustic wave propagation and on the PAT image reconstruction. We studied the photoacoustic acoustic wave aberration due to the acoustic impedance mismatch at the skull boundaries and the mode conversion between the longitudinal wave and shear wave. The wave's reverberation within the skull was investigated for both longitudinal and shear modes. In the inverse process, we reconstructed the transcranial photoacoustic computed tomography (PACT) and photoacoustic microscopy (PAM) images of a point target enclosed by the mouse skull, showing the skull's different impacts on both modalities. Finally, we experimentally validated the simulations by imaging an in vitro mouse skull phantom using representative transcranial PAM and PACT systems. The experimental results agreed well with the simulations and confirmed the accuracy of our forward and inverse models. We expect that our results will provide better understanding of the impacts of the murine skull on transcranial photoacoustic brain imaging and pave the ways for future technical improvements., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Acoustic-resolution photoacoustic microscopy (AR-PAM) can provide penetration depths beyond the optical diffusion limit, but its lateral resolution is typically much worse than its axial resolution, resulting in a low-resolution isotropy. We herein present a dual-view AR-PAM (DV-AR-PAM) system that exploits two orthogonally arranged focused ultrasonic transducers. Taking advantage of their complementary acoustic detecting profiles, DV-AR-PAM allows the fusion of the dual-view signals, resulting in an enhancement in the resolution isotropy from 29% to 95% within one imaging plane, as demonstrated by simulation results on densely packed particles and experimental results on a single carbon fiber. The application of this method in vivo revealed distinct microvasculature structures in mouse skin that were previously indistinguishable with single-view detection, demonstrating the potential of DV-AR-PAM for vascular and neurological studies.

By acoustically detecting the optical absorption contrast, photoacoustic (PA) tomography (PAT) has broken the penetration limits of traditional high-resolution optical imaging. Through spectroscopic analysis of the target's optical absorption, PAT can identify a wealth of endogenous and exogenous molecules and thus is inherently capable of molecular imaging with high sensitivity. PAT's molecular sensitivity is uniquely accompanied by non-ionizing radiation, high spatial resolution, and deep penetration in biological tissues, which other optical imaging modalities cannot achieve yet. In this concise review, we summarize the most recent technological advancements in PA molecular imaging and highlight the novel molecular probes specifically made for PAT in deep tissues. We conclude with a brief discussion of the opportunities for future advancements., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A conventional photoacoustic microscopy (PAM) system typically has to make tradeoffs between its spatial resolution and penetration depth, by choosing a fixed configuration of optical excitation and acoustic detection. The single-scale imaging capability of PAM may limit its applications in biomedical studies. Here, we report a quad-mode photoacoustic microscopy (QM-PAM) system with four complementary spatial resolutions and maximum penetration depths. For this we first developed a ring-shaped focused ultrasound transducer that has two independent elements with respective central frequencies at 20 MHz and 40 MHz, providing complementary acoustically-determined spatial resolutions and penetration depths. To accommodate the dual-element ultrasound transducer, we implemented two optical excitation modes to provide tightly- and weakly-focused light illumination. The dual-element acoustic detection combined with the two optical focusing modes can thus provide four imaging scales in a single imaging device, with consistent contrast mechanisms and co-registered field of views. We have demonstrated the multiscale morphological, functional, and molecular imaging capability of QM-PAM in the mouse head, leg and ear in vivo. We expect the high scale flexibility of QM-PAM will enable broad applications in preclinical studies.

Photoacoustic (PA) computed tomography (PACT) benefits from genetically encoded probes with photochromic behavior, which dramatically increase detection sensitivity and specificity through photoswitching and differential imaging. Starting with a DrBphP bacterial phytochrome, we have engineered a near-infrared photochromic probe, DrBphP-PCM, which is superior to the full-length RpBphP1 phytochrome previously used in differential PACT. DrBphP-PCM has a smaller size, better folding, and higher photoswitching contrast. We have imaged both DrBphP-PCM and RpBphP1 simultaneously on the basis of their unique signal decay characteristics, using a reversibly switchable single-impulse panoramic PACT (RS-SIP-PACT) with a single wavelength excitation. The simple structural organization of DrBphP-PCM allows engineering a bimolecular PA complementation reporter, a split version of DrBphP-PCM, termed DrSplit. DrSplit enables PA detection of protein-protein interactions in deep-seated mouse tumors and livers, achieving 125-µm spatial resolution and 530-cell sensitivity in vivo. The combination of RS-SIP-PACT with DrBphP-PCM and DrSplit holds great potential for noninvasive multi-contrast deep-tissue functional imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) has demonstrated fast, label-free volumetric imaging of optical-absorption contrast within the quasiballistic regime of photon scattering. However, the limited numerical aperture of the ultrasonic transducer restricts the detectability of the photoacoustic waves, thus resulting in incomplete reconstructed features. To tackle the limited-view problem, we added an oblique illumination beam to the original coaxial optical-acoustic scheme to provide a complementary detection view. The virtual augmentation of the detection view was validated through numerical simulations and tissue-phantom experiments. More importantly, the combination of top and oblique illumination successfully imaged a mouse brain in vivo down to 1 mm in depth, showing detailed brain vasculature. Of special note, it clearly revealed the diving vessels that were long missing in images from original OR-PAM., ((2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).)

We report a photoacoustic computed tomography (PACT) system using a customized optical fiber with a cylindrical diffuser to internally illuminate deep targets. The traditional external light illumination in PACT usually limits the penetration depth to a few centimeters from the tissue surface, mainly due to strong optical attenuation along the light propagation path from the outside in. By contrast, internal light illumination, with external ultrasound detection, can potentially detect much deeper targets. Different from previous internal illumination PACT implementations using forward-looking optical fibers, our internal-illumination PACT system uses a customized optical fiber with a 3-cm-long conoid needle diffuser attached to the fiber tip, which can homogeneously illuminate the surrounding space and substantially enlarge the field of view. We characterized the internal illumination distribution and PACT system performance. We performed tissue phantom and in vivo animal studies to further demonstrate the superior imaging depth using internal illumination over external illumination. We imaged a 7.5-cm-deep leaf target embedded in optically scattering medium and the beating heart of a mouse overlaid with 3.7-cm-thick chicken tissue. Our results have collectively demonstrated that the internal light illumination combined with external ultrasound detection might be a useful strategy to improve the penetration depth of PACT in imaging deep organs of large animals and humans., ((2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).)

Optical-resolution photoacoustic microscopy (OR-PAM) has proven useful for anatomical and functional imaging with high spatial resolutions. However, the coherent signal generation and the desired reflection-mode detection in OR-PAM can result in a limited detectability of features aligned with the acoustic axis (ie, vertical structures). Here, we investigated the limited-view phenomenon in OR-PAM by simulating the generation and propagation of the acoustic pressure waves and determined the key optical parameters affecting the visibility of vertical structures. Proof-of-concept numerical experiments were performed with different illumination angles, optical foci and numerical apertures (NA) of the objective lens. The results collectively show that an NA of 0.3 can readily improve the visibility of vertical structures in a typical reflection-mode OR-PAM system. This conclusion was confirmed by numerical simulations on the cortical blood vessels in a mouse brain and by experiments in a suture-cross phantom and in a mouse brain in vivo., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Temperature mapping during thermotherapy can help precisely control the heating process, both temporally and spatially, to efficiently kill the tumor cells and prevent the healthy tissues from heating damage. Photoacoustic tomography (PAT) has been used for noninvasive temperature mapping with high sensitivity, based on the linear correlation between the tissue's Grüneisen parameter and temperature. However, limited by the tissue's unknown optical properties and thus the optical fluence at depths beyond the optical diffusion limit, the reported PAT thermometry usually takes a ratiometric measurement at different temperatures and thus cannot provide absolute measurements. Moreover, ratiometric measurement over time at different temperatures has to assume that the tissue's optical properties do not change with temperatures, which is usually not valid due to the temperature-induced hemodynamic changes. We propose an optical-diffusion-model-enhanced PAT temperature mapping that can obtain the absolute temperature distribution in deep tissue, without the need of multiple measurements at different temperatures. Based on the initial acoustic pressure reconstructed from multi-illumination photoacoustic signals, both the local optical fluence and the optical parameters including absorption and scattering coefficients are first estimated by the optical-diffusion model, then the temperature distribution is obtained from the reconstructed Grüneisen parameters. We have developed a mathematic model for the multi-illumination PAT of absolute temperatures, and our two-dimensional numerical simulations have shown the feasibility of this new method. The proposed absolute temperature mapping method may set the technical foundation for better temperature control in deep tissue in thermotherapy., ((2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).)

We report a photoacoustic thermal flowmetry based on optical-resolution photoacoustic microscopy (OR-PAM) using a single laser source for both thermal tagging and photoacoustic excitation. When an optically absorbing medium is flowing across the optical focal zone of OR-PAM, a small volume of the medium within the optical focus is repeatedly illuminated and heated by a train of laser pulses with a high repetition rate. The average temperature of the heated volume at each laser pulse is indicated by the photoacoustic signal excited by the same laser pulse due to the well-established linear relationship between the Grueneisen coefficient and the local temperature. The thermal dynamics of the heated medium volume, which are closely related to the flow speed, can therefore be measured from the time course of the detected photoacoustic signals. Here, we have developed a lumped mathematical model to describe the time course of the photoacoustic signals as a function of the medium's flow speed. We conclude that the rising time constant of the photoacoustic signals is linearly dependent on the flow speed. Thus, the flow speed can be quantified by fitting the measured photoacoustic signals using the derived mathematical model. We first performed proof-of-concept experiments using defibrinated bovine blood flowing in a plastic tube. The experiment results have demonstrated that the proposed method has high accuracy (∼±6%) and a wide range of measurable flow speeds. We further validated the method by measuring the blood flow speeds of the microvasculature in a mouse ear in vivo., ((2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).)

Genetically encoded contrast in photoacoustic imaging (PAI) is complementary to the intrinsic contrast provided by endogenous absorbing chromophores such as hemoglobin. The use of reporter genes expressing absorbing proteins opens the possibility of visualizing dynamic cellular and molecular processes. This is an enticing prospect but brings with it challenges and limitations associated with generating and detecting different types of reporters. The purpose of this review is to compare existing PAI reporters and signal detection strategies, thereby offering a practical guide, particularly for the nonbiologist, to choosing the most appropriate reporter for maximum sensitivity in the biological and technological system of interest.

We applied high-speed photoacoustic microscopy (PAM) for both cortical microenvironment studies and dynamic brain studies, with micrometer-level optical resolution and a millisecond-level cross-sectional imaging speed over a millimeter-level field of view. We monitored blood flow redistribution in mini-stroke mouse models and cerebral autoregulation induced by a vasoactive agent. Our results collectively suggest that high-speed PAM is a promising tool for understanding dynamic neurophysiological phenomena, complementing conventional imaging modalities., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Optical-resolution photoacoustic microscopy (OR-PAM) offers label-free

Using photoacoustic microscopy (PAM), we evaluated non-invasively oxygenation and vascularization in vivo due to multiple myeloma (MM) progression. Mice injected with MM.1S-GFP were monitored with a fluorescence microscope for tumor progression. In vivo PAM of the cerebral bone marrow quantified the total oxygen saturation (sO 2 ). At 28 days after the MM cell injection, the total sO 2 had decreased by half in the developing tumor regions, while in the non-tumor regions it had decreased by 20% compared with the value at one day post MM injection. The blood vessel density was reduced by 35% in the developing tumor regions, while in the non-tumor regions it was reduced by 8% compared with the value at one day post MM injection. Hence, PAM corroborated the development of hypoxia due to MM progression and demonstrated decreased vascularization surrounding the tumor areas.

The life sciences can benefit greatly from imaging technologies that connect microscopic discoveries with macroscopic observations. One technology uniquely positioned to provide such benefits is photoacoustic tomography (PAT), a sensitive modality for imaging optical absorption contrast over a range of spatial scales at high speed. In PAT, endogenous contrast reveals a tissue's anatomical, functional, metabolic, and histologic properties, and exogenous contrast provides molecular and cellular specificity. The spatial scale of PAT covers organelles, cells, tissues, organs, and small animals. Consequently, PAT is complementary to other imaging modalities in contrast mechanism, penetration, spatial resolution, and temporal resolution. We review the fundamentals of PAT and provide practical guidelines for matching PAT systems with research needs. We also summarize the most promising biomedical applications of PAT, discuss related challenges, and envision PAT's potential to lead to further breakthroughs.

Photoacoustic tomography (PAT) has become one of the fastest growing fields in biomedical optics. Unlike pure optical imaging, such as confocal microscopy and two-photon microscopy, PAT employs acoustic detection to image optical absorption contrast with high-resolution deep into scattering tissue. So far, PAT has been widely used for multiscale anatomical, functional, and molecular imaging of biological tissues. We focus on PAT’s basic principles, major implementations, imaging contrasts, and recent applications.

Myoglobin is an essential oxygen-binding hemoprotein in skeletal and cardiac muscles that buffers intracellular oxygen (O2) concentration in response to hypoxia or elevated muscle activities. We present a method that uses photoacoustic computed tomography to measure the distribution of myoglobin in tissue and the oxygen saturation of myoglobin (sO2-Mb ). From photoacoustic measurements of mice in different oxygenation states, we performed calibration-free quantification of the sO2-Mb change in the backbone muscle in vivo.

Elastography can noninvasively map the elasticity distribution in biological tissue, which can potentially be used to reveal disease conditions. In this Letter, we have demonstrated photoacoustic elastography by using a linear-array photoacoustic computed tomography system. The feasibility of photoacoustic elastography was first demonstrated by imaging the strains of single-layer and bilayer gelatin phantoms with various stiffness values. The measured strains agreed well with theoretical values, with an average error of less than 5.2%. Next, in vivo photoacoustic elastography was demonstrated on a mouse leg, where the fat and muscle distribution was mapped based on the elasticity contrast. We confirmed the photoacoustic elastography results by ultrasound elastography performed simultaneously.

Photoacoustic tomography (PAT) combines rich optical absorption contrast with the high spatial resolution of ultrasound at depths in tissue. The high scalability of PAT has enabled anatomical imaging of biological structures ranging from organelles to organs. The inherent functional and molecular imaging capabilities of PAT have further allowed it to measure important physiological parameters and track critical cellular activities. Integration of PAT with other imaging technologies provides complementary capabilities and can potentially accelerate the clinical translation of PAT., (© The Author(s) 2015.)

Since its invention in the 17th century, optical microscopy has revolutionized biomedical studies by scrutinizing the biological realm on cellular levels, taking advantage of its excellent light-focusing capability. However, most biological tissues scatter light highly. As light travels in tissue, cumulative scattering events cause the photons to lose their original propagation direction and, thus, their ability to be focused, which has largely limited the penetration depth of optical microscopy. Conventional planar optical microscopy can provide penetration of only ~100 ?m before photons begin to be scattered. The penetration of modern optical microcopy, such as confocal microscopy and multiphoton microscopy, is still limited to approximately the optical diffusion limit (~1 mm in the skin as approximated by one optical transport mean free path), where scattered photons retain a strong memory of the original propagation direction. So far, it still remains a challenge for pure optical methods to achieve high-resolution in vivo imaging beyond the diffusion limit (i.e., superdepth imaging).

We present fast functional photoacoustic microscopy (PAM) for three-dimensional high-resolution, high-speed imaging of the mouse brain, complementary to other imaging modalities. We implemented a single-wavelength pulse-width-based method with a one-dimensional imaging rate of 100 kHz to image blood oxygenation with capillary-level resolution. We applied PAM to image the vascular morphology, blood oxygenation, blood flow and oxygen metabolism in both resting and stimulated states in the mouse brain.

We report photoacoustic and ultrasonic endoscopic images of two intact rabbit esophagi. To investigate the esophageal lumen structure and microvasculature, we performed in vivo and ex vivo imaging studies using a 3.8-mm diameter photoacoustic endoscope and correlated the images with histology. Several interesting anatomic structures were newly found in both the in vivo and ex vivo images, which demonstrates the potential clinical utility of this endoscopic imaging modality. In the ex vivo imaging experiment, we acquired high-resolution motion-artifact-free three-dimensional photoacoustic images of the vasculatures distributed in the walls of the esophagi and extending to the neighboring mediastinal regions. Blood vessels with apparent diameters as small as 190 μm were resolved. Moreover, by taking advantage of the dual-mode high-resolution photoacoustic and ultrasound endoscopy, we could better identify and characterize the anatomic structures of the esophageal lumen, such as the mucosal and submucosal layers in the esophageal wall, and an esophageal branch of the thoracic aorta. In this paper, we present the first photoacoustic images showing the vasculature of a vertebrate esophagus and discuss the potential clinical applications and future development of photoacoustic endoscopy.

Despite its critical function in coordinating the egress of inflammatory and immune cells out of tissues and maintaining fluid balance, the causative role of lymphatic network dysfunction in pathological settings is still understudied. Engineered-animal models and better noninvasive high spatial-temporal resolution imaging techniques in both preclinical and clinical studies will help to improve our understanding of different lymphatic-related pathologic disorders. Our aim was to take advantage of our newly optimized noninvasive wide-field fast-scanning photoacoustic (PA) microcopy system to coordinately image the lymphatic vasculature and its flow dynamics, while maintaining high resolution and detection sensitivity. Here, by combining the optical-resolution PA microscopy with a fast-scanning water-immersible microelectromechanical system scanning mirror, we have imaged the lymph dynamics over a large field-of-view, with high spatial resolution and advanced detection sensitivity. Depending on the application, lymphatic vessels (LV) were spectrally or temporally differentiated from blood vessels. Validation experiments were performed on phantoms and in vivo to identify the LV. Lymphatic flow dynamics in nonpathological and pathological conditions were also visualized. These results indicate that our newly developed PA microscopy is a promising tool for lymphatic-related biological research.

Compared with visible light (380-700 nm), near-infrared light (700-1400 nm) undergoes weaker optical attenuation in biological tissue; thus, it can penetrate deeper. Herein, we demonstrate near-infrared optical-resolution photoacoustic microscopy (NIR-OR-PAM) with 1046 nm illumination. A penetration depth of 3.2 mm was achieved in chicken breast tissue ex vivo using optical fluence within the American National Standards Institute (ANSI) limit (100  mJ/cm2). Beyond ∼0.6  mm deep in chicken breast tissue, NIR-OR-PAM has shown finer resolution than the visible counterpart with 570 nm illumination. The deep imaging capability of NIR-OR-PAM was validated in both a mouse ear and a mouse brain. NIR-OR-PAM of possible lipid contrast was explored as well.

Optical-resolution photoacoustic microscopy (OR-PAM) is an imaging modality with superb penetration depth and excellent absorption contrast. Here we demonstrate, for the first time, that this technique can advance quantitative analysis of conventional chromogenic histochemistry. Because OR-PAM can quantify the absorption contrast at different wavelengths, it is feasible to spectrally resolve the specific biomolecules involved in a staining color. Furthermore, the tomographic capability of OR-PAM allows for noninvasive volumetric imaging of a thick sample without microtoming it. By immunostaining the sample with different chromogenic agents, we further demonstrated the ability of OR-PAM to resolve different types of cells in a coculture sample with imaging depths up to 1 mm. Taken together, the integration of OR-PAM with (immuno)histochemistry offers a simple and versatile technique with broad applications in cell biology, pathology, tissue engineering, and related biomedical studies., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We present an optically encoded photoacoustic (PA) flow imaging method based on optical-resolution PA microscopy. An intensity-modulated continuous-wave laser photothermally encodes the flowing medium, and a pulsed laser generates PA waves to image the encoded heat pattern. Flow speeds can be calculated by cross correlation. The method was validated in phantoms at flow speeds ranging from 0.23 to 11  mm/s. Venous blood flow speed in a mouse ear was also measured.

We propose a calibration-free photoacoustic (PA) method for transverse flow measurements. In this method, a pulsed periodically structured (i.e., grating patterned) optical beam is used to illuminate flowing absorptive particles in an optically scattering medium. The PA signal amplitudes measured over consecutive laser pulses carry an imprint of the illumination structure. The modulation frequency of the imprint is proportional to the component of the flow speed projected onto the normal axis of the striped illumination pattern. This method can tolerate high particle density, and is insensitive to the particle size, thus calibration-free. Bovine blood and microsphere phantoms were used to validate the proposed method. Blood flow in a mouse ear was measured in vivo as well.

Currently, laser fluence calibration is typically required for quantitative measurement of particle concentration in photoacoustic imaging. Here, we present a calibration-free method to quantify the absolute particle concentration by statistically analyzing photoacoustic signals. The proposed method is based on the fact that Brownian motion induces particle count fluctuation in the detection volume. If the count of particles in the detection volume is assumed to follow the Poisson distribution, its expected value can be calculated by the photoacoustic signal mean and variance. We first derived a theoretical model for photoacoustic signals. Then, we applied our method to quantitative measurement of different concentrations of various particles, including red blood cells. Finally, we performed in vivo experiments to demonstrate the potential of our method in biological applications. The experimental results agreed well with the predictions from the theoretical model suggesting that our method can be used for noninvasive measurement of absolute particle concentrations in deep tissue without fluence calibration.

Subdiffraction optical microscopy allows the imaging of cellular and subcellular structures with a resolution finer than the diffraction limit. Here, combining the absorption-based photoacoustic effect and intensity-dependent photobleaching effect, we demonstrate a simple method for subdiffraction photoacoustic imaging of both fluorescent and nonfluorescent samples. Our method is based on a double-excitation process, where the first excitation pulse partially and inhomogeneously bleaches the molecules in the diffraction-limited excitation volume, thus biasing the signal contributions from a second excitation pulse striking the same region. The differential signal between the two excitations preserves the signal contribution mostly from the center of the excitation volume, and dramatically sharpens the lateral resolution. Moreover, due to the nonlinear nature of the signal, our method offers an inherent optical sectioning capability, which is lacking in conventional photoacoustic microscopy. By scanning the excitation beam, we performed three-dimensional subdiffraction imaging of varied fluorescent and nonfluorescent species. As any molecules have absorption, this technique has the potential to enable label-free subdiffraction imaging, and can be transferred to other optical imaging modalities or combined with other subdiffraction methods.

Degradation is among the most important properties of biomaterial scaffolds, which are indispensable for regenerative medicine. The currently used method relies on the measurement of mass loss across different samples and cannot track the degradation of an individual scaffold in situ. Here we report, for the first time, the use of multiscale photoacoustic microscopy to non-invasively monitor the degradation of an individual scaffold. We could observe alterations to the morphology and structure of a scaffold at high spatial resolution and deep penetration, and more significantly, quantify the degradation of an individual scaffold as a function of time, both in vitro and in vivo. In addition, the remodeling of vasculature inside a scaffold can be visualized simultaneously using a dual-wavelength scanning mode in a label-free manner. This optoacoustic method can be used to monitor the degradation of individual scaffolds, offering a new approach to non-invasively analyze and quantify biomaterial-tissue interactions in conjunction with the assessment of in vivo vascular parameters., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Photoacoustic thermography is a promising tool for temperature measurement in deep tissue. Here we propose an absolute temperature measurement method based on the dual temperature dependences of the Grüneisen parameter and the speed of sound in tissue. By taking ratiometric measurements at two adjacent temperatures, we can eliminate the factors that are temperature irrelevant but difficult to correct for in deep tissue. To validate our method, absolute temperatures of blood-filled tubes embedded ~9 mm deep in chicken tissue were measured in a biologically relevant range from 28°C to 46°C. The temperature measurement accuracy was ~0.6°C. The results suggest that our method can be potentially used for absolute temperature monitoring in deep tissue during thermotherapy.

Blood flow speed is an important functional parameter. Doppler ultrasound flowmetry lacks sufficient sensitivity to slow blood flow (several to tens of millimeters per second) in deep tissue. To address this challenge, we developed ultrasonically encoded photoacoustic flowgraphy combining ultrasonic thermal tagging with photoacoustic imaging. Focused ultrasound generates a confined heat source in acoustically absorptive fluid. Thermal waves propagate with the flow and are directly visualized in pseudo color using photoacoustic computed tomography. The Doppler shift is employed to calculate the flow speed. This method requires only acoustic and optical absorption, and thus is applicable to continuous fluid. A blood flow speed as low as 0.24  mm·s(-1)} was successfully measured. Deep blood flow imaging was experimentally demonstrated under 5-mm-thick chicken breast tissue.

We report the development of photoacoustic flowmetry assisted by high-intensity focused ultrasound (HIFU). This novel method employs HIFU to generate a heating impulse in the flow medium, followed by photoacoustic monitoring of the thermal decay process. Photoacoustic flowmetry in a continuous medium remains a challenge in the optical diffusive regime. Here, both the HIFU heating and photoacoustic detection can focus at depths beyond the optical diffusion limit (~1 mm in soft tissue). This method can be applied to a continuous medium, i.e., a medium without discrete scatterers or absorbers resolvable by photoacoustic imaging. Flow speeds up to 41 mm·s-1 have been experimentally measured in a blood phantom covered by 1.5-mm-thick tissue.

Due to strong light scattering in tissue, both the spatial resolution and maximum penetration depth of optical-resolution photoacoustic microscopy (OR-PAM) deteriorate sharply with depth. To reduce tissue scattering, we propose to use glycerol as an optical clearing agent in OR-PAM. Our results show that the imaging performance of OR-PAM can be greatly enhanced by optical clearing both in vitro and in vivo.