Photoacoustic Tomography with Temporal Encoding Reconstruction (PATTERN) for cross-modal individual analysis of the whole brain.

Cross-modal analysis of the same whole brain is an ideal strategy to uncover brain function and dysfunction. However, it remains challenging due to the slow speed and destructiveness of traditional whole-brain optical imaging techniques. Here we develop a new platform, termed Photoacoustic Tomography with Temporal Encoding Reconstruction (PATTERN), for non-destructive, high-speed, 3D imaging of ex vivo rodent, ferret, and non-human primate brains. Using an optimally designed image acquisition scheme and an accompanying machine-learning algorithm, PATTERN extracts signals of genetically-encoded probes from photobleaching-based temporal modulation and enables reliable visualization of neural projection in the whole central nervous system with 3D isotropic resolution. Without structural and biological perturbation to the sample, PATTERN can be combined with other whole-brain imaging modalities to acquire the whole-brain image with both high resolution and morphological fidelity. Furthermore, cross-modal transcriptome analysis of an individual brain is achieved by PATTERN imaging. Together, PATTERN provides a compatible and versatile strategy for brain-wide cross-modal analysis at the individual level.

Innovative methods for microplastic characterization and detection: Deep learning supported by photoacoustic imaging and automated pre-processing data.

Plastic products' widespread applications and their non-biodegradable nature have resulted in the continuous accumulation of microplastic waste, emerging as a significant component of ecological environmental issues. In the field of microplastic detection, the intricate morphology poses challenges in achieving rapid visual characterization of microplastics. In this study, photoacoustic imaging technology is initially employed to capture high-resolution images of diverse microplastic samples. To address the limited dataset issue, an automated data processing pipeline is designed to obtain sample masks while effectively expanding the dataset size. Additionally, we propose Vqdp2, a generative deep learning model with multiple proxy tasks, for predicting six forms of microplastics data. By simultaneously constraining model parameters through two training modes, outstanding morphological category representations are achieved. The results demonstrate Vqdp2's excellent performance in classification accuracy and feature extraction by leveraging the advantages of multi-task training. This research is expected to be attractive for the detection classification and visual characterization of microplastics.

Multispectral optoacoustic tomography of benign parotid tumors in vivo: a prospective observational pilot study.

Parotid lumps are a heterogeneous group of mainly benign but also malignant tumors. Preoperative imaging does not allow a differentiation between tumor types. Multispectral optoacoustic tomography (MSOT) may improve the preoperative diagnostics. In this first prospective pilot trial the ability of MSOT to discriminate between the two most frequent benign parotid tumors, pleomorphic adenoma (PA) and Warthin tumor (WT) as well as to normal parotid tissue was explored. Six wavelengths (700, 730, 760, 800, 850, 900 nm) and the parameters deoxygenated (HbR), oxygenated (HbO2), total hemoglobin (HbT), and saturation of hemoglobin (sO2) were analyzed. Ten patients with PA and fourteen with WT were included (12/12 female/male; median age: 51 years). For PA, the mean values for all measured wave lengths as well as for the hemoglobin parameters were different for the tumors compared to the healthy parotid (all p < 0.05). The mean MSOT parameters were all significantly higher (all p < 0.05) in the WT compared to healthy parotid gland except for HbT and sO2. Comparing both tumors directly, the mean values of MSOT parameters were not different between PA and WT (all p > 0.05). Differences were seen for the maximal MSOT parameters. The maximal tumor values for 900 nm, HbR, HbT, and sO2 were lower in PA than in WT (all p < 0.05). This preliminary MSOT parotid tumor imaging study showed clear differences for PA or WT compared to healthy parotid tissue. Some MSOT characteristics of PA and WT were different but needed to be explored in larger studies.

Doping Engineering To Modulate Surface Plasmon Resonance and Enzyme-like Activities for Enhancing Photoacoustic Imaging-Guided Targeted Cancer Therapy in the Second Near-Infrared Window.

Biological imaging-guided targeted tumor therapy has been a soughtafter goal in the field of cancer diagnosis and treatment. To this end, we proposed a strategy to modulate surface plasmon resonance and endow WO3-x nanoparticles (NPs) with enzyme-like catalytic properties by doping Fe2+ in the structure of the NPs. Doping of the Fe2+ introduced oxygen vacancies into the structure of the NPs, inducing a red shift of the maximum absorption wavelength into the near-infrared II (NIR-II) region and enhancing the photoacoustic (PA) and photothermal properties of the NPs for more effective imaging-guided cancer therapy. Under NIR-II laser irradiation, the Fe-WO3-x NPs produced very strong NIR-II PA and photothermal effects, which significantly enhanced the PA imaging and photothermal treatment effects. On the other hand, Fe2+ in Fe-WO3-x could undergo Fenton reactions with H2O2 in the tumor tissue to generate ·OH for chemodynamic therapy. In addition, Fe-WO3-x can also catalyze the above reactions to produce more reactive oxygen species (ROS) and induce the oxidation of NADH to interfere with intracellular adenosine triphosphate (ATP) synthesis, thereby further improving the efficiency of cancer therapy. Specific imaging of tumor tissue and targeted synergistic therapy was achieved after ligation of a MUC1 aptamer to the surface of the Fe-WO3-x NPs by the complexing of -COOH in MUC1 with tungsten ions on the surface of the NPs. These results demonstrated that Fe-WO3-x NPs could be a promising diagnosis and therapeutic agent for cancer. Such a study opens up new avenues into the rational design of nanodiagnosis and treatment agents for NIR-II PA imaging and cancer therapy.

Polymerized Network-Based Artificial Peroxisome Reprogramming Macrophages for Photoacoustic Imaging-Guided Treatment of Rheumatoid Arthritis.

Artificial peroxisomes (AP) with enzyme-mimetic catalytic activity and recruitment ability have drawn a great deal of attention in fabricating protocell systems for scavenging reactive oxygen species (ROS), modulating the inflammatory microenvironment, and reprogramming macrophages, which is of great potential in treating inflammatory diseases such as rheumatoid arthritis (RA). Herein, a macrophage membrane-cloaked Cu-coordinated polyphthalocyanine-based AP (CuAP) is prepared with a macrocyclic conjugated polymerized network and embedded Cu-single atomic active center, which mimics the catalytic activity and coordination environment of natural superoxide dismutase and catalase, possesses the inflammatory recruitment ability of macrophages, and performs photoacoustic imaging (PAI)-guided treatment. The results of both in vitro cellular and in vivo animal experiments demonstrated that the CuAP under ultrasound and microbubbles could efficiently scavenge excess ROS in cells and tissues, modulate microenvironmental inflammatory cytokines such as interleukin-1ß, tumor necrosis factor-α, and arginase-1, and reprogram macrophages by polarization of M1 (proinflammatory phenotype) to M2 (anti-inflammatory phenotype). We believe this study offers a proof of concept for engineering multifaceted AP and a promising approach for a PAI-guided treatment platform for RA.

Compressed Sensing for Biomedical Photoacoustic Imaging: A Review.

Photoacoustic imaging (PAI) is a rapidly developing emerging non-invasive biomedical imaging technique that combines the strong contrast from optical absorption imaging and the high resolution from acoustic imaging. Abnormal biological tissues (such as tumors and inflammation) generate different levels of thermal expansion after absorbing optical energy, producing distinct acoustic signals from normal tissues. This technique can detect small tissue lesions in biological tissues and has demonstrated significant potential for applications in tumor research, melanoma detection, and cardiovascular disease diagnosis. During the process of collecting photoacoustic signals in a PAI system, various factors can influence the signals, such as absorption, scattering, and attenuation in biological tissues. A single ultrasound transducer cannot provide sufficient information to reconstruct high-precision photoacoustic images. To obtain more accurate and clear image reconstruction results, PAI systems typically use a large number of ultrasound transducers to collect multi-channel signals from different angles and positions, thereby acquiring more information about the photoacoustic signals. Therefore, to reconstruct high-quality photoacoustic images, PAI systems require a significant number of measurement signals, which can result in substantial hardware and time costs. Compressed sensing is an algorithm that breaks through the Nyquist sampling theorem and can reconstruct the original signal with a small number of measurement signals. PAI based on compressed sensing has made breakthroughs over the past decade, enabling the reconstruction of low artifacts and high-quality images with a small number of photoacoustic measurement signals, improving time efficiency, and reducing hardware costs. This article provides a detailed introduction to PAI based on compressed sensing, such as the physical transmission model-based compressed sensing method, two-stage reconstruction-based compressed sensing method, and single-pixel camera-based compressed sensing method. Challenges and future perspectives of compressed sensing-based PAI are also discussed.

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.

Semiconducting Polymers Based on Asymmetric Thiadiazoloquinoxaline for Augmented In Vivo NIR-II Photoacoustic Imaging.

A photoacoustic (PA) imaging technique using the second near-infrared (NIR-II) window has attracted more and more attention because of its merits of deeper penetration depth and higher signal-to-noise (S/N) ratio than that using the first near-infrared (NIR-I) one. However, the design and development of high-performance PA imaging contrast agents in the NIR-II window is still a challenge. A semiconducting polymer, constructed by asymmetric units, exhibits regiorandom characteristics that effectively increase the distortion of the backbone. This increase in the degree of twist can regulate the twisted intramolecular charge transfer (TICT) effect, resulting in an enhancement of the PA signal. In this paper, an asymmetric structural acceptor strategy is developed to improve the PA signals of the resulting semiconducting polymer (PATQ-MP) in the NIR-II window with improved brightness, higher S/N ratio, and better photothermal conversion efficiency compared to polymers with the same main-chain structure containing a symmetric acceptor. DFT analysis showed that PATQ-MP containing an asymmetric acceptor monomer had a larger dihedral angle, which effectively improved the PA signal intensity by enhancing the TICT effect. The PEG-encapsulated PATQ-MP nanoparticles exhibit promising performance in the PA imaging of mouse tumors in vivo, demonstrating the clear identification of microvessels as small as 100 µm along with rapid metabolism within a span of 5 h. Therefore, this work provides a unique molecular design strategy for improving the signal intensity of PA imaging in the NIR-II window.

Programmable spin and transport of a living shrimp egg through photoacoustic pressure.

In the fields of biomedicine and microfluidics, the non-contact capture, manipulation, and spin of micro-particles hold great importance. In this study, we propose a programmable non-contact manipulation technique that utilizes photoacoustic effect to spin and transport living shrimp eggs. By directing a modulated pulsed laser toward a liquid-covered stainless-steel membrane, we can excite patterned Lamb waves within the membrane. These Lamb waves occur at the interface between the membrane and the liquid, enabling the manipulation of nearby particles. Experimental results demonstrate the successful capture, spin, and transport of shrimp eggs in diameter of 220 µm over a distance of about 5 mm. Calculations indicate that the acoustic radiation force and torque generated by our photoacoustic manipulation system are more than 299.5 nN and 41.0 nN·mm, respectively. The system surpasses traditional optical tweezers in terms of force and traditional acoustic tweezers in terms of flexibility. Consequently, this non-contact manipulation system significantly expands the possibilities for applications in various fields, including embryo screening, cell manipulation, and microfluidics.

Thermal-tagging photoacoustic remote sensing flowmetry.

Ultrasound coupling is one of the critical challenges for traditional photoacoustic (or optoacoustic) microscopy (PAM) techniques transferred to the clinical examination of chronic wounds and open tissues. A promising alternative potential solution for breaking the limitation of ultrasound coupling in PAM is photoacoustic remote sensing (PARS), which implements all-optical non-interferometric photoacoustic measurements. Functional imaging of PARS microscopy was demonstrated from the aspects of histopathology and oxygen metabolism, while its performance in hemodynamic quantification remains unexplored. In this Letter, we present an all-optical thermal-tagging flowmetry approach for PARS microscopy and demonstrate it with comprehensive mathematical modeling and ex vivo and in vivo experimental validations. Experimental results demonstrated that the detectable range of the blood flow rate was from 0 to 12 mm/s with a high accuracy (measurement error:±1.2%) at 10-kHz laser pulse repetition rate. The proposed all-optical thermal-tagging flowmetry offers an effective alternative approach for PARS microscopy realizing non-contact dye-free hemodynamic imaging.

Novel activated NIR-II fluorescence/Ratio photoacoustic probe for dual-modality accurate imaging of palladium ions overload in mouse liver.

Palladium contaminants can pose risks to human health and the natural environment. Once Pd2+ enters the body, it can bind with DNA, proteins, and other macromolecules, disrupting cellular processes and causing serious harm to health. Therefore, it becomes critical to develop simple, highly selective and precise methods for detecting Pd2+in vivo. Here, we have successfully developed the first activated second near-infrared region fluorescence (NIR-II FL) and ratio photoacoustic (PA) probe NYR-1 for dual-modal accurate detection of Pd2+ levels. NYR-1 is capable of rapidly (< 60 s) and sensitively detection of Pd2+ in solution, providing switched on NIR-II FL920 and ratio PA808/PA720 dual-mode signal change. More notably, the probe NYR-1 was successfully used for non-invasive imaging of Pd2+ overload in mouse liver by NIR-II FL/Ratio PA dual-modality imaging technology for the first time. Thus, this work opens up a promising dual-modal detection method for the precise detection of Pd2+ in organisms and in the environment.

Influence mechanism of the temporal duration of laser irradiation on photoacoustic technique: a review.

Significance: In the photoacoustic (PA) technique, the laser irradiation in the time domain (i.e., laser pulse duration) governs the characteristics of PA imaging-it plays a crucial role in the optical-acoustic interaction, the generation of PA signals, and the PA imaging performance. Aim: We aim to provide a comprehensive analysis of the impact of laser pulse duration on various aspects of PA imaging, encompassing the signal-to-noise ratio, the spatial resolution of PA imaging, the acoustic frequency spectrum of the acoustic wave, the initiation of specific physical phenomena, and the photothermal-PA (PT-PA) interaction/conversion. Approach: By surveying and reviewing the state-of-the-art investigations, we discuss the effects of laser pulse duration on the generation of PA signals in the context of biomedical PA imaging with respect to the aforementioned aspects. Results: First, we discuss the impact of laser pulse duration on the PA signal amplitude and its correlation with the lateral resolution of PA imaging. Subsequently, the relationship between the axial resolution of PA imaging and the laser pulse duration is analyzed with consideration of the acoustic frequency spectrum. Furthermore, we examine the manipulation of the pulse duration to trigger physical phenomena and its relevant applications. In addition, we elaborate on the tuning of the pulse duration to manipulate the conversion process and ratio from the PT to PA effect. Conclusions: We contribute to the understanding of the physical mechanisms governing pulse-width-dependent PA techniques. By gaining insight into the mechanism behind the influence of the laser pulse, we can trigger the pulse-with-dependent physical phenomena for specific PA applications, enhance PA imaging performance in biomedical imaging scenarios, and modulate PT-PA conversion by tuning the pulse duration precisely.

Phase-locked µPIV analysis of flow dynamics in a simulated root canal with different laser-activated irrigations.

PURPOSE: To measure the dynamic characteristics of the flow field in a complex root canal model activated by two laser-activated irrigation (LAI) modalities at different activation energy outputs: photon-induced photoacoustic streaming (PIPS) and microshort pulse (MSP). METHODS: A phase-locked micro-scale Particle Image Velocimetry (µPIV) system was employed to characterise the temporal variations of LAI-induced velocity fields in the root canal following a single laser pulse. The wall shear stress (WSS) in the lateral root canal was subsequently estimated from the phase-averaged velocity fields. RESULTS: Both PIPS and MSP were able to generate the 'breath mode' of the irrigant current under all tested conditions. The transient irrigation flush in the root canal peaked at speeds close to 6 m/s. However, this intense flushing effect persisted for only about 2000 µs (or 3% of a single laser-pulse activation cycle). For MSP, the maximum WSS magnitude was approximately 3.08 Pa at an activation energy of E = 20 mJ/pulse, rising to 9.01 Pa at E = 50 mJ/pulse. In comparison, PIPS elevated the WSS to 10.63 Pa at E = 20 mJ/pulse. CONCLUSION: Elevating the activation energy can boost the peak flushing velocity and the maximum WSS, thereby enhancing irrigation efficiency. Given the same activation energy, PIPS outperforms MSP. Additionally, increasing the activation frequency may be an effective strategy to improve irrigation performance further.

Hyaluronic acid functionalized iron-platinum nanoparticles for photothermal therapy and photoacoustic imaging.

This study represents an innovative approach to construct multi-functional nanoplatforms for cancer diagnosis and therapy by combining hyaluronic acid (HA) with iron-platinum nanoparticles (FePt NPs). These HA-coated FePt NPs, referred to as FePt@HA NPs, demonstrated remarkable biocompatibility, high absorption, and excellent light-to-heat conversion properties in the near-infrared (NIR) region, making them ideal candidates for photothermal therapy (PTT). In vitro studies revealed their effective cancer cell eradication under NIR laser irradiation, while in vivo experiments on mice showcased their superior heating capabilities. Moreover, FePt@HA NPs exhibited a distinct and strong photoacoustic (PA) signal, facilitating enhanced and precise intra-tumoral PA imaging. Our results highlight the potential of FePt@HA NPs as promising photothermal agents for future PTT applications. They offer high selectivity, precision, and minimal side effects in cancer treatment, along with their valuable PA imaging application for tumor localization and characterization.

NIR Fluorescent/Photoacoustic Bimodal Imaging of Ferroptosis in Pancreatic Cancer Using Biothiols-Activable Probes.

Ferroptosis modulation is a powerful therapeutic option for pancreatic ductal adenocarcinoma (PDAC) with a low 5-year survival rate and lack of effective treatment methods. However, due to the dual role of ferroptosis in promoting and inhibiting pancreatic tumorigenesis, regulating the degree of ferroptosis is very important to obtain the best therapeutic effect of PDAC. Biothiols are suitable as biomarkers of imaging ferroptosis due to the dramatic decreases of biothiol levels in ferroptosis caused by the inhibited synthesis pathway of glutathione (GSH) and the depletion of biothiol by reactive oxygen species. Moreover, a very recent study reported that cysteine (Cys) depletion can lead to pancreatic tumor ferroptosis in mice and may be employed as an effective therapeutic strategy for PDAC. Therefore, visualization of biothiols in ferroptosis of PDAC will be helpful for regulating the degree of ferroptosis, understanding the mechanism of Cys depletion-induced pancreatic tumor ferroptosis, and further promoting the study and treatment of PDAC. Herein, two biothiol-activable near-infrared (NIR) fluorescent/photoacoustic bimodal imaging probes (HYD-BX and HYD-DX) for imaging of pancreatic tumor ferroptosis were reported. These two probes show excellent bimodal response performances for biothiols in solution, cells, and tumors. Subsequently, they have been employed successfully for real-time visualization of changes in concentration levels of biothiols during the ferroptosis process in PDAC cells and HepG2 cells. Most importantly, they have been further applied for bimodal imaging of ferroptosis in pancreatic cancer in mice, with satisfactory results. The development of these two probes provides new tools for monitoring changes in concentration levels of biothiols in ferroptosis and will have a positive impact on understanding the mechanism of Cys depletion-induced pancreatic tumor ferroptosis and further promoting the study and treatment of PDAC.

Speckle Variance Photoacoustic Microscopy for Microhemodynamic Imaging.

Relying on the strong optical absorption of hemoglobin to pulsed laser energy, photoacoustic microscopy provides morphological and functional information on microvasculature label-freely. Here, we propose speckle variance photoacoustic microscopy (SV-PAM), which harnesses intrinsic imaging contrast from temporal-varied photoacoustic signals of moving red blood cells in blood vessels, for recovering three-dimension hemodynamic images down to capillary-level resolution within the microcirculatory tissue beds in vivo. Calculating the speckle variance of consecutive photoacoustic B-scan frames acquired at the same lateral position enables accurate identification of blood perfusion and occlusion, which provides interpretations of dynamic blood flow in the microvasculature, in addition to the microvascular anatomic structures. We demonstrate high-resolution hemodynamic imaging of vascular occlusion and reperfusion in the microvasculature of mice ears in vivo. The results suggest that our SV-PAM is potentially invaluable for biomedical hemodynamic investigations, for example, imaging ischemic stroke and hemorrhagic stroke.

An AND-Gate Photoacoustic Probe for Cys and H2S Precise Photoacoustic Sensing in Localized Tumors.

Photoacoustic (PA) tomography has shown many promising aspects in noninvasive and precise imaging of deep-localized biomarkers. However, these traditional single-locked PA probes always face challenges in precise PA imaging with high specificity. Here, we report a novel AND-gate photoacoustic probe, BAE, to improve tumor imaging accuracy via the combination of two tumor-associated biomarkers, cysteine (Cys) and hydrogen sulfide (H2S). Only when Cys and H2S are concurrently introduced into the detection system does the absorption of BAE red-shift from the initial 680 to 810 nm, thereby showing a 5.29-fold enhancement in its PA signal at 810 nm. The good specificity of BAE is proven, since an obvious PA signal could be observed only in the solution containing both Cys and H2S and was not affected by other reactive sulfur species. After being taken up by tumors with the assistance of a nanomicelle, the AND-gate PA probe BAE was applied for dynamic real-time monitoring of Cys and H2S in vivo, achieving precise identification of tumors. This AND-gate PA probe provides a potential technical tool for precise sensing analysis of deep-seated diseases.

Design, implementation, and analysis of a compressed sensing photoacoustic projection imaging system.

Significance: Compressed sensing (CS) uses special measurement designs combined with powerful mathematical algorithms to reduce the amount of data to be collected while maintaining image quality. This is relevant to almost any imaging modality, and in this paper we focus on CS in photoacoustic projection imaging (PAPI) with integrating line detectors (ILDs). Aim: Our previous research involved rather general CS measurements, where each ILD can contribute to any measurement. In the real world, however, the design of CS measurements is subject to practical constraints. In this research, we aim at a CS-PAPI system where each measurement involves only a subset of ILDs, and which can be implemented in a cost-effective manner. Approach: We extend the existing PAPI with a self-developed CS unit. The system provides structured CS matrices for which the existing recovery theory cannot be applied directly. A random search strategy is applied to select the CS measurement matrix within this class for which we obtain exact sparse recovery. Results: We implement a CS PAPI system for a compression factor of 4:3, where specific measurements are made on separate groups of 16 ILDs. We algorithmically design optimal CS measurements that have proven sparse CS capabilities. Numerical experiments are used to support our results. Conclusions: CS with proven sparse recovery capabilities can be integrated into PAPI, and numerical results support this setup. Future work will focus on applying it to experimental data and utilizing data-driven approaches to enhance the compression factor and generalize the signal class.

A Nanographene-Porphyrin Hybrid for Near-Infrared-Ii Phototheranostics.

Photoacoustic imaging (PAI)-guided photothermal therapy (PTT) in the second near-infrared (NIR-II, 1000-1700 nm) window has been attracting attention as a promising cancer theranostic platform. Here, it is reported that the π-extended porphyrins fused with one or two nanographene units (NGP-1 and NGP-2) can serve as a new class of NIR-responsive organic agents, displaying absorption extending to ≈1000 and ≈1400 nm in the NIR-I and NIR-II windows, respectively. NGP-1 and NGP-2 are dispersed in water through encapsulation into self-assembled nanoparticles (NPs), achieving high photothermal conversion efficiency of 60% and 69%, respectively, under 808 and 1064 nm laser irradiation. Moreover, the NIR-II-active NGP-2-NPs demonstrated promising photoacoustic responses, along with high photostability and biocompatibility, enabling PAI and efficient NIR-II PTT of cancer in vivo.

Multispectral Optoacoustic Tomography Enables In Vivo Anatomical and Functional Assessment of Human Tendons.

Tendon injuries resulting from accidents and aging are increasing globally. However, key tendon functional parameters such as microvascularity and oxygen perfusion remain inaccessible via the currently available clinical diagnostic tools, resulting in disagreements on optimal treatment options. Here, a new noninvasive method for anatomical and functional characterization of human tendons based on multispectral optoacoustic tomography (MSOT) is reported. Healthy subjects are investigated using a hand-held scanner delivering real-time volumetric images. Tendons in the wrist, ankle, and lower leg are imaged in the near-infrared optical spectrum to utilize endogenous contrast from Type I collagen. Morphology of the flexor carpi ulnaris, carpi radialis, palmaris longus, and Achilles tendons are reconstructed in full. The functional roles of the flexor digitorium longus, hallicus longus, and the tibialis posterior tendons have been visualized by dynamic tracking during toe extension-flexion motion. Furthermore, major vessels and microvasculature near the Achilles tendon are localized, and the global increase in oxygen saturation in response to targeted exercise is confirmed by perfusion studies. MSOT is shown to be a versatile tool capable of anatomical and functional tendon assessments. Future studies including abnormal subjects can validate the method as a viable noninvasive clinical tool for tendinopathy management and healing monitoring.