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Although photothermal therapy (PTT) has demonstrated its clinical value and adaptability, it still requires imaging guidance to motivate the development of precise and effective treatment. For PTT, high-resolution visualization of tumor microvasculature and accurate location of nanoparticles distribution are crucial for the therapeutic outcome. Here, a wavelength-switchable photoacoustic microscopy (PAM) was developed to noninvasively investigate the tumor microvasculature and the accumulation of nanoparticles for accurately guiding PTT and evaluating the therapeutic effect. In a tumor model, PAM was used to continuously monitor the tumor microenvironment in vivo, and the proportion of microvessels in tumor site was found increased by 10%, and the diameters of the draining veins were doubled on day 7. In addition, quantitative parameters such as tumor volume and vascular density can also be demonstrated by the PAM. Meanwhile, the concentration of Den-RGD/Cy7 at the tumor site reached its maximum at 8 h by PA mapping after intravenous injection, which was used to determine the optimal irradiation timing. After treatment, photoacoustic monitoring showed that PTT can precisely kill the tumors and minimize damage to surrounding normal tissues, which was consistent with the pathological slides. The experimental results proved that PAM can provide an auxiliary means for precision PTT.

Photoacoustic microscopy is an in vivo imaging technology based on the photoacoustic effect. It is widely used in various biomedical studies because it can provide high-resolution images while being label-free, safe, and harmless to biological tissue. Polygon-scanning is an effective scanning method in photoacoustic microscopy that can realize fast imaging of biological tissue with a large field of view. However, in polygon-scanning, fluctuations of the rotating motor speed and the geometric error of the rotating mirror cause image distortions, which seriously affect the photoacoustic-microscopy imaging quality. To improve the image quality of photoacoustic microscopy using polygon-scanning, an image correction method is proposed based on accurate ultrasound positioning. In this method, the photoacoustic and ultrasound imaging data of the sample are simultaneously obtained, and the angle information of each mirror used in the polygon-scanning is extracted from the ultrasonic data to correct the photoacoustic images. Experimental results show that the proposed method can significantly reduce image distortions in photoacoustic microscopy, with the image dislocation offset decreasing from 24.774 to 10.365 μm.

As a hybrid optical microscopic imaging technology, photoacoustic microscopy images the optical absorption contrasts and takes advantage of low acoustic scattering of biological tissues to achieve high-resolution anatomical and functional imaging. When combined with other imaging modalities, photoacoustic microscopy-based multimodal technologies can provide complementary contrast mechanisms to reveal complementary information of biological tissues. To achieve intrinsically and precisely registered images in a multimodal photoacoustic microscopy imaging system, either the ultrasonic transducer or the light source can be shared among the different imaging modalities. These technologies are the major focus of this minireview. It also covered the progress of the recently developed penta-modal photoacoustic microscopy imaging system featuring a novel dynamic focusing technique enabled by OCT contour scan.

We have developed a multimodal imaging system, which integrated optical resolution photoacoustic microscopy, optical coherence tomography, optical coherence tomography angiography, and confocal fluorescence microscopy in one platform. The system is able to image complementary features of a biological sample by combining different contrast mechanisms. We achieved fast imaging and large field of view by combining optical scanning with mechanical scanning, similar to our previous publication. We have demonstrated the capability of the multimodal imaging system by imaging a mouse ear in vivo. Impact statement Photoacoustic microscopy-based multimodal imaging technology can provide high-resolution complementary information for biological tissues in vivo. It will potentially bring significant impact on the research and diagnosis of diseases by providing combined structural and functional information.

International audience; In the paper, combined bi-modal microscopic visualization of biological objects based on reflection-mode optical and photoacoustic measurements was presented. Gas-microphone configuration was chosen for the registration of photothermal response. Precise positioning of the scanning laser beam with modulated intensity was performed employing acousto-optic deflectors. Photoacoustic images are shown to give complementary details to the optical images obtained in the reflected light. Specifically, the photoacoustic imaging mode allows much better visualization of the features with enhanced heat localization due to the reduced heat outflow. For example, the application of the photoacoustic imaging mode was especially successful to visualize Drosophila fly's micro-hairs. Furthermore, the photoacoustic image quality was shown to be adjusted through modulation frequency.

Photoacoustic imaging (PAI) is a hybrid non-invasive imaging technique used to merge high optical contrast and high acoustic resolution in deep tissue. PAI has been extensively developed by utilizing its advantages that include deep imaging depth, high resolution, and label-free imaging. As a representative implementation of PAI, photoacoustic microscopy (PAM) has been used in preclinical and clinical studies for its micron-scale spatial resolution capability with high optical absorption contrast. Several handheld and portable PAM systems have been developed that improve its applicability to several fields, making it versatile. In this study, we developed a laboratory-customized, two-axis, waterproof, galvanometer scanner-based handheld PAM (WP-GVS-HH-PAM), which provides an extended field of view (14.5 × 9 mm2) for wide-range imaging. The fully waterproof handheld probe enables free movement for imaging regardless of sample shape, and volume rate and scanning region are adjustable per experimental conditions. Results of WP-GVS-HH-PAM-based phantom and in vivo imaging of mouse tissues (ear, iris, and brain) confirm the feasibility and applicability of our system as an imaging modality for various biomedical applications.

A dual modality microscopy with the highest imaging resolution reported so far based on reflection-mode photoacoustic and confocal fluorescence is presented in this study. The unique design of the imaging head of the microscope makes it highly convenient for scalable high-resolution imaging by simply switching the optical objectives. The submicron resolution performance of the system is demonstrated via in vivo imaging of zebrafish, normal mouse ear, and a xenograft tumor model inoculated in the mouse ear. The imaging results confirm that the presented dual-modality microscopy imaging system could play a vital role in observing model organism, studying tumor angiogenesis and assessment of antineoplastic drugs. MSC: 00-01, 99-00, Keywords: Photoacoustic microscopy, Confocal microscopy, Multimodal imaging

Pathological aggregation of Aβ peptides results in the deposition of amyloid in the brain parenchyma (senile plaques in Alzheimer’s disease [AD]) and around cerebral microvessels (cerebral amyloid angiopathy [CAA]). Our current understanding of the amyloid-induced microvascular changes has been limited to the structure and hemodynamics—leaving the oxygen-metabolic aspect unattended. In this Letter, we report a dual-contrast photoacoustic microscopy (PAM) technique, which integrates the molecular contrast of dichroism PAM and the physiological contrast of multi-parametric PAM for simultaneous, intravital imaging of amyloid deposition and cerebrovascular function in a mouse model that develops AD and CAA. This technique opens up new opportunities to study the spatiotemporal interplay between amyloid deposition and vascular-metabolic dysfunction in AD and CAA.

Photoacoustic microscopy (PAM) is an emerging imaging method combining light and sound. However, limited by the laser's repetition rate, state-of-the-art high-speed PAM technology often sacrifices spatial sampling density (i.e., undersampling) for increased imaging speed over a large field-of-view. Deep learning (DL) methods have recently been used to improve sparsely sampled PAM images; however, these methods often require time-consuming pre-training and large training dataset with ground truth. Here, we propose the use of deep image prior (DIP) to improve the image quality of undersampled PAM images. Unlike other DL approaches, DIP requires neither pre-training nor fully-sampled ground truth, enabling its flexible and fast implementation on various imaging targets. Our results have demonstrated substantial improvement in PAM images with as few as 1.4$\%$ of the fully sampled pixels on high-speed PAM. Our approach outperforms interpolation, is competitive with pre-trained supervised DL method, and is readily translated to other high-speed, undersampling imaging modalities.

Photoacoustic (PA) microscopy is being increasingly used to visualize the microcirculation of the brain cortex at the micron level in living rodents. By combining it with long-term cranial window techniques, vasculature can be monitored over a period of days extending to months through a field of view. To fulfill the requirements of long-term in vivo PA imaging, the cranial window must involve a simple and rapid surgical procedure, biological compatibility, and sufficient optical-acoustic transparency, which are major challenges. Recently, several cranial window techniques have been reported for longitudinal PA imaging. Here, the development of chronic cranial windows for PA imaging is reviewed and its technical details are discussed, including window installation, imaging quality, and longitudinal stability.

Fiber laser technology has experienced a rapid growth over the past decade owing to increased applications in precision measurement and optical testing, medical care, and industrial applications, including laser welding, cleaning, and manufacturing. A fiber laser can output laser pulses with high energy, a high repetition rate, a controllable wavelength, low noise, and good beam quality, making it applicable in photoacoustic imaging. Herein, recent developments in fiber-laser-based photoacoustic microscopy (PAM) are reviewed. Multispectral PAM can be used to image oxygen saturation or lipid-rich biological tissues by applying a Q-switched fiber laser, a stimulated Raman scattering-based laser source, or a fiber-based supercontinuum source for photoacoustic excitation. PAM can also incorporate a single-mode fiber laser cavity as a high-sensitivity ultrasound sensor by measuring the acoustically induced lasing-frequency shift. Because of their small size and high flexibility, compact head-mounted, wearable, or hand-held imaging modalities and better photoacoustic endoscopes can be enabled using fiber-laser-based PAM.

Optical-resolution photoacoustic microscopy (OR-PAM) is a promising noninvasive biomedical imaging technology with label-free optical absorption contrasts. Performance of OR-PAM is usually closely related to the optical-acoustic combiner. In this study, we propose an optical-acoustic combiner based on a flat acoustic reflector and an off-axis parabolic acoustic mirror with a conical bore. Quantitative simulation and experiments demonstrated that this combiner can provide better acoustic focusing performance and detection sensitivity. Moreover, OR-PAM is based on the combiner suffer low optical disorders, which guarantees the good resolution. In vivo experiments of the mouse brain and the iris were also conducted to show the practicability of the combiner in biomedicine. This proposed optical-acoustic combiner realizes a high-quality optical-acoustic confocal alignment with minimal optical disorders and acoustic insertion loss, strong acoustic focusing, and easy implementation. These characteristics might be useful for improving the performance of OR-PAM.

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 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 were 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 were 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 blood pressure, vessel size, and blood oxygenation. This article is protected by copyright. All rights reserved.

Photoacoustic (PA) imaging has been widely used in biomedical research and preclinical studies during the past two decades. It has also been explored for nondestructive testing and evaluation (NDT/E) and for industrial applications. This paper describes the basic principles of PA technology for NDT/E and its applications in recent years. PA technology for NDT/E includes the use of a modulated continuous-wave laser and a pulsed laser for PA wave excitation, PA-generated ultrasonic waves, and all-optical PA wave excitation and detection. PA technology for NDT/E has demonstrated broad applications, including the imaging of railway cracks and defects, the imaging of Li metal batteries, the measurements of the porosity and Young’s modulus, the detection of defects and damage in silicon wafers, and a visualization of underdrawings in paintings.

Vascular damage is one of the therapeutic mechanisms of photodynamic therapy (PDT). In particular, short-term PDT treatments can effectively destroy malignant lesions while minimizing damage to nonmalignant tissue. In this study, we investigate the feasibility of label-free quantitative photoacoustic microscopy (PAM) for monitoring the vasculature changes under the effect of PDT in mouse ear melanoma tumors. In particular, quantitative vasculature evaluation was conducted based on Hessian filter segmentation. Three-dimensional morphological PAM and depth-resolved images before and after PDT treatment were acquired. In addition, five quantitative vasculature parameters, including the PA signal, vessel diameter, vessel density, perfused vessel density, and vessel complexity, were analyzed to evaluate the influence of PDT on four different areas: Two melanoma tumors, and control and normal vessel areas. The quantitative and qualitative results successfully demonstrated the potential of the proposed PAM-based quantitative approach to evaluate the effectiveness of the PDT method.

This study demonstrates that chlorophosphonazo III (CPZ III) can be used as a contrast agent for photoacoustic calcium imaging. CPZ III can pass across the plasma membrane for labeling intracellular Ca2+ without cytotoxicity. In optical-resolution photoacoustic microscopy (OR-PAM), the photoacoustic (PA) signal intensity was strongly correlated with the presence of CPZ III and Ca2+ at various concentrations. The sensitivity of PA signal reception was enhanced by using an 8 MHz single-element focused ultrasound detector due to their matched frequency characteristics. Differences in the PA signal intensity were successfully found between the core and margin areas of tumorspheres in three-dimensional cell cultures. These findings indicate that CPZ III can serve as a novel PA contrast agent for functional Ca2+ imaging using OR-PAM.

Optical imaging technologies have enabled outstanding analysis of biomedical tissues through providing detailed functional and morphological contrast. Leveraging the valuable information provided by these modalities can help us build an understanding of tissues’ characteristics. Among various optical imaging technologies, photoacoustic imaging (PAI) and optical coherence tomography (OCT) naturally complement each other in terms of contrast mechanism, penetration depth, and spatial resolution. The rich and unique molecular-specified absorption contrast offered by PAI would be well complemented by detailed scattering information of OCT. Together these two powerful imaging modalities can extract important characteristic of tissue such as depth-dependent scattering profile, volumetric structural information, chromophore concentration, flow velocity, polarization properties, and temperature distribution map. As a result, multimodal PAI-OCT imaging could impact a broad range of clinical and preclinical imaging applications including but not limited to oncology, neurology, dermatology, and ophthalmology. This review provides an overview of the technical specs of existing dual-modal PAI-OCT imaging systems, their applications, limitations, and future directions.

Laser diodes (LDs) have been considered as cost-effective and compact excitation sources to overcome the requirement of costly and bulky pulsed laser sources that are commonly used in photoacoustic microscopy (PAM). However, the spatial resolution and/or imaging speed of previously reported LD-based PAM systems have not been optimized simultaneously. In this paper, we developed a high-speed and high-resolution LD-based PAM system using a continuous wave LD, operating at a pulsed mode, with a repetition rate of 30 kHz, as an excitation source. A hybrid scanning mechanism that synchronizes a one-dimensional galvanometer mirror and a two-dimensional motorized stage is applied to achieve a fast imaging capability without signal averaging due to the high signal-to-noise ratio. By optimizing the optical system, a high lateral resolution of 4.8 μm has been achieved. In vivo microvasculature imaging of a mouse ear has been demonstrated to show the high performance of our LD-based PAM system.

We report wide-field polygon-scanning functional OR-PAM that for the first time achieves 1-MHz A-line rate of oxygen saturation in vivo. We address two technical challenges. The first is a 1-MHz dual-wavelength pulsed laser that has sufficient pulse energy and ultrafast wavelength switching. The second is a polygon-scanning imaging probe that has a fast scanning speed, a large field of view, and great sensitivity. The OR-PAM system offers a B-scan rate of 477.5 Hz in a 12-mm range and a volumetric imaging rate of ∼1 Hz over a 12 × 5 mm2 scanning area. We image microvasculature and blood oxygen saturation in a 12 × 12 mm2 scanning area in 5 s. Dynamic imaging of oxygen saturation in the mouse ear is demonstrated to monitor fast response to epinephrine injection. The new wide-field fast functional imaging ability broadens the biomedical application of OR-PAM.

Photoacoustic imaging (PAI) has achieved remarkable growth in the past few decades since it takes advantage of both optical and ultrasound (US) imaging. In order to better promote the wide clinical applications of PAI, many miniaturized and portable PAI systems have recently been proposed. Most of these systems utilize fiber optic technologies. Here, we overview the fiber optic technologies used in PAI. This paper discusses three different fiber optic technologies: fiber optic light transmission, fiber optic US transmission, and fiber optic US detection. These fiber optic technologies are analyzed in different PAI modalities including photoacoustic microscopy (PAM), photoacoustic computed tomography (PACT), and minimally invasive photoacoustic imaging (MIPAI).

Label-free chemical bond imaging is of great importance in biology and medicine. Photoacoustic imaging at the third near-infrared windows (1600-1870 nm, nearinfrared-III) provides a stable molecular vibrational imaging tool for lipid-rich tissue owing to the first overtone transition of the C-H bond at 1.7 _m. However, lacking high-energy pulsed laser sources at 1.7 _m and the strong water absorption significantly limit the signal-to-noise ratio of the lipid imaging, especially for thin lipid tissues. To circumvent this barrier, we develop near-infrared-III double-illumination optical-resolution photoacoustic microscopy (DIOR-PAM) for improving the sensitivity of label-free lipid imaging. Using the same laser, DIOR-PAM can enhance the sensitivity by nearly 100%, which we prove in the Monte Carlo simulation.We experimentally demonstrated 50%i100% sensitivity enhancement on non-biological and biological lipid-rich samples. This article is protected by copyright. All rights reserved.

Although photoacoustic microscopy (PAM) and optical coherence tomography (OCT) allow visualization of the retinal microvasculature, distinguishing early neovascularization from adjacent vessels remains challenging. Herein, gold nanostars (GNSs) functionalized with an RGD peptide were utilized as multimodality contrast agents for both PAM and OCT. GNSs have great absorption and scattering characteristics in the near-infrared region where most vasculature and tissue generates a less intrinsic photoacoustic signal while having a small size, excellent biocompatibility in vivo, and great photostability under nanosecond pulsed laser illumination. This enabled visualization and differentiation of individual microvasculature in vivo using multimodal PAM and OCT imaging. Detailed three-dimensional imaging of GNSs was achieved in an important choroidal neovascularization model in living rabbits. Through the administration of GNSs, PA contrast increased up to 17-fold and OCT intensities increased 167%. This advanced molecular-imaging platform with GNSs provides a unique tool for detailed mapping of the pathogenesis of the microvasculature.

Correct guiding of the catheter is a critical issue in almost all balloon catheter applications, including arterial stenosis expansion, coronary arterial diseases, and gastrointestinal tracking. To achieve safe and precise guiding of the balloon catheter, a novel imaging method with high-resolution, sufficient depth of penetration, and real-time display is required. Here, we present a new balloon catheter guiding method using fast photoacoustic microscopy (PAM) technique for precise balloon catheter tracking and visualization as a feasibility study. We implemented ex vivo and in vivo experiments with three different medium conditions of balloon catheter: no air, air, and water. Acquired cross-sectional, maximum amplitude projection (MAP), and volumetric 3D PAM images demonstrated its capability as a new imaging guiding tool for balloon catheter tracking and visualization.

Photoacoustic imaging (PAI) is drawing extensive attention and gaining rapid development as an emerging biomedical imaging technology because of its high spatial resolution, large imaging depth, and rich optical contrast. PAI has great potential applications in endoscopy, but the progress of endoscopic PAI was hindered by the challenges of manufacturing and assembling miniature imaging components. Over the last decade, microelectromechanical systems (MEMS) technology has greatly facilitated the development of photoacoustic endoscopes and extended the realm of applicability of the PAI. As the key component of photoacoustic endoscopes, micromachined ultrasound transducers (MUTs), including piezoelectric MUTs (pMUTs) and capacitive MUTs (cMUTs), have been developed and explored for endoscopic PAI applications. In this article, the recent progress of pMUTs (thickness extension mode and flexural vibration mode) and cMUTs are reviewed and discussed with their applications in endoscopic PAI. Current PAI endoscopes based on pMUTs and cMUTs are also introduced and compared. Finally, the remaining challenges and future directions of MEMS ultrasound transducers for endoscopic PAI applications are given.

Optical resolution photoacoustic microscopy (OR-PAM) can achieve an optical-dependent high lateral resolution (

Photoacoustic microscopy (PAM) provides high resolution and contrast imaging of microstructures such as blood vessels and various optical contrast agents. Acoustic-resolution PAM (AR-PAM) is capable of reaching sub-centimeter of penetration depth and is optimized for tissue samples and small animals. However, the state-of-art AR-PAMs are large in size and expensive in cost, which hinders their democratization. While previous studies have focused on replacing laser sources or data acquisition devices, bulky and costly high precision linear translation stages are used to actuate the sensor to build a 3D PAM image. Here, we focused on substituting these linear translation stages with delta configuration actuators adapted from a low-cost commercially available off-the-shelf 3D printer to miniaturize the size of the system and to drastically reduce the cost. We introduce an economical PAM system that integrates the unique sensor actuation hardware and the software approach of obtaining the high-resolution image through 3D reconstruction. The preliminary phantom study demonstrated the applicability of low-cost delta configuration actuators for AR-PAM imaging.

Photoacoustic imaging (PAI) takes advantage of both optical and ultrasound imaging properties to visualize optical absorption with high resolution and contrast. Photoacoustic microscopy (PAM) is usually categorized with all-optical microscopy techniques such as optical coherence tomography or confocal microscopes. Despite offering high sensitivity, novel imaging contrast, and high resolution, PAM is not generally an all-optical imaging method unlike the other microscopy techniques. One of the significant limitations of photoacoustic microscopes arises from their need to be in physical contact with the sample through a coupling media. This physical contact, coupling, or immersion of the sample is undesirable or impractical for many clinical and pre-clinical applications. This also limits the flexibility of photoacoustic techniques to be integrated with other all-optical imaging microscopes for providing complementary imaging contrast. To overcome these limitations, several non-contact photoacoustic signal detection approaches have been proposed. This paper presents a brief overview of current non-contact photoacoustic detection techniques with an emphasis on all-optical detection methods and their associated physical mechanisms.

Multispectral photoacoustic microscopy uses a wavelength-dependent absorption difference as a contrast mechanism to image the target molecule. In this paper, we present a novel multispectral pulsed fiber laser source, which selectively alternates the excitation wavelengths between green and red colors based on the stimulated Raman scattering (SRS) effect for imaging. This laser has a high pulse repetition rate (PRR) of 300 kHz and high pulse energy of more than 200 nJ meeting the real-time requirements of optical-resolution photoacoustic microscopy imaging. By switching the polarization state of the pump light and optical paths of the pump light, the operating wavelengths of the light source can be selectively alternated at the same fast PRR for any two SRS peak wavelengths between 545 and 655 nm. At 545 nm excitation wavelength, molecular photoacoustic signals from both blood vessels and gold nanorods were obtained simultaneously. However, at 655 nm, the photoacoustic signals of gold nanorods were dominant because the absorption of light by the blood vessels decreased drastically in the spectral region over 600 nm. Thus the multispectral photoacoustic system designed using the novel laser source implemented here could simultaneously monitor the time-dependent fast movement of two molecules independently, having different wavelength-dependent absorption properties at a high repetition rate of 0.49 frames per second (fps).

Faster and better wound healing is a critical medical issue. Because the repair process of wounds is closely related to revascularization, accurate early assessment and postoperative monitoring are very important for establishing an optimal treatment plan. Herein, we present an extended depth-of-field photoacoustic microscopy system (E-DOF-PAM) that can achieve a constant spatial resolution and relatively uniform excitation efficiency over a long axial range. The superior performance of the system was verified by phantom and in vivo experiments. Furthermore, the system was applied to the imaging of normal and trauma sites of volunteers, and the experimental results accurately revealed the morphological differences between the normal and traumatized skin of the epidermis and dermis. These results demonstrated that the E-DOF-PAM is a powerful tool for observing and understanding the pathophysiology of cutaneous wound healing. [Image: see text]

Optical-resolution photoacoustic microscopy (OR-PAM) has been shown to be an excellent tool for high-resolution imaging of microvasculature, and quantitative analysis of the microvasculature can provide valuable information for the early diagnosis and treatment of various vascular-related diseases. In order to address the characteristics of weak signals, discontinuity and small diameters in photoacoustic microvascular images, we propose a method adaptive to the microvascular segmentation in photoacoustic images, including Hessian matrix enhancement and the morphological connection operators. The accuracy of our vascular segmentation method is quantitatively evaluated by the multiple criteria. To obtain more precise and continuous microvascular skeletons, an improved skeleton extraction framework based on the multistencil fast marching (MSFM) method is developed. We carried out in vivo OR-PAM microvascular imaging in mouse ears and subcutaneous hepatoma tumor model to verify the correctness and superiority of our proposed method. Compared with the previous methods, our proposed method can extract the microvascular network more completely, continuously and accurately, and provide an effective solution for the quantitative analysis of photoacoustic microvascular images with many small branches.

Significance: The development of ultralow energy photoacoustic microscopy (PAM) on the clinically relevant pigmented rabbit eye model paves a road toward translation of the emerging PAM technology in ophthalmology clinics. Aim: Since the eye is particularly vulnerable to laser damage, we aim to develop an ultralow energy PAM system to significantly improve the laser safety of PAM by increasing the sensitivity of the system and reducing the incident laser energy for imaging. Approach: A multichannel data acquisition circuit with two-stage signal amplification was specially designed, which, in combination with the application of 3 by 3 median filter in the spatial domain, significantly improved the signal-to-noise ratio of the PAM system. The safety of this system was validated by histopathology, fluorescein angiography, and fundus photography. Results: Experiments performed on pigmented rabbits demonstrated that, when using this ultralow energy PAM system, satisfactory image quality can be achieved in the eye with an incident laser fluence that is only 1% of the American National Standards Institute safety limit. Fundus photography, fluorescein angiography, and histopathology were performed after the imaging procedure, and no retinal or ocular damage was observed. Conclusions: The proposed ultralow energy PAM system has excellent safety and holds potential to be developed into a clinical tool for ocular 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.

Capable of imaging blood perfusion, oxygenation, and flow simultaneously at the microscopic level, multi-parametric photoacoustic microscopy (PAM) has quickly emerged as a powerful tool for studying hemodynamic and metabolic changes due to physiological stimulations or pathological processes. However, the low scanning speed poised by the correlation-based blood flow measurement impedes its application in studying rapid microvascular responses. To address this challenge, we have developed a new, to the best of our knowledge, multi-parametric PAM system. By extending the optical scanning range with a cylindrically focused ultrasonic transducer (focal zone, 76 µ m × 4.5 m m ) for simultaneous acquisition of 500 B-scans, the new system is 112 times faster than our previous multi-parametric system that uses a spherically focused transducer (focal diameter, 40 µm) and enables high-resolution imaging of blood perfusion, oxygenation, and flow over an area of 4.5 × 1 m m 2 at a frame rate of 1 Hz. We have demonstrated the feasibility of this system in the living mouse ear. Further development of this system into reflection mode will enable real-time cortex-wide imaging of hemodynamics and metabolism in the mouse brain.

In this study, a photoacoustic microscopy (PAM) system based on a multifocal point (MFP) transducer was fabricated to produce a large depth-of-field tissue image. The customized MFP transducer has seven focal points, distributed along with the transducer&rsquo, s axis, fabricated by separate spherically-focused surfaces. These surfaces generate distinct focal zones that are overlapped to extend the depth-of-field. This design allows extending the focal zone of 10 mm for the 11 MHz MFP transducer, which is a great improvement over the 0.48 mm focal zone of the 11 MHz single focal point (SFP) transducer. The PAM image penetration depths of a chicken-hemoglobin phantom using SFP and MFP transducers were measured as 5 mm and 8 mm, respectively. The significant increase in the PAM image-based penetration depth of the chicken-hemoglobin phantom was a result of using the customized MFP transducer.

We present a dual modality functional optical coherence tomography and photoacoustic microscopy (OCT-PAM) system. The photoacoustic modality employs an akinetic optical sensor with a large imaging window. This imaging window enables direct reflection mode operation, and a seamless integration of optical coherence tomography (OCT) as a second imaging modality. Functional extensions to the OCT-PAM system include Doppler OCT (DOCT) and spectroscopic PAM (sPAM). This functional and non-invasive imaging system is applied to image zebrafish larvae, demonstrating its capability to extract both morphological and hemodynamic parameters in vivo in small animals, which are essential and critical in preclinical imaging for physiological, pathophysiological and drug response studies.

Photoacoustic microscopy (PAM) provides a new method for the imaging of small-animals with high-contrast and deep-penetration. However, the established PAM systems have suffered from a limited field-of-view or imaging speed, which are difficult to both monitor wide-field activity of organ and record real-time change of local tissue. Here, we reported a dual-raster-scanned photoacoustic microscope (DRS-PAM) that integrates a two-dimensional motorized translation stage for large field-of-view imaging and a two-axis fast galvanometer scanner for real-time imaging. The DRS-PAM provides a flexible transition from wide-field monitoring the vasculature of organs to real-time imaging of local dynamics. To test the performance of DRS-PAM, clear characterization of angiogenesis and functional detail was illustrated, hemodynamic activities of vasculature in cerebral cortex of a mouse were investigated. Furthermore, response of tumor to treatment were successfully monitored during treatment. The experimental results demonstrate the DRS-PAM holds the great potential for biomedical research of basic biology.

Vessel development in the yolk sac is important for the embryo development and the malfunction of which can lead to cardiac dysfunction, embryonic malformation and miscarriage. Although substantial emphasis has been placed on the yolk sac vascular remodeling, no detailed three-dimensional (3D) imaging and quantitative analysis of this process has been described. Herein, we explored the development of the vascular system in the visceral yolk sac (VYS) on E11.5, E12.5 and E13.5 mouse embryos using a home-built large field-of-view (FOV) optical-resolution photoacoustic microscopy (OR-PAM). The results showed that OR-PAM can be used as a label-free imaging tool for studying the 2D/3D morphology changes of the vascular system during organogenesis. In addition, after a quantitative analysis the results showed that the microvascular density in the VYS gradually reduced along with the embryo growth. Vascular density in the VYS of E11.5 mouse embryos was almost 6-fold than that of E13.5. Hovever, the averaged vessel diameter of the entire VYS membrane increased gradually with the development of embryos. This study suggests that OR-PAM is a potential tool for acquiring the hemodynamic parameters of mammalian embryos, which could be further used for studying diseases related with the vascular remodeling such as vascular malformations and heart defects. Keywords: Optical-resolution photoacoustic microscopy, Embryo development, Visceral yolk sac vascular, Vascular remodeling, Vascular density

Human skin sections affected by various degrees of photoaging were imaged using photoacoustic microscopy. The photoacoustic signal intensity from the upper dermis increased with aging progress. The feasibility of quantitative human skin evaluation was demonstrated. © 2020 HATTORI Hiroki, NAMITA Takeshi, KONDO Kengo, YAMAKAWA Makoto, and SHIINA Tsuyoshi

The early detection of microvascular changes in cancer diagnosis is needed in the clinic. A change in the vascular bifurcation density is a biomarker for the sprouting activity. Here, Optical-Resolution PhotoAcoustic Microscopy is used for quantitative vascular bifurcation mapping in 2D after the creation of Virtual Tubes out of Bifurcations. In stacks of OR-PAM images of the hemoglobin distribution, bifurcations become tubes and are selected by the 3D tubeness filter. These fast analyses will be compared to a classical approach and are easier to implement for functional analysis of the vascular bifurcation density in healthy and diseased tissues.

The current study is presenting a non-invasive and high resolution multimodal photoacoustic microscopy (PAM) and optical coherence tomography (OCT) system for visualization of choroidal vascular lesions in living rabbits.

Among the numerous endogenous biological molecules, information on lipids is highly coveted for understanding both aspects of developmental biology and research in fatal chronic diseases. Due to the pronounced absorption features of lipids in the extended near-infrared region (1650−1850 nm), visualisation and identification of lipids become possible using multi-spectral photoacoustic (optoacoustic) microscopy. However, the spectroscopic studies in this spectral region require lasers that can produce high pulse energies over a broad spectral bandwidth to efficiently excite strong photoacoustic signals. The most well-known laser sources capable of satisfying the multi-spectral photoacoustic microscopy requirements (tunability and pulse energy) are tunable nanosecond optical parametric oscillators. However, these lasers have an inherently large footprint, thus preventing their use in compact microscopy systems. Besides, they exhibit low-repetition rates. Here, we demonstrate a compact all-fibre, high pulse energy supercontinuum laser that covers a spectral range from 1440 to 1870 nm with a 7 ns pulse duration and total energy of 18.3 μJ at a repetition rate of 100 kHz. Using the developed high-pulse energy source, we perform multi-spectral photoacoustic microscopy imaging of lipids, both ex vivo on adipose tissue and in vivo to study the development of Xenopus laevis tadpoles, using six different excitation bands over the first overtone transition of C–H vibration bonds (1650−1850 nm).

We demonstrate a high-energy gain-switched TDFL for volumetric PA microscopy of lipids at 1700, 1725, and 1750 nm. Pulse energies are 58.2, 66.8, and 75.3 μJ, respectively, with a 16.7 ns duration at 10 kHz. © 2020 The Author(s)

Bone microvasculature plays a paramount role in bone marrow maintenance, development, and hematopoiesis. Studies of calvarian vascular patterns within living mammalian skull with the available intravital microscopy techniques are limited to small scale observations. We developed an optical-resolution optoacoustic microscopy method combined with ultrasound biomicroscopy in order to reveal and discern the intricate networks of calvarian and cerebral vasculature over large fields of view covering majority of the murine calvaria. The vasculature segmentation method is based on an angle-corrected homogeneous model of the rodent skull, generated using simultaneously acquired three-dimensional pulse-echo ultrasound images. The hybrid microscopy design along with the appropriate skull segmentation method enable high throughput studies of a living bone while facilitating correct anatomical interpretation of the vasculature images acquired with optical resolution optoacoustic microscopy., Photoacoustics, 19

[EN] We explore the use of a beamforming method intended for large-area scanning in optical-resolution photoacoustic microscopy. It has been evaluated in a experimental setup that comprises a low-cost laser diode and a phase array with a 128-elements linear probe. Three different beamforming strategies are discussed: no-beamforming, static beamforming and dynamic beamforming. The method has been tested in gelatine-based phantoms as well as ex-vivo organs. Results show that, compared with the other two, dynamic beamforming increases up to 15dB and homogenizes signal-to-noise ratio (SNR) along images of roughly 1 cm2. The method and system presented here could be the baseline for more advanced array-based systems that leverage the low-cost laser sources for clinical applications., This research has been supported by the Spanish Ministry of Science, Innovation and Universities through grant Juan de la Cierva - Incorporacion (IJC2018-037897-I), and program Proyectos I+D+i 2019 (PID2019-111436RB-C22). Action co-financed by the European Union through the Programa Operativo del Fondo Europeo de Desarrollo Regional (FEDER) of the Comunitat Valenciana 2014-2020 (IDIFEDER/2018/022). A.C. received financial support from Generalitat Valenciana and Universitat Politecnica de Val ` encia through the grants APOSTD/2018/229 and program PAID-10-19, respectively.

Optoacoustic imaging is a medical imaging modality that uses optical excitation and acoustic detection to generate images of tissue structures based up optical absorption within a tissue sample. This imaging modality has been widely explored as a tool for a number of clinical applications, including cancer diagnosis and wound healing tracking. Recently, the optoacoustic imaging community has published a number of reports of hand-held optoacoustic imaging devices and platforms; these hand-held configurations improve the modality’s potential for commercial clinical implementation. Here, we review recent advancements in hand-held optoacoustic imaging platforms and methods, including recent pre-clinical applications, and we present an overview of the remaining limitations in optoacoustic imaging that must be addressed to increase the translation of the modality into commercial and clinical use. Keywords: Hand-held, Optoacoustic tomography, Photoacoustic microscopy, Multispectral, Raster scanning

Photoacoustic microscopy (PAM) has become an increasingly popular technology for biomedical applications, providing anatomical, functional, and molecular information. In this concise review, we first introduce the basic principles and typical system designs of PAM, including optical-resolution PAM and acoustic-resolution PAM. The major imaging characteristics of PAM, i.e. spatial resolutions, penetration depth, and scanning approach are discussed in detail. Then, we introduce the major biomedical applications of PAM, including anatomical imaging across scales from cellular level to organismal level, label-free functional imaging using endogenous biomolecules, and molecular imaging using exogenous contrast agents. Lastly, we discuss the technical and engineering challenges of PAM in the translation to potential clinical impacts.

We report on the use of a single supercontinuum (SC) source for multimodal imaging. The 2-octave bandwidth (475–2300 nm) makes the SC source suitable for optical coherence tomography (OCT) as well as for multispectral photoacoustic microscopy (MPAM). The IR band centered at 1310 nm is chosen for OCT to penetrate deeper into tissue with 8 mW average power on the sample. The 500–840 nm band is used for MPAM. The source has the ability to select the central wavelength as well as the spectral bandwidth. An energy of more than 35 nJ within a less than 50 nm bandwidth is achieved on the sample for wavelengths longer than 500 nm. In the present paper, we demonstrate the capabilities of such a multimodality imaging instrument based on a single optical source. In vitro mouse ear B-scan images are presented. Keywords: Multimodal imaging, Multispectral imaging, Photoacoustic, Optical coherence tomography, Supercontinuum

Photoacoustic imaging acquires the absorption contrast of biological tissue with ultrasound resolution. It has been broadly investigated in biomedicine for animal and clinical studies. Recently, a micro-electro-mechanical system (MEMS) scanner has been utilized in photoacoustic imaging systems to enhance their performance and extend the realm of applications. The review provides a recap of recent developments in photoacoustic imaging using MEMS scanner, from instrumentation to applications. The topics include the design of MEMS scanner, its use in photoacoustic microscopy, miniature imaging probes, development of dual-modality systems, as well as cutting-edge bio-imaging studies.

Graphical abstract, Photoacoustic microscopy (PAM) is an important imaging tool that can noninvasively visualize the anatomical structure of living animals. However, the limited scanning area restricts traditional PAM systems for scanning a large animal. Here, we firstly report a dual-channel PAM system based on a custom-made slider-crank scanner. This novel scanner allows us to stably capture an ultra-widefield scanning area of 24 mm at a high B-scan speed of 32 Hz while maintaining a high signal-to-noise ratio. Our system's spatial resolution is measured at ∼3.4 μm and ∼37 μm for lateral and axial resolution, respectively. Without any contrast agent, a dragonfly wing, a nude mouse ear, an entire rat ear, and a portion of mouse sagittal are successfully imaged. Furthermore, for hemodynamic monitoring, the mimicking circulating tumor cells using magnetic contrast agent is rapidly captured in vitro. The experimental results demonstrated that our device is a promising tool for biological applications.

In this work, a novel fiber optic sensor based on Fabry-Pérot interferometry is adopted in an optical coherence photoacoustic microscopy (OC-PAM) system to enable high-resolution in vivo imaging. The complete OC-PAM system is characterized using the fiber optic sensor for photoacoustic measurement. After characterization, the performance of the system is evaluated by imaging zebrafish larvae in vivo. With a lateral resolution of 3.4 μm and an axial resolution of 3.7 μm in air, the optical coherence microscopy subsystem visualizes the anatomy of the zebrafish larvae. The photoacoustic microscopy subsystem reveals the vasculature of the zebrafish larvae with a lateral resolution of 1.9 μm and an axial resolution of 37.3 μm. As the two modalities share the same sample arm, we obtain inherently co-registered morphological and vascular images. This OC-PAM system provides comprehensive information on the anatomy and vasculature of the zebrafish larvae. Featuring compactness, broad detection bandwidth, and wide detection angle, the fiber optic sensor enables a large field of view with a static sensor position. We verified the feasibility of the fiber optic sensor for dual-modality in vivo imaging. The OC-PAM system, as a non-invasive imaging method, demonstrates its superiority in the investigation of zebrafish larvae, an animal model with increasing significance in developmental biology and disease research. This technique can also be applied for functional as well as longitudinal studies in the future. Nanyang Technological University Published version This work was supported by the Joint Ph.D. Program Medical University of Vienna/NTU Singapore “Kooperation Singapur” (Grant No. SO10300010), the European Commission Horizon 2020 LEIT Information and Communication Technologies under Grant Agreement No. 732720 (ESOTRAC), the FETOPEN-01-2018-2019-2020-FET-Open Project SWIMMOT under Grant Agreement No. 899612, the H2020-ICT-2020-2 Project REAP under Grant Agreement No. 101016964, the H2020-MSCA-IF-2019 Project SkinOptima under Grant Agreement No. 894325, the European Research Council under Advanced Grant No. 741149, and the Austrian Research Promotion Agency (FFG) under Grant No. 7940628 (Danio4Can).