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The need for faster and more accessible alternatives to laboratory microscopy is driving many innovations throughout the image and data acquisition chain in the biomedical field. Benchtop microscopes are bulky, lack communications capabilities, and require trained personnel for analysis. New technologies, such as compact 3D-printed devices integrated with the Internet of Things (IoT) for data sharing and cloud computing, as well as automated image processing using deep learning algorithms, can address these limitations and enhance the conventional imaging workflow. This review reports on recent advancements in microscope miniaturization, with a focus on emerging technologies such as photoacoustic microscopy and more established approaches like smartphone-based microscopy. The potential applications of IoT in microscopy are examined in detail. Furthermore, this review discusses the evolution of image processing in microscopy, transitioning from traditional to deep learning methods that facilitate image enhancement and data interpretation. Despite numerous advancements in the field, there is a noticeable lack of studies that holistically address the entire microscopy acquisition chain. This review aims to highlight the potential of IoT and artificial intelligence (AI) in combination with portable microscopy, emphasizing the importance of a comprehensive approach to the microscopy acquisition chain, from portability to image analysis. [ABSTRACT FROM AUTHOR]

Photoacoustic microscopy (PAM) is a high‐resolution and non‐invasive imaging modality that provides optical absorption contrast. By employing dual‐ or multiple‐wavelength excitation, PAM extends its capabilities to offer valuable spectroscopic information. To achieve efficient multispectral PAM imaging, an essential requirement is a light source characterized by a high repetition rate and switching rate, a ≈microjoule pulse energy, and a ≈nanosecond pulse duration. However, there exists a notable deficiency in suitable light sources, particularly in the near‐infrared‐II window. In this study, a custom‐built all‐fiber‐based light source is reported that provides >3 µJ, 2 ns pulses with a repetition rate of 200 kHz. Digitally addressed semiconductor seed lasers, followed by stimulated Raman scattering amplification, enabled arbitrary sequences of pulses having wavelengths of either 1168.4 or 1202.1 nm. In a switching mode of operation, a 100 kHz switching rate is used to alternate between these wavelengths in even/odd pulses. Furthermore, a high‐resolution multispectral photoacoustic microscopy of three polymer samples is demonstrated with the proposed light source. [ABSTRACT FROM AUTHOR]

Photoacoustic microscopy (PAM) enables label‐free imaging of the 3D vasculature and functional information with 2D lateral scan. The unique capacity in probing metabolism makes it ideal for animal research and clinical application. However, the high‐excitation power impedes the high‐speed monitoring of hemodynamics due to thermal accumulation and photon damage. To address this challenge, a self‐supervised photoacoustic single volume denoising (PSVD) approach, which combines 3D random sampling and noise augmentation to achieve 6 dB signal‐to‐noise‐ratio and contrast‐to‐noise‐ratio increases for the customized optical‐resolution photoacoustic microscope, is developed. Using PSVD, high‐quality PAM images of the mouse ear are acquired with only 10% fluence of normal excitation. Functional imaging is validated with this PSVD‐empowered low‐fluence PAM. Accurate oxygen saturation maps and high‐contrast flow kymographs are obtained. Moreover, the capability of this approach in the live mouse ear under hypercapnia is demonstrated. Further transformation into clinical imaging with low fluence will broaden the application of PAM. [ABSTRACT FROM AUTHOR]

The incorporation of gold nanoparticles (GNPs) into retinal imaging signifies a notable advancement in ophthalmology, offering improved accuracy in diagnosis and patient outcomes. This review explores the synthesis and unique properties of GNPs, highlighting their adjustable surface plasmon resonance, biocompatibility, and excellent optical absorption and scattering abilities. These features make GNPs advantageous contrast agents, enhancing the precision and quality of various imaging modalities, including photoacoustic imaging, optical coherence tomography, and fluorescence imaging. This paper analyzes the unique properties and corresponding mechanisms based on the morphological features of GNPs, highlighting the potential of GNPs in retinal disease diagnosis and management. Given the limitations currently encountered in clinical applications of GNPs, the approaches and strategies to overcome these limitations are also discussed. These findings suggest that the properties and efficacy of GNPs have innovative applications in retinal disease imaging. [ABSTRACT FROM AUTHOR]

Bone diseases deteriorate the microstructure of bone tissue. Optical-resolution photoacoustic microscopy (OR-PAM) enables high spatial resolution of imaging bone tissues. However, the spatiotemporal trade-off limits the application of OR-PAM. The purpose of this study was to improve the quality of OR-PAM images without sacrificing temporal resolution. In this study, we proposed the Photoacoustic Dense Attention U-Net (PADA U-Net) model, which was used for reconstructing full-scanning images from under-sampled images. Thereby, this approach breaks the trade-off between imaging speed and spatial resolution. The proposed method was validated on resolution test targets and bovine cancellous bone samples to demonstrate the capability of PADA U-Net in recovering full-scanning images from under-sampled OR-PAM images. With a down-sampling ratio of [4, 1], compared to bilinear interpolation, the Peak Signal-to-Noise Ratio and Structural Similarity Index Measure values (averaged over the test set of bovine cancellous bone) of the PADA U-Net were improved by 2.325 dB and 0.117, respectively. The results demonstrate that the PADA U-Net model reconstructed the OR-PAM images well with different levels of sparsity. Our proposed method can further facilitate early diagnosis and treatment of bone diseases using OR-PAM. [ABSTRACT FROM AUTHOR]

In this paper, we design a multimodal visible/near-infrared photoacoustic microscopy and optical coherence tomography (VIS/NIR-PAM-OCT) system for imaging both retina and retinal pigment epithelium (RPE)/choroid complex layer. F127 and DSPE-PEG-cRGD encapsulated IR-1048 nanoparticles (FINPs) exhibiting absorption peak up to 1,064 nm were utilized as contrast agents to enhance NIR-PAM for in vivo imaging of fundus tissues. The fundus structure and vessels are clearly visualized by the multimodal imaging, and their parameters were quantitatively analyzed. NIR-PAM and OCT imaging of fundus were time-serially monitored over 60 min following the intravenous injection of FINPs into rats. The results indicated a 134 % increase in image signals in PAM at 1 min, along with an 8.23 % intensity enhancement in OCT. Moreover, laser-induced choroidal neovascularization (CNV) was specifically detected and accurately quantified using VIS/NIR-PAM-OCT. Lastly, FINPs demonstrated excellent biocompatibility in hematology analysis and pathology testing.

Optical‐resolution photoacoustic microscopy (OR‐PAM) has been increasingly utilized for in vivo imaging of biological tissues, offering structural, functional, and molecular information. In OR‐PAM, it is often necessary to make a trade‐off between imaging depth, lateral resolution, field of view, and imaging speed. To improve the lateral resolution without sacrificing other performance metrics, we developed a virtual‐point‐based deconvolution algorithm for OR‐PAM (VP‐PAM). VP‐PAM has achieved a resolution improvement ranging from 43% to 62.5% on a single‐line target. In addition, it has outperformed Richardson‐Lucy deconvolution with 15 iterations in both structural similarity index and peak signal‐to‐noise ratio on an OR‐PAM image of mouse brain vasculature. When applied to an in vivo glass frog image obtained by a deep‐penetrating OR‐PAM system with compromised lateral resolution, VP‐PAM yielded enhanced resolution and contrast with better‐resolved microvessels. [ABSTRACT FROM AUTHOR]

Photoacoustic imaging (PAI) is an emerging hybrid imaging modality that combines high-contrast optical imaging with high-spatial-resolution ultrasound imaging. PAI can provide a high spatial resolution and significant imaging depth by utilizing the distinctive spectroscopic characteristics of tissue, which gives it a wide variety of applications in biomedicine and preclinical research. In addition, it is non-ionizing and non-invasive, and photoacoustic (PA) signals are generated by a short-pulse laser under thermal expansion. In this study, we describe the basic principles of PAI, recent advances in research in human and animal tissues, and future perspectives. [ABSTRACT FROM AUTHOR]

Blood vessel imaging provides crucial information for physiological monitoring and disease diagnosis. Optical‐resolution photoacoustic microscopy (PAM) is gaining intense attention in high‐resolution in vivo imaging of blood vessels. However, its applicability in clinical settings is hindered by the requirement of bulky and costly pulsed lasers as well as complex alignment procedures. Here, as a novel approach to high‐quality and cost‐effective microvasculature imaging, a dual‐wavelength fiber‐coupled laser diode‐based PAM (FC‐LD‐PAM) system is demonstrated on the mouse ear and brain in vivo. Furthermore, assisted with a deep learning method, the proposed technique effectively combines the advantages when using both small and large core‐size multimode fibers—successfully enhances the resolution of blurry mouse microvascular images obtained using a large core‐size fiber, revealing fine details that are comparable to those achieved using a small core‐size fiber; significantly reduces the overall time for data acquisition up to fourfold, improving the efficiency significantly in the imaging process. In addition, the proposed approach can achieve accurate and high‐resolution mapping of oxygen saturation in vivo, providing functional insight on living tissue. Therefore, the FC‐LD‐PAM can serve as a translational tool for high‐resolution imaging with enhanced accessibility and versatility in the biomedical field. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) affects the morphological structure and causes significant degradation in kidney function, leading to renal replacement treatment in affected individuals. Vascular rarefaction is thought to be an important factor in accelerating kidney damage in CKD patients, therefore, the assessment of renal morphology and vasculature is crucial in nephrology. The objective of this study was to evaluate the morphological and vascular changes caused by CKD in mice kidneys. In this study, dual photoacoustic microscopy (PAM) and optical coherence microscopy (OCM) oriented wide-field high-resolution imaging modalities were employed for diseased renal imaging. The unilateral ureteral obstruction (UUO) model was used to prepare renal samples with CKD, and the developed wide-field dual imaging system was used to image both control and CKD-affected kidneys for assessing vascular and morphological changes during CKD progression. The obtained results reveal a gradual alteration in vascular intensity and pelvis space with the progress of UUO disease. Furthermore, a quantitative micro-vessel analysis was performed based on the node, junction, and mesh of the vessel, which provides details on the increasing microvascular-related characteristics in the peripheral area as the disease progresses. Thus, by concurrently employing the advantages of each optical imaging technique, the proposed method of assessing the OCM-based morphological and PAM-based vascular properties of the renal sample using a wide-field multimodal imaging system can be an efficient technique for whole volume analysis without any exogenous contrast agents in kidney histopathology. [ABSTRACT FROM AUTHOR]

Actively Q‐switched (AQS) fiber laser and solid‐state laser (SSL) are widely used for photoacoustic microscopy (PAM). In contrast, passively Q‐switched (PQS) SSL not only maintains most of the merits of AQS lasers, but also exhibits unique advantages, including the pulse width (PW), pulse repetition rate (PRR) tunability, wavelength, compactness, and cost. These advantages all benefit the PAM. However, there are few reports demonstrating the performance of PQS‐SSL on PA imaging. Here, we demonstrate a compact PQS‐SSL for fast and efficient PA imaging. The laser uniquely maintains a constant PW (~2 ns) and pulse energy (~3 μJ) during the PRR variation (30–100 kHz), which is valuable for preserving a stabilized imaging performance at different scanning rates. The PA imaging performance is compared by a resolution target and showcased by whole‐body scanning of an embryonic zebrafish in vivo. The performance indicates that PQS‐SSL is a promising candidate for PAM. [ABSTRACT FROM AUTHOR]

Photoacoustic microscopy is expected to have clinical applications as a noninvasive and three-dimensional (3D) method of observing intradermal structures. Investigate the applicability of a photoacoustic microscope equipped with two types of pulsed lasers that can simultaneously recognize hemoglobin and melanin. 16 skin lesions including erythema, pigmented lesions, vitiligo and purpura, were analyzed to visualize 3D structure of melanin granule distribution and dermal blood vessels. 13 cases of livedo racemosa in cutaneous polyarteritis nodosa (cPN) were further analyzed to visualize the 3D structure of dermal blood vessels in detail. Vascular structure was also analyzed in the biopsy specimens obtained from tender indurated erythema of cPN by CD34 immunostaining. Hemoglobin-recognition signal clearly visualized the 3D structure of dermal blood vessels and melanin-recognition signal was consistently reduced in vitiligo. In livedo racemosa, the hemoglobin-recognition signal revealed a relatively thick and large reticular structure in the deeper layers that became denser and finer toward the upper layers. The numerical analysis revealed that the number of dermal blood vessels was 1.29-fold higher (p<0.05) in the deeper region of the lesion than that of normal skin. The CD34 immunohistochemical analysis in tender indurated erythema revealed an increased number of dermal vessels compared with normal skin in 88.9% (8/9) of the cases, suggesting that vascular network remodeling had occurred in cPN. The photoacoustic system has an advantage in noninvasively detecting dermal blood vessel structures that are difficult to recognize by two-dimensional histopathology specimen examination and is worth evaluating in various skin diseases. • Applicability of photoacoustic microscopy detecting hemoglobin and melanin. • Noninvasive visualization of three-dimensional structure of dermal blood vessels. • Visualization of consistent reduction of melanin granules in vitiligo lesion. • Identification of dermal vasculature remodeling in cutaneous polyarteritis nodosa. [ABSTRACT FROM AUTHOR]

To identify the vascular alteration by photodynamic therapy (PDT), the utilization of high-resolution, high-speed, and wide-field photoacoustic microscopy (PAM) has gained enormous interest. The rapid changes in vasculature during PDT treatment and monitoring of tumor tissue activation in the orthotopic pancreatic cancer model have received limited attention in previous studies. Here, a fully two-axes waterproof galvanometer scanner-based photoacoustic microscopy (WGS-PAM) system was developed for in vivo monitoring of dynamic variations in micro blood vessels due to PDT in an orthotopic pancreatic cancer mouse model. The photosensitizer (PS), Chlorin e6 (Ce6), was utilized to activate antitumor reactions in response to the irradiation of a 660 nm light source. Microvasculatures of angiogenesis tissue were visualized on a 40 mm2 area using the WGS-PAM system at 30 min intervals for 3 h after the PDT treatment. The decline in vascular intensity was observed at 24.5% along with a 32.4% reduction of the vascular density at 3 h post-PDT by the analysis of PAM images. The anti-vascularization effect was also identified with fluorescent imaging. Moreover, Ce6-PDT increased apoptotic and necrotic markers while decreasing vascular endothelial growth factor (VEGF) expression in MIA PaCa-2 and BxPC-3 pancreatic cancer cell lines. The approach of the WGS-PAM system shows the potential to investigate PDT effects on the mechanism of angiographic dynamics with high-resolution wide-field imaging modalities. [ABSTRACT FROM AUTHOR]

A fast scanner of optical-resolution photoacoustic microscopy is inherently vulnerable to perturbation, leading to severe image distortion and significant misalignment among multiple 2D or 3D images. Restoration and registration of these images is critical for accurately quantifying dynamic information in long-term imaging. However, traditional registration algorithms face a great challenge in computational throughput. Here, we develop an unsupervised deep learning based registration network to achieve real-time image restoration and registration. This method can correct artifacts from B-scan distortion and remove misalignment among adjacent and repetitive images in real time. Compared with conventional intensity based registration algorithms, the throughput of the developed algorithm is improved by 50 times. After training, the new deep learning method performs better than conventional feature based image registration algorithms. The results show that the proposed method can accurately restore and register the images of fast-scanning photoacoustic microscopy in real time, offering a powerful tool to extract dynamic vascular structural and functional information.

Photoacoustic microscopy (PAM) has gained increasing popularity in biomedical imaging, providing new opportunities for tissue monitoring and characterization. With the development of deep learning techniques, convolutional neural networks have been used for PAM image resolution enhancement and denoising. However, there exist several inherent challenges for this approach. This work presents a Unified PhotoAcoustic Microscopy image reconstruction Network (UPAMNet) for both PAM image super-resolution and denoising. The proposed method takes advantage of deep image priors by incorporating three effective attention-based modules and a mixed training constraint at both pixel and perception levels. The generalization ability of the model is evaluated in details and experimental results on different PAM datasets demonstrate the superior performance of the method. Experimental results show improvements of 0.59 dB and 1.37 dB, respectively, for 1/4 and 1/16 sparse image reconstruction, and 3.9 dB for image denoising in peak signal-to-noise ratio.

Breast cancer often necessitates surgical interventions such as breast-conserving surgery or mastectomy. In these surgeries, sentinel lymph node (SLN) samples are often excised for histopathological examination to ascertain the presence of cancer metastasis. Despite its importance, traditional hematoxylin and eosin (H&E) staining, considerably prolongs the operation because of its complex processing requirements. Ultraviolet photoacoustic microscopy (UV-PAM) has emerged as a solution for bypassing the necessity of tissue staining or sectioning. However, its clinical application has been hindered by imaging speed. To overcome this challenge, we have developed a fast-scanning, reflection-mode UV-PAM designed for histopathology without staining based on high-sensitivity, wide-vision optical ultrasound detection. A specimen area of around 12 mm2 can be scanned in 8 min with a lateral resolution of 1.5 μm. To enhance imaging speed, multi-focal PAM was implemented, resulting in a fourfold acceleration. This PAM technique has been utilized in SLN biopsy to differentiate cancerous tissue in breast cancer patients.

The need for faster and more accessible alternatives to laboratory microscopy is driving many innovations throughout the image and data acquisition chain in the biomedical field. Benchtop microscopes are bulky, lack communications capabilities, and require trained personnel for analysis. New technologies, such as compact 3D-printed devices integrated with the Internet of Things (IoT) for data sharing and cloud computing, as well as automated image processing using deep learning algorithms, can address these limitations and enhance the conventional imaging workflow. This review reports on recent advancements in microscope miniaturization, with a focus on emerging technologies such as photoacoustic microscopy and more established approaches like smartphone-based microscopy. The potential applications of IoT in microscopy are examined in detail. Furthermore, this review discusses the evolution of image processing in microscopy, transitioning from traditional to deep learning methods that facilitate image enhancement and data interpretation. Despite numerous advancements in the field, there is a noticeable lack of studies that holistically address the entire microscopy acquisition chain. This review aims to highlight the potential of IoT and artificial intelligence (AI) in combination with portable microscopy, emphasizing the importance of a comprehensive approach to the microscopy acquisition chain, from portability to image analysis.

Photoacoustic microscopy (PAM) is a medical-imaging technique with the merits of high contrast and resolution. Nevertheless, conventional PAM scans specimens in a diameter-by-diameter fashion, resulting in a time-consuming process. Furthermore, deep-learning-based PAM image enhancement necessitates acquiring ground-truth data for training purposes. In this paper, we built an optical-resolution photoacoustic microscopy system and introduced an innovative unsupervised-learning algorithm. First, we enhanced the rotational-scanning method, transitioning from a diameter-by-diameter approach to a sector-by-sector one, significantly reducing imaging time (from 280 s to 109 s). Second, by establishing a metric for unsupervised learning, we eliminated the need for collecting reliable and high-quality ground truth, which is a challenging task in photoacoustic microscopy. A total of 324 pairs of datasets (mouse ears) were collected for unsupervised learning, with 274 for training and 50 for testing. Additionally, carbon-fiber data were sampled for lateral resolution and contrast evaluation, as well as the effective rate evaluation of the algorithm. The enhanced images demonstrated superior performance compared with that of maximum projection, both subjectively and objectively. A 76% improvement in the lateral resolution was observed. The effective rate of the algorithm was measured to be 100%, which was tested on 50 random samples. The technique presented in this paper holds substantial potential for image postprocessing and opens new avenues for unsupervised learning in photoacoustic microscopy. [ABSTRACT FROM AUTHOR]

Background: Skin autografts have been broadly used to manage the skin and soft tissue defects. It is important for surgeons to assess the vitality of skin autografts via observing the angiogenesis. However, there is lack of reliable approach for giving the quantitative angiogenesis information on the skin autografts. Recently, photoacoustic microscopy imaging has attracted much attention based on its good performance in angiography. Methods: In this study, we aim to monitor angiogenesis in skin autografts via PAM, and further verify its clinical potential for the early prediction of skin autografts clinical outcome. Results and Conclusions: The results indicate that PAM is a feasible, precise, high‐resolution, noninvasive technique for the early prediction of necrosis of skin autografts via monitoring the angiogenesis, providing a promising tool for surgeons to use this surgical technology. [ABSTRACT FROM AUTHOR]

This research paper discusses the development of a spiral laser scanning photoacoustic microscopy (sLS-PAM) system for functional brain imaging in rats. The aim of the study was to create a high-resolution, fast frame-rate, and wide field-of-view (FOV) PAM system that can capture hemodynamic changes in the brain. The sLS-PAM system was able to image cerebral hemodynamics in response to whisker and electrical stimulation, as well as vascular imaging of a modeled brain injury. The researchers believe that this simple-to-implement PAM system could become an affordable functional neuroimaging tool for researchers. [Extracted from the article]

The incorporation of gold nanoparticles (GNPs) into retinal imaging signifies a notable advancement in ophthalmology, offering improved accuracy in diagnosis and patient outcomes. This review explores the synthesis and unique properties of GNPs, highlighting their adjustable surface plasmon resonance, biocompatibility, and excellent optical absorption and scattering abilities. These features make GNPs advantageous contrast agents, enhancing the precision and quality of various imaging modalities, including photoacoustic imaging, optical coherence tomography, and fluorescence imaging. This paper analyzes the unique properties and corresponding mechanisms based on the morphological features of GNPs, highlighting the potential of GNPs in retinal disease diagnosis and management. Given the limitations currently encountered in clinical applications of GNPs, the approaches and strategies to overcome these limitations are also discussed. These findings suggest that the properties and efficacy of GNPs have innovative applications in retinal disease imaging.

Photoacoustic imaging (PAI) is an emerging hybrid imaging modality that combines high-contrast optical imaging with high-spatial-resolution ultrasound imaging. PAI can provide a high spatial resolution and significant imaging depth by utilizing the distinctive spectroscopic characteristics of tissue, which gives it a wide variety of applications in biomedicine and preclinical research. In addition, it is non-ionizing and non-invasive, and photoacoustic (PA) signals are generated by a short-pulse laser under thermal expansion. In this study, we describe the basic principles of PAI, recent advances in research in human and animal tissues, and future perspectives.

Photoacoustic microscopy (PAM), due to its deep penetration depth and high contrast, is playing an increasingly important role in biomedical imaging. PAM imaging systems equipped with conventional ultrasound transducers have demonstrated excellent imaging performance. However, these opaque ultrasonic transducers bring some constraints to the further development and application of PAM, such as complex optical path, bulky size, and difficult to integrate with other modalities. To overcome these problems, ultrasonic transducers with high optical transparency have appeared. At present, transparent ultrasonic transducers are divided into optical-based and acoustic-based sensors. In this paper, we mainly describe the acoustic-based piezoelectric transparent transducers in detail, of which the research advances in PAM applications are reviewed. In addition, the potential challenges and developments of transparent transducers in PAM are also demonstrated. [ABSTRACT FROM AUTHOR]

To identify the vascular alteration by photodynamic therapy (PDT), the utilization of high-resolution, high-speed, and wide-field photoacoustic microscopy (PAM) has gained enormous interest. The rapid changes in vasculature during PDT treatment and monitoring of tumor tissue activation in the orthotopic pancreatic cancer model have received limited attention in previous studies. Here, a fully two-axes waterproof galvanometer scanner-based photoacoustic microscopy (WGS-PAM) system was developed for in vivo monitoring of dynamic variations in micro blood vessels due to PDT in an orthotopic pancreatic cancer mouse model. The photosensitizer (PS), Chlorin e6 (Ce6), was utilized to activate antitumor reactions in response to the irradiation of a 660 nm light source. Microvasculatures of angiogenesis tissue were visualized on a 40 mm2 area using the WGS-PAM system at 30 min intervals for 3 h after the PDT treatment. The decline in vascular intensity was observed at 24.5% along with a 32.4% reduction of the vascular density at 3 h post-PDT by the analysis of PAM images. The anti-vascularization effect was also identified with fluorescent imaging. Moreover, Ce6-PDT increased apoptotic and necrotic markers while decreasing vascular endothelial growth factor (VEGF) expression in MIA PaCa-2 and BxPC-3 pancreatic cancer cell lines. The approach of the WGS-PAM system shows the potential to investigate PDT effects on the mechanism of angiographic dynamics with high-resolution wide-field imaging modalities.

Photoacoustic microscopy (PAM), due to its deep penetration depth and high contrast, is playing an increasingly important role in biomedical imaging. PAM imaging systems equipped with conventional ultrasound transducers have demonstrated excellent imaging performance. However, these opaque ultrasonic transducers bring some constraints to the further development and application of PAM, such as complex optical path, bulky size, and difficult to integrate with other modalities. To overcome these problems, ultrasonic transducers with high optical transparency have appeared. At present, transparent ultrasonic transducers are divided into optical-based and acoustic-based sensors. In this paper, we mainly describe the acoustic-based piezoelectric transparent transducers in detail, of which the research advances in PAM applications are reviewed. In addition, the potential challenges and developments of transparent transducers in PAM are also demonstrated.

Photoacoustic imaging has emerged as a promising biomedical imaging technique that enables visualization of the optical absorption characteristics of biological tissues in vivo. Among the different photoacoustic imaging system configurations, optical-resolution photoacoustic microscopy stands out by providing high spatial resolution using a tightly focused laser beam, which is typically transmitted through optical fibers. Achieving high-quality images depends significantly on optical fluence, which is directly proportional to the signal-to-noise ratio. Hence, optimizing the laser-fiber coupling is critical. Conventional coupling systems require manual adjustment of the optical path to direct the laser beam into the fiber, which is a repetitive and time-consuming process. In this study, we propose an automated laser-fiber coupling module that optimizes laser delivery and minimizes the need for manual intervention. By incorporating a motor-mounted mirror holder and proportional derivative control, we successfully achieved efficient and robust laser delivery. The performance of the proposed system was evaluated using a leaf-skeleton phantom in vitro and a human finger in vivo, resulting in high-quality photoacoustic images. This innovation has the potential to significantly enhance the quality and efficiency of optical-resolution photoacoustic microscopy. [ABSTRACT FROM AUTHOR]

Organs maintain their unique functions through a variety of distinctive vessel systems. However, examination of multiset vessels over the entire organ still remains challenging due to the difficulties of assigning multiple vessel systems and labor‐intensive imaging reconstruction technologies. Here, an organ‐level photoacoustic microscopic imaging platform, named Organ‐PAM, with a micron‐scale spatial resolution and ultra‐large field of view (FOV), which can achieve a lateral and axial FOV of up to ≈81 cm2 and ≈30 mm, respectively, is presented. With the assistance of whole‐organ decellularization, optical clearing, and a specifically designed photoacoustic gradient concentration differential angiographic pipeline, the visualization of up to four sets of vessel systems inside diversified organs is successfully achieved with multiple scales. In addition, quantitative analyses of vessel systems in both healthy livers and kidneys as well as cancerous livers with exogenous transplanted tumors are conducted at different stages. Thus, the platform enables high‐efficiency multiset vessel imaging, recognition, and quantification of different organs, providing critical insights into distinct vessel systems under varying pathological conditions. [ABSTRACT FROM AUTHOR]

Photoacoustic microscopy (PAM) is a high‐resolution imaging modality that has been mainly implemented with small field of view applications. Here, we developed a fast PAM system that utilizes a unique spiral laser scanning mechanism and a wide acoustic detection unit. The developed system can image an area of 12.5 cm2 in 6.4 s. The system has been characterized using highly detailed phantoms. Finally, the imaging capabilities of the system were further demonstrated by imaging a sheep brain ex vivo and a rat brain in vivo. [ABSTRACT FROM AUTHOR]

Photoacoustic microscopy provides a new dimension of observation in microscopic life science. However, due to the high complexity of building a photoacoustic microscopy system, for many life science practitioners, it usually takes several years to build a stable photoacoustic microscopy system. For the above situation, in this article, a productized strategy of photoacoustic (532 and 1064 nm) and ultrasonic coscanning microscopy for in vivo imaging on small animals is presented. A 532 nm laser is applied to image blood vessels and pigments in label‐free manner, whereas 1064 nm laser is applied to image pigments and some novel probes developed for NIR‐II windows. Ultrasound is applied to assist photoacoustic imaging to accurately locate its imaging site in tissues. All 3D results are obtained with one single scan. The strategy presented here will help life science practitioners to build a stable photoacoustic microscopy platform. [ABSTRACT FROM AUTHOR]

In this paper, a portable photoacoustic microscopy (PAM) system is proposed based on a large stroke electrothermal micromirror to achieve high resolution and fast imaging. The crucial micromirror in the system realizes a precise and efficient 2-axis control. Two different designs of electrothermal actuators with "O" and "Z" shape are evenly located around the four directions of mirror plate. With a symmetrical structure, the actuator realized single direction drive only. The finite element modelling of both two proposed micromirror has realized a large displacement over 550 μm and the scan angle over ±30.43° at 0–10 V DC excitation. In addition, the steady-state and transient-state response show a high linearity and quick response respectively, which can contribute to a fast and stable imaging. Using the Linescan model, the system achieves an effective imaging area of 1 mm × 3 mm in 14 s and 1 mm × 4 mm in 12 s for the "O" and "Z" types, respectively. The proposed PAM systems have advantages in image resolution and control accuracy, indicating a significant potential in the field of facial angiography. [ABSTRACT FROM AUTHOR]

Photoacoustic microscopy is advancing with research on utilizing ultraviolet and visible light. Dual-wavelength approaches are sought for observing DNA/RNA- and vascular-related disorders. However, the availability of high numerical aperture lenses covering both ultraviolet and visible wavelengths is severely limited due to challenges such as chromatic aberration in the optics. Herein, we present a groundbreaking proposal as a pioneering simulation study for incorporating multilayer metalenses into ultraviolet-visible photoacoustic microscopy. The proposed metalens has a thickness of 1.4 µm and high numerical aperture of 0.8. By arranging cylindrical hafnium oxide nanopillars, we design an achromatic transmissive lens for 266 and 532 nm wavelengths. The metalens achieves a diffraction-limited focal spot, surpassing commercially available objective lenses. Through three-dimensional photoacoustic simulation, we demonstrate high-resolution imaging with superior endogenous contrast of targets with ultraviolet and visible optical absorption bands. This metalens will open new possibilities for downsized multispectral photoacoustic microscopy in clinical and preclinical applications.

Pathology interpretations of tissue rely on the gold standard of histology imaging, potentially hampering timely access to critical information for diagnosis and management of neoplasms because of tedious sample preparations. Slide-free capture of cell nuclei in unprocessed specimens without staining is preferable; however, inevitable irregular surfaces in fresh tissues results in limitations. An ultraviolet metasurface with the ability to generate an ultraviolet optical focus maintaining

In addition to offering morphological visualizations via capture of the spatial distributions of optical absorption, photoacoustic imaging technology can reveal abundant physical information about biological particles, including their orientation, density, and viscoelasticity, through analysis of the pressure transients in the spectral domain. However, the low-amplitude wideband photoacoustic signals of intrinsic microscopic optically-absorbing objects under the action of confined photoacoustic excitation power continue to hinder simultaneous photoacoustic structural imaging and spectroscopic analysis of the nonfluorescent chromophores in living biological tissues because of the inadequate responses to photoacoustic impulses observed in most photoacoustic imaging setups that include piezoelectric transducers. Building upon a recently-developed optical evanescent wave sensor that can respond to ultrasound with high sensitivity over a broad frequency range, we propose in vivo spatial-spectral photoacoustic microscopy for recovery of structural imaging in three dimensions and characterization of anatomical features in the acoustic frequency domain. Label-free photoacoustic images of a living zebrafish are acquired in which spectroscopically-resolved differentiation of the microarchitecture is accessed, along with isometric micrometer-scale volumetric visualizations. The proposed imaging technology could potentially provide more comprehensive evaluations of the physiopathological status of living small animals.

Neurovascular imaging is essential for investigating neurodegenerative diseases. However, the existing neurovascular imaging technology suffers from a trade-off between a field of view (FOV) and resolution in the whole brain, resulting in an inhomogeneous resolution and lack of information. Here, homogeneous-resolution arched-scanning photoacoustic microscopy (AS-PAM), which has an ultrawide FOV to cover the entire mouse cerebral cortex, was developed. Imaging of the neurovasculature was performed with a homogenous resolution of 6.9 µm from the superior sagittal sinus to the middle cerebral artery and caudal rhinal vein in an FOV of 12 × 12 mm2. Moreover, using AS-PAM, vascular features of the meninges and cortex were quantified in early Alzheimer’s disease (AD) and wild-type (WT) mice. The results demonstrated high sensitivity to the pathological progression of AD on tortuosity and branch index. The high-fidelity imaging capability in large FOV enables AS-PAM to be a promising tool for precise brain neurovascular visualization and quantification.

Photoacoustic imaging (PAI) has emerged as a molecular-selective imaging technology based on optical absorption contrast. Dichroism-sensitive photoacoustic (DS-PA) imaging has been reported, where the absorption coefficient has a vector characteristic, featuring dimensions of contrast in polarization and wavelength. Herein, we present a DS-PA microscopy (DS-PAM) system that implements optical anisotropy contrast and molecular selectivity. Moreover, we propose mathematical solutions to fully derive dichroic properties. A wavelength for the PAI of collagenous tissue was used, and the proposed algorithms were validated using linear dichroic materials. We successfully mapped dichroic information in fibrous tissue imaging based on the degree of anisotropy and axis orientation, and also deduced mechanical assessment from the tissue arrangement. The proposed DS-PAM system and algorithms have great potential in various diagnostic fields using polarimetry, such as musculoskeletal and cardiovascular systems.

Multi-modality imaging is significant for biomedical applications. We propose a dual-sensitive sensor to simultaneously detect optical and ultrasonic signals. Based upon the classical piezoelectric structure, we attach a photosensitive layer made of carbon nanotubes-polydimethylsiloxane (CNTs-PDMS) composite to the surface. The photosensitive layer absorbs light and converts it into ultrasound, while allowing acoustic energy to transmit through concurrently. After optimizing the ratio of PDMS to CNTs, we increase the sensor’s light detection sensitivity and maintain the ultrasound detection sensitivity. Finally, the successful implementation in mouse ear optical attenuation–photoacoustic imaging demonstrates the dual-sensitive sensor’s potential application in multi-modality imaging.

We report an adaptive energy‐compensated synthetic aperture focusing technique (eC‐SAFT) for improving the imaging performance of photoacoustic microscopy (PAM) in terms of depth of field (DOF), spatial resolution (both axial and lateral), and SNR. In addition to coherency and time‐delay (in conventional SAFT), our beamforming‐based reconstruction algorithm takes into account acoustic energy loss—a primary physical parameter in acoustic wave propagation—following Beer‐Lambert's law. Experimental validation studies were performed in tissue‐mimicking (Agar) phantoms, complex leaf veins, and chicken breast tissues. Results demonstrate that our proposed eC‐SAFT+CF outperforms conventional SAFT+CF to improve axial resolution (up to ∼5%), lateral resolution (up to ∼5%), SNR (up to ∼6%) and CR (up to ∼8%). [ABSTRACT FROM AUTHOR]

Accurate volumetric photoacoustic image reconstruction requires ultrasonic detection with sufficient sensitivity to pressure transients over a broad spectral bandwidth. By using the ultrafast temporal dynamics and high‐sensitivity responses to refractive index changes of optical surface waves, a structureless ultrasonic sensor is proposed for photoacoustic impulse measurement that is manufactured simply by covering a ubiquitous bare glass with several drops of water. Ultrasensitive photoacoustic detection with a broadband frequency response is achieved by converting the photoacoustically modulated phase shift in the optical reflection into a time‐varying polarization. This sensor achieves noise‐equivalent pressure sensitivity at ≈93 Pa while maintaining an acoustic bandwidth of ≈174 MHz. Incorporation of the transparent sensor into an optical‐resolution photoacoustic microscope enables reflection‐mode 3D imaging with isometric spatial resolution on the micrometer scale. Label‐free volumetric visualizations of adult zebrafish are obtained in vivo. The favorable performance of the proposed sensor offers considerable potential for advancement of biomedical photoacoustic investigations. [ABSTRACT FROM AUTHOR]

Optical‐resolution photoacoustic microscopy (OR‐PAM) enables both high‐resolution and high‐contrast imaging of optical chromophores ranging from biological tissues to inorganic samples. The lateral spatial resolution of OR‐PAM depends on its optical configuration and is primarily determined by the numerical aperture of the objective lens. This study demonstrates a novel, lens‐free, shear‐force photoacoustic microscopy system using a tapered fiber, serving as a proof‐of‐concept toward the implementation of super‐resolution, near‐field scanning photoacoustic microscopy. An uncoated tapered fiber is attached to a quartz tuning fork, thereby maintaining the near‐field distance between the fiber and sample surface via a shear‐force detection mechanism. Light‐field simulation confirms an evanescent wave at the end of the uncoated, tapered fiber. Based on the photoacoustic simulation and 2D photoacoustic scanning experimental results, targets are imaged with high‐lateral resolutions of the order of 1.0 ± 0.3 µm. These results demonstrate the existence of near‐field photoacoustic signals and the potential for future development of super‐resolution, near‐field, scanning photoacoustic microscopy. [ABSTRACT FROM AUTHOR]

Blood vessels that deliver nutrients and oxygen over the entire body is essential for bone homeostasis. Especially, for the bone recovery, long‐term in vivo vascular imaging is desirable. Here, we propose an optical and ultrasonic transparent bone window, which allows repeated, chronic monitoring of bone angiogenesis in mouse tibia defect. A metal ring with an outer diameter of 2 mm and an inner diameter of 1 mm is bonded with a silicone‐based polydimethylsiloxane (PDMS) film and cover the bone surface, which can effectively eliminate the inflammation caused by repeated wound opening before imaging. We make a bone defect model in mouse tibia, and employ an optical resolution photoacoustic microscopy (ORPAM) to provide a high‐resolution, label‐free, long‐term, in vivo observation of the bone vascularization during the bone defect healing. The results suggest that the artificial bone window can remain stable for inspection and play positive role for bone repair. [ABSTRACT FROM AUTHOR]

Acoustically detecting the optical absorption contrast, photoacoustic imaging (PAI) is a highly versatile imaging modality that can provide anatomical, functional, molecular, and metabolic information of biological tissues. PAI is highly scalable and can probe the same biological process at various length scales ranging from single cells (microscopic) to the whole organ (macroscopic). Using hemoglobin as the endogenous contrast, PAI is capable of label-free imaging of blood vessels in the brain and mapping hemodynamic functions such as blood oxygenation and blood flow. These imaging merits make PAI a great tool for studying ischemic stroke, particularly for probing into hemodynamic changes and impaired cerebral blood perfusion as a consequence of stroke. In this narrative review, we aim to summarize the scientific progresses in the past decade by using PAI to monitor cerebral blood vessel impairment and restoration after ischemic stroke, mostly in the preclinical setting. We also outline and discuss the major technological barriers and challenges that need to be overcome so that PAI can play a more significant role in preclinical stroke research, and more importantly, accelerate its translation to be a useful clinical diagnosis and management tool for human strokes. [ABSTRACT FROM AUTHOR]

In this paper, we propose an efficient label-free in vivo photoacoustic (PA) imaging of melanoma using a condensed near infrared-I (NIR-I) supercontinuum light source. Although NIR-II spectral window is advantageous such as longer penetration depth compared to the NIR-I region, supercontinuum light sources emitting both NIR-I and NIR-II region could lower the efficiency to target melanoma because of low optical power density in the melanoma’s absorption spectra. To exploit efficient in vivo PA imaging of melanoma, we demonstrated the light source emitting from visible (532–600 nm) to NIR-I (600–1000 nm) by optimizing stimulated Raman scattering induced supercontinuum generation. The melanoma’s structure is successfully differentiated from blood vessels at a high pulse energy of 2.5 µJ and a flexible pulse repetition rate (PRR) of 5–50 kHz. The proposed light source with the microjoules energies and tens of kHz of PRR can potentially accelerate clinical trials such as early diagnosis of melanoma.

Spatial sampling density and data size are important determinants of the imaging speed of photoacoustic microscopy (PAM). Therefore, undersampling methods that reduce the number of scanning points are typically adopted to enhance the imaging speed of PAM by increasing the scanning step size. Since undersampling methods sacrifice spatial sampling density, by considering the number of data points, data size, and the characteristics of PAM that provides three-dimensional (3D) volume data, in this study, we newly reported deep learning-based fully reconstructing the undersampled 3D PAM data. The results of quantitative analyses demonstrate that the proposed method exhibits robustness and outperforms interpolation-based reconstruction methods at various undersampling ratios, enhancing the PAM system performance with 80-times faster-imaging speed and 800-times lower data size. The proposed method is demonstrated to be the closest model that can be used under experimental conditions, effectively shortening the imaging time with significantly reduced data size for processing.

Optical-resolution photoacoustic microscopy offers high-resolution, label-free hemodynamic and functional imaging to many biomedical applications. However, long-standing technical barriers, such as limited field of view, bulky scanning probes, and slow imaging speed, have limited the application of optical-resolution photoacoustic microscopy. Here, we present freehand scanning photoacoustic microscopy (FS-PAM) that can flexibly image various anatomical sites. We develop a compact handheld photoacoustic probe to acquire 3D images with high speed, and great flexibility. The high scanning speed not only enables video camera mode imaging but also allows for the first implementation of simultaneous localization and mapping (SLAM) in photoacoustic microscopy. We demonstrate fast in vivo imaging of some mouse organs, and human oral mucosa. The high imaging speed greatly reduces motion artifacts and distortions from tissue moving, breathing, and unintended handshaking. We demonstrate small-lesion localization in a large region of the brain. FS-PAM offers a flexible high-speed imaging tool with an extendable field of view, enabling more biomedical imaging applications.

During tumor resection, doctors use intraoperative biopsies to determine the tumor margin. However, the pathological procedures of traditional diagnostic methods, such as imprint cytology and frozen section analysis, are complicated and time-consuming. As this is not conducive to surgeries, their applications are limited to a large extent. Therefore, novel fast microscopy imaging technologies with resolutions comparable to those of pathological tissue sections are necessary. Stimulated Raman scattering (SRS), photoacoustic microscopy (PAM), multiphoton microscopy (MPM), and optical coherence microscopy (OCM) exhibit the advantages of high spatial resolution, large imaging depth, avoiding damage to biological tissues, label-free detection, and the availability of biochemical information of tissues. Additionally, they are superior to intraoperative biopsies owing to their fast imaging speeds. Therefore, they possess broad application prospects in tumor resection surgeries and the diagnosis of other diseases. This study briefly introduces the basic principles, structural characteristics, advantages and disadvantages, and the existing research status of SRS, PAM, MPM, and OCM in biomedicine. Furthermore, we propose a multi-mode hybrid detection technology that can be used for surgeries. The combination of the proposed technology with deep learning-based artificial intelligence can form the basis for intraoperative diagnosis in the future.

The ischemic stroke animal model evaluates the efficacy of reperfusion and neuroprotective strategies for ischemic injuries. Various conventional methods have been reported to induce the ischemic models; however, controlling specific neurological deficits, mortality rates, and the extent of the infarction is difficult as the size of the affected region is not precisely controlled. In this paper, we report a single laser-based localized target ischemic stroke model development method by simultaneous vessel monitoring and photothrombosis induction using photoacoustic microscopy (PAM), which has minimized the infarct size at precise location with high reproducibility. The proposed method has significantly reduced the infarcted region by illuminating the precise localization. The reproducibility and validity of suggested method have been demonstrated through repeated experiments and histological analyses. These results demonstrate that our method can provide the ischemic stroke model closest to the clinical pathology for brain ischemia research from inducement, occurrence mechanisms to the recovery process.

Photoacoustic imaging has emerged as a promising biomedical imaging technique that enables visualization of the optical absorption characteristics of biological tissues in vivo. Among the different photoacoustic imaging system configurations, optical-resolution photoacoustic microscopy stands out by providing high spatial resolution using a tightly focused laser beam, which is typically transmitted through optical fibers. Achieving high-quality images depends significantly on optical fluence, which is directly proportional to the signal-to-noise ratio. Hence, optimizing the laser-fiber coupling is critical. Conventional coupling systems require manual adjustment of the optical path to direct the laser beam into the fiber, which is a repetitive and time-consuming process. In this study, we propose an automated laser-fiber coupling module that optimizes laser delivery and minimizes the need for manual intervention. By incorporating a motor-mounted mirror holder and proportional derivative control, we successfully achieved efficient and robust laser delivery. The performance of the proposed system was evaluated using a leaf-skeleton phantom in vitro and a human finger in vivo, resulting in high-quality photoacoustic images. This innovation has the potential to significantly enhance the quality and efficiency of optical-resolution photoacoustic microscopy.

We propose a high-speed all-optic dual-modal system that integrates spectral-domain optical coherence tomography and photoacoustic microscopy (PAM). A 3 ∗ 3 coupler-based interferometer is used to remotely detect the surface vibration caused by photoacoustic (PA) waves. Three outputs of the interferometer are acquired simultaneously with a multi-channel data acquisition card. One channel data with the highest PA signal detection sensitivity is selected for sensitivity compensation. Experiment on the phantom demonstrates that the proposed method can successfully compensate for the loss of intensity caused by sensitivity variation. The imaging speed of the PAM is improved compared to our previous system. The total time to image a sample with 2 5 6 × 2 5 6 pixels is ∼ 2 0 s. Using the proposed system, the microvasculature in the mouse auricle is visualized and the blood flow state is accessed. [ABSTRACT FROM AUTHOR]

The high-quality imaging of vascular networks in biological tissue is significant to accurate cancer diagnosis with acoustic-resolution-based photoacoustic microscopy (AR-PAM). So far, many new back-projection (BP) models have been proposed to improve the image quality of AR-PAM in the off-focal regions. However, many essential arguments are still open regarding the effectiveness of these methods. To settle these remaining questions and explore the potential and adaptability of these BP methods in vascular network imaging, we conducted extensive simulations of a complex vascular network based on a GPU-based data generation framework. Results show that the SAFT-CF algorithm effectively improves the reconstructed image but mainly highlights point targets. In contrast, the STR-BP algorithm can effectively balance the computational cost, signal-to-noise ratio (SNR), and consistency of target intensity for both point and line targets. Results proved that data interpolation for more A-line numbers would not improve the image quality due to information lost. Thus, the detector number in the scan should be sufficiently large. Results also showed that the STR-BP method improved the PSNR of the image by 4.7 to 7.5 dB, which helps the image withstand a noise level of higher than 25%. The proposed simulation framework and the intuitive findings will guide the design of AR-PAM systems and image reconstruction. [ABSTRACT FROM AUTHOR]