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Abstract The miniature optical fiber ultrasound sensor with high sensitivity and bandwidth is important for the field of ultrasonic detection. In this study, a unique fiber ultrasound sensor via integrating optical microrings on a multicore fiber facet is reported. The results demonstrate this sensor can be used in liquid environment and has high sensitivity with broadband bandwidth. The detector's equivalent noise pressure (NEP) can reach 13.2 mPa Hz−1/2 with a broadband ultrasound response over 250 MHz (103 MHz in −6 dB). Besides, this sensor is resistant to environmental impurities. The sensor is successfully applied to photoacoustic microscopy (PAM) with a lateral resolution of 5 µm and an axial resolution of 6.6 µm. Furthermore, an integrated transceiver structure for ultrasound/photoacoustic detection is proposed, which has great application prospects for using in constrained tiny spaces, such as endoscopy, or closely scanning over uneven surfaces.

Topical corticosteroids manage inflammatory skin conditions via their action on the immune system. An effect of application of corticosteroids to the skin is skin blanching caused by peripheral vasoconstriction. This has been used to characterize, in some cases relative potency and also as a way to compare skin penetration. Chromameters have been used to assess skin blanching—the outcome of vasoconstriction caused by topical corticosteroids—but do not directly measure vasoconstriction. Here, we demonstrate quantitative volumetric photoacoustic microscopy (PAM) as a tool for directly assessing the vasoconstriction followed by topical corticosteroid application, noninvasively visualizing skin vasculature without any exogeneous contrast agent. We photoacoustically differentiated the vasoconstrictive ability of four topical corticosteroids in small animals through multiparametric analyses, offering detailed 3D insights into vasoconstrictive mechanisms across different skin depths. Our findings highlight the potential of PAM as a noninvasive tool for measurement of comparative vasoconstriction with potential for clinical, pharmaceutical, and bioequivalence applications.

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.

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 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.

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

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.

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.

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.

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.

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.

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.

Photoacoustic imaging (PAI) is a noninvasive biomedical imaging technology capable of multiscale imaging of biological samples from organs down to cells. Multiscale PAI requires different ultrasound transducers that are flat or focused because the current widely-used piezoelectric transducers are rigid and lack the flexibility to tune their spatial ultrasound responses. Inspired by the rapidly-developing flexible photonics, we exploited the inherent flexibility and low-loss features of optical fibers to develop a flexible fiber-laser ultrasound sensor (FUS) for multiscale PAI. By simply bending the fiber laser from straight to curved geometry, the spatial ultrasound response of the FUS can be tuned for both wide-view optical-resolution photoacoustic microscopy at optical diffraction-limited depth (~1 mm) and photoacoustic computed tomography at optical dissipation-limited depth of several centimeters. A radio-frequency demodulation was employed to get the readout of the beat frequency variation of two orthogonal polarization modes in the FUS output, which ensures low-noise and stable ultrasound detection. Compared to traditional piezoelectrical transducers with fixed ultrasound responses once manufactured, the flexible FUS provides the freedom to design multiscale PAI modalities including wearable microscope, intravascular endoscopy, and portable tomography system, which is attractive to fundamental biological/medical studies and clinical applications.

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.

We propose a high-speed all-optic dual-modal system that integrates spectral-domain optical coherence tomography and photoacoustic microscopy (PAM). A [Formula: see text] 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 [Formula: see text] pixels is [Formula: see text][Formula: see text]s. Using the proposed system, the microvasculature in the mouse auricle is visualized and the blood flow state is accessed.

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.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract 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.

In most multispectral optical-resolution photoacoustic microscopy (OR-PAM), spatial scanning is repeated for each excitation wavelength, which decreases throughput and causes motion artifacts during spectral processing. This study proposes a new spectroscopic OR-PAM technique to acquire information on the photoacoustic signal intensity and excitation wavelength from single spatial scans. The technique involves irradiating an imaging target with two broadband optical pulses with and without wavelength-dependent time delays. The excitation wavelength of the sample is then calculated by measuring the time delay between the photoacoustic signals generated by the two optical pulses. This technique is validated by measuring the excitation wavelengths of dyes in tubes. Furthermore, we demonstrate the three-dimensional spectroscopic OR-PAM of cells stained with suitable dyes. Although the tradeoff between excitation efficiency and excitation bandwidth must be adjusted based on the application, combining the proposed technique with fast spatial scanning methods can significantly contribute to recent OR-PAM applications, such as monitoring quick biological events and microscale tracking of moving materials.

Histological images can reveal rich cellular information of tissue sections, which are widely used by pathologists in disease diagnosis. However, the gold standard for histopathological examination is based on thin sections on slides, which involves inevitable time-consuming and labor-intensive tissue processing steps, hindering the possibility of intraoperative pathological assessment of the precious patient specimens. Here, by incorporating ultraviolet photoacoustic microscopy (UV-PAM) with deep learning, we show a rapid and label-free histological imaging method that can generate virtually stained histological images (termed Deep-PAM) for both thin sections and thick fresh tissue specimens. With the tissue non-destructive nature of UV-PAM, the imaged intact specimens can be reused for other ancillary tests. We demonstrated Deep-PAM on various tissue preparation protocols, including formalin-fixation and paraffin-embedding sections (7-µm thick) and frozen sections (7-µm thick) in traditional histology, and rapid assessment of intact fresh tissue (~ 2-mm thick, within 15 min for a tissue with a surface area of 5 mm × 5 mm). Deep-PAM potentially serves as a comprehensive histological imaging method that can be simultaneously applied in preoperative, intraoperative, and postoperative disease diagnosis.

Optical-resolution photoacoustic microscopy (OR-PAM) enjoys superior spatial resolution and has received intense attention in recent years. The application, however, has been limited to shallow depths because of strong scattering of light in biological tissues. In this work, we propose to achieve deep-penetrating OR-PAM performance by using deep learning enabled image transformation on blurry living mouse vascular images that were acquired with an acoustic-resolution photoacoustic microscopy (AR-PAM) setup. A generative adversarial network (GAN) was trained in this study and improved the imaging lateral resolution of AR-PAM from 54.0 µm to 5.1 µm, comparable to that of a typical OR-PAM (4.7 µm). The feasibility of the network was evaluated with living mouse ear data, producing superior microvasculature images that outperforms blind deconvolution. The generalization of the network was validated with in vivo mouse brain data. Moreover, it was shown experimentally that the deep-learning method can retain high resolution at tissue depths beyond one optical transport mean free path. Whilst it can be further improved, the proposed method provides new horizons to expand the scope of OR-PAM towards deep-tissue imaging and wide applications in biomedicine.

Near-infrared photoacoustics receives increasing interest as an intravital modality to sense key biomolecules. One of the most central types of biomolecules of interest are lipids as they constitute essential bio-hallmarks of cardiovascular and metabolic diseases and their in-vivo detection holds insightful information about disease progression and treatment monitoring. However, the full potential of near-infrared photoacoustic for high-resolution and high-sensitivity biomedical studies of lipids has so far not been exploited due a lack of appropriate excitation sources delivering short-pulses at high-repetition-rate, high-pulse-energy, and wavelength around 1200 nm. Here, we demonstrate a custom-built SRS fiber amplifier that provides optical excitations at 1192.8 nm, repetition rates of 200 kHz, pulse durations below 2 ns, and pulse energies beyond 5 μJ. We capitalize on the performance of our excitation source and show near-infrared photoacoustics resolving intrinsic lipid contrast in biomedically relevant specimens ranging from single cells to lipid-rich tissue with subcellular resolution.

Ultraviolet photoacoustic microscopy (UV-PAM) has been investigated to provide label-free and registration-free volumetric histological images for whole organs, offering new insights into complex biological organs. However, because of the high UV absorption of lipids and pigments in tissue, UV-PAM suffers from low image contrast and shallow image depth, hindering its capability for revealing various microstructures in organs. To improve the UV-PAM imaging contrast and imaging depth, here we propose to implement a state-of-the-art optical clearing technique, CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis), to wash out the lipids and pigments from tissues. Our results show that the UV-PAM imaging contrast and quality can be significantly improved after tissue clearing. With the cleared tissue, multilayers of cell nuclei can also be extracted from time-resolved PA signals. Tissue clearing-enhanced UV-PAM can provide fine details for organ imaging.

The investigation of neuronal activity in non-human primate models is of critical importance due to their genetic similarity to human brains. In this study, we tested the feasibility of using photoacoustic imaging for the detection of cortical and subcortical responses due to peripheral electrical stimulation in a squirrel monkey model. Photoacoustic computed tomography and photoacoustic microscopy were applied on squirrel monkeys for real-time deep subcortical imaging and optical-resolution cortical imaging, respectively. The electrically evoked hemodynamic changes in primary somatosensory cortex, premotor cortices, primary motor cortex, and underlying subcortical areas were measured. Hemodynamic responses were observed in both cortical and subcortical brain areas at the cortices during external stimulation, demonstrating the feasibility of photoacoustic technique for functional imaging of non-human primate brain.

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.

In recent years, many methods have been investigated to improve imaging speed in photoacoustic microscopy (PAM). These methods mainly focused upon three critical factors contributing to fast PAM: laser pulse repetition rate, scanning speed, and computing power of the microprocessors. A high laser repetition rate is fundamentally the most crucial factor to increase the PAM speed. In this paper, we review methods adopted for fast PAM systems in detail, specifically with respect to light sources. To the best of our knowledge, ours is the first review article analyzing the fundamental requirements for developing high-speed PAM and their limitations from the perspective of light sources.

High-speed optical-resolution photoacoustic microscopy (OR-PAM), integrating the merits of high spatial resolution and fast imaging acquisition, can observe dynamic processes of the optical absorption-based molecular specificities. However, it remains challenging for the evaluation to morphological and physiological parameters that are closely associated with photoacoustic spectrum due to the inadequate ultrasonic frequency response of the routinely-employed piezoelectric transducer. By utilizing the galvanometer for fast optical scanning and our previously-developed surface plasmon resonance sensor as an unfocused broadband ultrasonic detector, high-speed spectroscopic photoacoustic imaging was accessed in the OR-PAM system, achieving an acoustic bandwidth of ∼125 MHz and B-scan rate at ∼200 Hz over a scanning range of ∼0.5 mm. Our system demonstrated the dynamic imaging of the moving phantoms’ structures and the simultaneous characterization of their photoacoustic spectra over time. Further, fast volumetric imaging and spectroscopic analysis of microanatomic features of a zebrafish eye ex vivo was obtained label-freely.

Optical-resolution photoacoustic microscopy (OR-PAM) can provide functional, anatomical, and molecular images at micrometer level resolution with an imaging depth of less than 1 mm in tissue. However, the imaging speed of traditional OR-PAM is often low due to the point-by-point mechanical scanning and cannot capture time-sensitive dynamic information. In this work, we demonstrate a recent effort in improving the imaging speed of OR-PAM, using a newly developed water-immersible two-axis scanner. Driven by water-compatible electromagnetic actuation force, the new scanning mirror employs a novel torsion-bending mechanism to achieve fast 2D scanning. The torsion scanning along the fast-axis works in the resonant model, and the bending scanning along the slow-axis operate at the quasi-static mode. The scanning speed and scanning range along the two axes can be independently adjusted. Steered by the two-axis torsion-bending scanning mirror immersed in water, the focused excitation light and the generated acoustic wave can be confocally aligned over the entire imaging area. Thus, a high imaging speed can be achieved without sacrificing the detection sensitivity. Equipped with the torsion-bending scanner, the high-speed OR-PAM system has achieved a cross-sectional frame rate of 400 Hz, and a volumetric imaging speed of 1 Hz over a field of view of 1.5 × 2.5 mm2. We have also demonstrated high-speed OR-PAM of the hemodynamic changes in response to pharmaceutical and physiological challenges in small animal models in vivo. We expect the torsion-bending scanner based OR-PAM will find matched biomedical studies of tissue dynamics.

Photoacoustic (PA) microscopy (PAM) has achieved remarkable progress in biomedicine in the past decade. It is a fast-rising imaging modality with diverse applications, such as hemodynamics, oncology, metabolism, and neuroimaging. Combining optical excitation and acoustic detection, the hybrid nature of PAM provides advantages of rich contrast and deep penetration. In recent years, high-speed PAM has flourished and enabled high‐speed wide‐field imaging of functional activity. In this review, we summarize the most recent advances in high-speed PAM technologies, including high-repetition-rate multi-wavelength laser development, fast scanning techniques, and novel PA signal acquisition strategies.

Functional imaging of microvascular dynamics in extremities delivers intuitive information for early detection, diagnosis, and prognosis of vascular diseases. High-resolution and high-speed photoacoustic microscopy (PAM) visualizes and measures multiparametric information of microvessel networks in vivo such as morphology, flow, oxygen saturation, and metabolic rate. Here, we demonstrate high-resolution photoacoustic monitoring of vascular dynamics in human fingers. We photoacoustically monitored the position displacement of blood vessels associated with arterial pulsation in human fingers. Then, during and after arterial occlusion, we photoacoustically quantified oxygen consumption and blood perfusion in the fingertips. The results demonstrate that high-resolution functional PAM could be a vital tool in peripheral vascular examination for measuring heart rate, oxygen consumption, and/or blood perfusion.

The point-by-point scanning mechanism of photoacoustic microscopy (PAM) results in low-speed imaging, limiting the application of PAM. In this work, we propose a method to improve the quality of sparse PAM images using convolutional neural networks (CNNs), thereby speeding up image acquisition while maintaining good image quality. The CNN model utilizes attention modules, residual blocks, and perceptual losses to reconstruct the sparse PAM image, which is a mapping from a 1/4 or 1/16 low-sampling sparse PAM image to a latent fully-sampled one. The model is trained and validated mainly on PAM images of leaf veins, showing effective improvements quantitatively and qualitatively. Our model is also tested using in vivo PAM images of blood vessels of mouse ears and eyes. The results suggest that the model can enhance the quality of the sparse PAM image of blood vessels in several aspects, which facilitates fast PAM and its clinical applications.

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 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.

Determination of the precise location and the degree of the Choroidal neovascularization (CNV) lesion is essential for diagnosation Neovascular age-related macular degeneration (AMD) and evaluation the efficacy of treatment. Noninvasive imaging techniques with specific contrast for CNV evaluation are demanded. In this paper, two noninvasive imaging techniques, namely Optical coherence tomography (OCT) and Photoacoustic microscopy (PAM), are combined to provide specific detection of CNV for their complimentary contrast mechanisms. In vivo time-serial evaluation of Laser-induced CNV in rats is present at days 1, 3, 5, 7, 14, 21 after laser photocoagulation is applied to the rat fundus. Both OCT and PAM show that the CNV increases to its maximum at day 7 and decreases at day 14. Quantification of CNV area and CNV thickness is given. The dual-modal information of CNV is consistent with the histologic evaluation by hematoxylin and eosin (H&E) staining.

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.

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.

The goal of this study is to further develop a multi-modality eye imaging system and evaluate its feasibility of acquiring images of different modalities simultaneously. An integrated multimodality imaging system combining spectral-domain optical coherence tomography (SD-OCT), photoacoustic microscopy (PAM), and fluorescence microscopy (FM) was developed, and its performance for eye imaging was validated on multiple clinically-relevant retinal disease models in vivo in rabbits. OCT imaging allows for visualization of the different anatomic retinal layers with high axial resolution. PAM can be used to image vasculature, angiogenesis, and hemorrhages. The leakage of neovascularization can be verified with FM and fluorescein dye. Simultaneous imaging with OCT, PAM, and FM ensures co-registration of the three modalities without being affected by motion artifacts caused by breathing, body or eye movements, and heartbeat. This simultaneous multi-modality eye imaging system could be a new tool for applications both in ophthalmology and other fields.

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.