Optical-resolution Photoacoustic Microscopy Research Articles

Non-telecentric photoacoustic microscopy for multi-scale imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) excels in precisely imaging a biological tissue based on absorption contrast. However, existing OR-PAMs are confined by fixed compromises between spatial resolution and field of view (FOV), preventing the integration of large FOV and local high-resolution within one system. Here, we present a non-telecentric OR-PAM (nTC-PAM) that empowers efficient adaptation of FOV and spatial resolution to match the multi-scale requirement of diverse biological imaging. Our method allows for a large-scale transformation in FOV and even surpassing the nominal FOV of the objective with minimal marginal degradation of the lateral resolution. We demonstrate the advantage of nTC-PAM through multi-scale imaging of the leaf phantom, mouse ear, and cortex. The results reveal that nTC-PAM can switch the FOV and spatial resolution to meet the requirements of different biological tissues, such as large-scale imaging of the whole cerebral cortex and high-resolution imaging of microvascular structures in local brain regions.

Testbed mimicking intracellular environment for optical-resolution photoacoustic microscopy

Photoacoustic imaging (PAI) represents an innovative method that utilizes optics for transmission and acoustic for sensing. Within the realm of PAI modalities, optical-resolution photoacoustic microscopy (OR-PAM) stands out for its ability to achieve high-resolution cellular and intracellular imaging. However, an optical testbed setup emulating the intracellular environment and accelerating the development of cellular-level OR-PAM systems has not been established. In this study, we present a testbed setup designed to mimic intracellular conditions. Assessment of this testbed setup efficacy involved embedding photoacoustic contrasts and imaging them using an OR-PAM system. The imaging results show that our testbed setup can provide instrumental in accurately measuring photoacoustic signals within an intracellular aqueous environment, avoiding risks of the contamination and degradation of the acoustic transducer.

WSA-MP-Net: Weak-signal-attention and multi-scale perception network for microvascular extraction in optical-resolution photoacoustic microcopy

The unique advantage of optical-resolution photoacoustic microscopy (OR-PAM) is its ability to achieve high-resolution microvascular imaging without exogenous agents. This ability has excellent potential in the study of tissue microcirculation. However, tracing and monitoring microvascular morphology and hemodynamics in tissues is challenging because the segmentation of microvascular in OR-PAM images is complex due to the high density, structure complexity, and low contrast of vascular structures. Various microvasculature extraction techniques have been developed over the years but have many limitations: they cannot consider both thick and thin blood vessel segmentation simultaneously, they cannot address incompleteness and discontinuity in microvasculature, there is a lack of open-access datasets for DL-based algorithms. We have developed a novel segmentation approach to extract vascularity in OR-PAM images using a deep learning network incorporating a weak signal attention mechanism and multi-scale perception (WSA-MP-Net) model. The proposed WSA network focuses on weak and tiny vessels, while the MP module extracts features from different vessel sizes. In addition, Hessian-matrix enhancement is incorporated into the pre-and post-processing of the input and output data of the network to enhance vessel continuity. We constructed normal vessel (NV-ORPAM, 660 data pairs) and tumor vessel (TV-ORPAM, 1168 data pairs) datasets to verify the performance of the proposed method. We developed a semi-automatic annotation algorithm to obtain the ground truth for our network optimization. We applied our optimized model successfully to monitor glioma angiogenesis in mouse brains, thus demonstrating the feasibility and excellent generalization ability of our model. Compared to previous works, our proposed WSA-MP-Net extracts a significant number of microvascular while maintaining vessel continuity and signal fidelity. In quantitative analysis, the indicator values of our method improved by about 1.3% to 25.9%. We believe our proposed approach provides a promising way to extract a complete and continuous microvascular network of OR-PAM and enables its use in many microvascular-related biological studies and medical diagnoses.

Photoacoustic microscopy for real-time monitoring of near-infrared optical absorbers inside biological tissue.

We developed a high-speed optical-resolution photoacoustic microscopy (OR-PAM) system using a high-repetition-rate supercontinuum (SC) light source and a two-axes Galvano scanner. The OR-PAM system enabled real-time imaging of optical absorbers inside biological tissues with excellent excitation wavelength tunability. In the near-infrared (NIR) wavelength range, high-speed OR-PAM faces limitations due to the lack of wavelength-tunable light sources. Our study aimed to enable high-speed OR-PAM imaging of various optical absorbers, including NIR contrast agents, and validate the performance of high-speed OR-PAM in the detection of circulating tumor cells (CTCs). A high-repetition nanosecond pulsed SC light source was used for OR-PAM. The excitation wavelength was adjusted by bandpass filtering of broadband light pulses produced by an SC light source. Phantom and in vivo experiments were performed to detect tumor cells stained with an NIR contrast agent within flowing blood samples. The newly developed high-speed OR-PAM successfully detected stained cells both in the phantom and in vivo. The phantom experiment confirmed the correlation between the tumor cell detection rate and tumor cell concentration in the blood sample. The high-speed OR-PAM effectively detected stained tumor cells. Combining high-speed OR-PAM with molecular probes that stain tumor cells in vivo enables in vivo CTC detection.

Unsupervised Learning for Enhanced Computed Photoacoustic Microscopy

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.

Photoacoustic expansion microscopy of melanosomes.

Optical resolution photoacoustic microscopy (OR-PAM) is a hybrid imaging method for visualizing organelles due to the high spatial resolution and abundant optical contrast. Usually, OR-PAM employs high numerical aperture (NA) objectives and high-frequency ultrasonic detectors to resolve three-dimensional (3D) microstructures of cells. Expansion microscopy (ExM) provides a nanoscale resolution by isotropically enlarging cells instead of utilizing ultrahigh NA objectives. In this Letter, we report the development of photoacoustic expansion microscopy (PA-ExM) that combines the advantages of OR-PAM and ExM for 3D organelle imaging using near-infrared light. We evaluate the performance of PA-ExM using label-free melanoma cells, where the image quality of melanosome distributions in expanded cells using a 40× objective is comparable to that of unexpanded cells using an oil-immersed 100× objective. The results suggest that PA-ExM possesses the great potential to study organelles.

Highly sensitive self-focused ultrasound transducer with a bionic back-reflector for multiscale-resolution photoacoustic microscopy.

In this study, we designed a self-focused ultrasonic transducer made of polyvinylidene fluoride (PVDF). This transducer involves a back-reflector, which is modeled after tapetum lucidum in the eyes of some nocturnal animals. The bionic structure reflects the ultrasound, which passes through the PVDF membrane, back to PVDF and provides a second chance for the PVDF to convert the ultrasound to electric signals. This design increases the amount of ultrasound absorbed by the PVDF, thereby improving the detection sensitivity. Both ultrasonic and photoacoustic (PA) experiments were conduct to characterize the performance of the transducer. The results show that the fabricated transducer has a center frequency of 13.07 MHz, and a bandwidth of 96% at -6 dB. With an acoustic numerical aperture (NA) of 0.64, the transducer provides a lateral resolution of 140µm. Importantly, the bionic design improves the detection sensitivity of the transducer about 30%. Finally, we apply the fabricated transducer to optical-resolution (OR) and acoustic-resolution photoacoustic microscopy (AR-PAM) to achieve multiscale-resolution PA imaging. Imaging of the bamboo leaf and the leaf skeleton demonstrates that the proposed transducer can provide high spatial resolution, better imaging intensity and contrast. Therefore, the proposed transducer design will be useful to enhance the performance of multiscale-resolution PAM.

Utilization of Curvelet Transform in Reconstructing Cellular Images for Undersampled Optical-resolution Photoacoustic Microscopy.

We address the problem of limited temporal resolution in optical-resolution microscopy (OR-PAM) for cellular imaging by undersampling and reconstruction. A curvelet transform method in a compressed sensing framework (CS-CVT) was devised to specifically reconstruct the boundary and separability of cells object in an image. The performance of CS-CVT approach was justified by comparisons with the natural neighbor interpolation (NNI) followed by smoothing filters on various imaging objects. In addition, a full-raster scanned image was provided as reference. In terms of structure, CS-CVT produces cellular images with smoother boundary but less aberration. We found the strength of CS-CVT in recovering high frequency that is important in representing sharp edges which often missing in typical smoothing filter. In a noisy environment, CS-CVT was less affected by the noise compared to NNI with smoothing filter. Furthermore, CS-CVT could attenuate noise beyond the full raster scanned image. By considering the finest structure in the cellular image, CS-CVT was performing well with minimum range of undersampling around 5% to 15%. In practice, this undersampling were easily translates into 8- to 4-fold faster OR-PAM imaging. In summary, our approach improves temporal resolution of OR-PAM without significant trade-off in image quality.

High-speed adaptive photoacoustic microscopy

Optical-resolution photoacoustic microscopy (OR-PAM) is capable of observing the distribution of optical absorbers inside bio-tissues with a high spatial resolution of micrometers. Unfortunately, due to the employment of a tight optical focus, it suffers from a limited depth of field (DOF), making it challenging to achieve high-resolution imaging of targets with arbitrary surfaces. Here, we propose a high spatiotemporal adaptive photoacoustic focusing mechanism through integrating a high-speed optical focuser, a time-of-flight contour deriving algorithm, and the rotary-scanning photoacoustic microscopy. The developed system, named high-speed adaptive photoacoustic microscopy (HA-PAM), features an ultrashort focus-shifting time of 5 ms and an enlarged DOF of up to 5 mm. With the assistance of the proposed mechanism, we can achieve a homogeneous lateral resolution of 6 μm over a 10 mm circular imaging domain within 5 s. We demonstrate the advantages of HA-PAM through imaging phantoms with curved surfaces, subcutaneous tumor-bearing mice, resected rabbit kidneys, and pulsating mouse brains. The imaging results suggest that this approach provides a high and consistent spatial resolution for imaging bio-tissues with arbitrary surfaces without sacrificing the imaging speed, and has the potential to extend the fundamental and clinical applications of OR-PAM.

In vivo structural and functional imaging of human nailbed microvasculature using photoacoustic microscopy.

Monitoring microvascular structure and function is of great significance for the diagnosis of many diseases. In this study, we demonstrate the feasibility of OR-PAM to nailbed microcirculation detection as a new, to the best of our knowledge, application scenario in humans. We propose a dual-wavelength optical-resolution photoacoustic microscopy (OR-PAM) with improved local-flexible coupling to image human nailbed microvasculature. Microchip lasers with 532 nm wavelength are employed as the pump sources. The 558 nm laser is generated from the 532 nm laser through the stimulated Raman scattering effect. The flowing water, circulated by a peristaltic pump, maintains the acoustic coupling between the ultrasonic transducer and the sample. These designs improve the sensitivity, practicality, and stability of the OR-PAM system for human in vivo experiments. The imaging of the mouse ear demonstrates the ability of our system to acquire structural and functional information. Then, the system is applied to image human nailbed microvasculature. The imaging results reveal that the superficial capillaries are arranged in a straight sagittal pattern, approximately parallel to the long axis of the finger. The arterial and venular limbs are distinguished according to their oxygen saturation differences. Additionally, the images successfully discover the capillary loops with single or multiple twists, the oxygen release at the end of the capillary loop, and the changes when the nailbed is abnormal.

Noise insensitive volumetric fusion method for enhanced photoacoustic microscopy.

Photoacoustic imaging is an emerging imaging modality that combines the high contrast of optical imaging and the high penetration of acoustic imaging. However, the strong focusing of the laser beam in optical-resolution photoacoustic microscopy (OR-PAM) leads to a limited depth-of-field (DoF). Here, a volumetric photoacoustic information fusion method was proposed to achieve large volumetric photoacoustic imaging at low cost. First, the initial decision map was built through the focus detection based on the proposed three-dimensional Laplacian operator. Majority filter-based consistency verification and Gaussian filter-based map smoothing were then utilized to generate the final decision map for the construction of photoacoustic imaging with extended DoF. The performance of the proposed method was tested to show that our method can expand the limited DoF by a factor of 1.7 without the sacrifice of lateral resolution. Four sets of multi-focus vessel data at different noise levels were fused to verify the effectiveness and robustness of the proposed method. The proposed method can efficiently extend the DoF of OR-PAM under different noise levels.

Long-term assessment of cutaneous inflammation and treatment using optical resolution photoacoustic microscopy.

Cutaneous inflammation is an acute skin disease characterized by edema and vascular hyperplasia. Longitudinal monitoring of vasculature is crucial for studying the development of inflammation and evaluating the therapeutic efficacy of drugs. Optical-resolution photoacoustic microscopy (OR-PAM) is a hybrid imaging tool for non-invasive and label-free visualization of microcirculations with a capillary-scale spatial resolution. In this study, we assess the feasibility of OR-PAM for long-term monitoring of vascular changes in 12-O-Tetradecanoylphorbol-13-Acetate (TPA)-induced mouse models, as well as the corresponding treatment process. Quantitative vascular evaluation is conducted based on derived key parameters, including vessel length, branchpoint number, vessel area fraction, vessel diameter, fractal dimension, vessel tortuosity and ear thickness, which reveal that vascular morphological changes are highly dependent on the concentration of TPA and existence of therapeutic drugs. Furthermore, the results show the potential of OR-PAM in the clinical management of inflammation and as an effective tool to evaluate vascular responses to pharmacological interventions in vivo.

Noise-insensitive defocused signal and resolution enhancement for optical-resolution photoacoustic microscopy via deep learning.

Optical-resolution photoacoustic microscopy suffers from narrow depth of field and a significant deterioration in defocused signal intensity and spatial resolution. Here, a method based on deep learning was proposed to enhance the defocused resolution and signal-to-noise ratio. A virtual optical-resolution photoacoustic microscopy based on k-wave was used to obtain the datasets of deep learning with different noise levels. A fully dense U-Net was trained with randomly distributed sources to improve the quality of photoacoustic images. The results show that the PSNR of defocused signal was enhanced by more than 1.2 times. An over 2.6-fold enhancement in lateral resolution and an over 3.4-fold enhancement in axial resolution of defocused regions were achieved. The large volumetric and high-resolution imaging of blood vessels further verified that the proposed method can effectively overcome the deterioration of the signal and the spatial resolution due to the narrow depth of field of optical-resolution photoacoustic microscopy.

Automated Laser-Fiber Coupling Module for Optical-Resolution Photoacoustic Microscopy.

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.

High-resolution in vivo imaging of human nailbed microvasculature by using photoacoustic microscopy.

Microcirculation imaging has significantly clinical value in early diagnosis and curative effect judgment of various diseases. The most superficial layer of the nailbed is rich in capillaries, which is suitable as a window on the microcirculation. However, few techniques can noninvasively observe the blood supply distribution of the nailbed, especially for high-resolution imaging of capillaries. In this study, we adapted an optical-resolution photoacoustic microscopy (OR-PAM) to image the nailbed microvasculature. The imaging sensitivity was significantly improved by hydration pretreatment of the nail. In vitro phantom experiments demonstrate that the sensitivity was improved about 3.5 times after hydration. In vivo imaging experiments of the nailbed microvasculature were conducted to further examine the enhanced sensitivity and practicability of OR-PAM. Moreover, the quantitative analysis of capillary loops showed that OR-PAM can extract the detection indicators including vascular morphology, diameter, and length, which provides a basis for clinical microcirculation detection using OR-PAM. This article is protected by copyright. All rights reserved.

A fiber-coupled laser diode design for reflection mode optical resolution photoacoustic microscopy

Pulsed Laser Diodes (PLD) are compact and high pulse repetition rate laser sources that show a great potential for low-cost Optical Resolution Photoacoustic Microscopes (OR-PAM). Nevertheless, their non-uniform multimode laser beams are of low quality so that high lateral resolutions with tightly focused beams are difficult to realize at long focusing distances, as required for reflection mode OR-PAM devices of clinical application. A new strategy based on homogenizing and shaping the laser diode beam with a square-core multimode optical fiber allowed to attain competitive lateral resolutions while keeping one centimeter working distance. The theoretical expressions for the laser spot size, determining optical lateral resolution, and the depth of focus are also written for general multimode beams. An OR-PAM system was built in confocal reflection mode with a linear phased-array as the ultrasound receiver in order to test its performance, first, on a resolution test target and, afterwards, on ex vivo rabbit ears to show the system potential for subcutaneous imaging of blood vessels and hair follicles.

In vivo quantitative photoacoustic monitoring of corticosteroid-induced vasoconstriction.

Corticosteroids-commonly prescribed medications for skin diseases-inhibit the secretion of vasodilators, such as prostaglandin, thereby exerting anti-inflammatory action by constricting capillaries in the dermis. The effectiveness of corticosteroids is determined by the degree of vasoconstriction followed by skin whitening, namely, the blanching effect. However, the current method of observing the blanching effect indirectly evaluates the effects of corticosteroids. In this study, we employed optical-resolution photoacoustic (PA) microscopy (OR-PAM) to directly visualize the blood vessels and quantitatively evaluate vasoconstriction. Using OR-PAM, the vascular density in mice skin was monitored for 60min after performing each experimental procedure for four groups, and the vasoconstriction was quantified. Volumetric PA data were segmented into the papillary dermis, reticular dermis, and hypodermis based on the vascular characteristics obtained through OR-PAM. The vasoconstrictive effect of each skin layer was quantified according to the dermatological treatment method. In the case of corticosteroid topical application, vasoconstriction was observed in the papillary ( ) and reticular ( ) dermis. For corticosteroid subcutaneous injection, constriction was observed solely in the reticular ( ) dermis. In contrast, no vasoconstrictions were observed with nonsteroidal topical application. Our results indicate that OR-PAM can quantitatively monitor the vasoconstriction induced by corticosteroids, thereby validating OR-PAMs potential as a practical evaluation tool for predicting the effectiveness of corticosteroids in dermatology.

High resolution intravital photoacoustic microscopy reveals VEGF-induced bone regeneration in mouse tibia.

Osteogenesis and angiogenesis are essential for bone homeostasis and repair. Newly formed vessels convey osteogenic progenitors during bone regeneration. However, the lack of continuous and label-free visualization of the bone microvasculature has resulted in little understanding of the neovascular dynamics. Here, we take advantage of optical-resolution photoacoustic microscopy (ORPAM) for label-free, intravital, long-term observation of the bone vascular dynamics, including angiogenesis, remodeling and quantified angiogenic effect of locally-applied vascular endothelial growth factor (VEGF) in the murine tibial defect model. We employed ex vivo confocal microscopy and micro-computed tomography (micro-CT) imaging to verify the positive role of VEGF treatment. VEGF treatment increased the concentration of total hemoglobin, vascular branching, and vascular density, which correlated with more osteoprogenitors and increased bone formation within the defect. These data demonstrated ORPAM as a useful imaging tool that detected functional capillaries to understand hemodynamics, and revealed the effectiveness of locally delivered therapeutic agents with sufficient sensitivity, contributing to the understanding of spatiotemporal regulatory mechanisms on blood vessels during bone regeneration.

Single-Pixel Photoacoustic Microscopy with Speckle Illumination

Wide-field optical-resolution microscopy with structured illumination and single-pixel detection has been the topic of a number of research investigations. Its advantages over point scanning approaches are many and include a faster acquisition rate for sparse samples, sectioning, and super-resolution features. Initially introduced for fluorescence imaging, structured illumination approaches have been adapted and developed for many other imaging modalities. In this paper, we illustrate how speckle illumination, as a particular type of structured illumination, can be exploited to perform optical-resolution photoacoustic microscopy with a single-pixel imaging approach. We first introduce the principle of single-pixel detection applied to photoacoustic imaging and then illustrate in 2 different situations how photoacoustic images may be computationally reconstructed from speckle illumination: In the first situation where the speckle patterns are known through a prior calibration, various reconstruction approaches may be implemented, which are demonstrated experimentally through both scattering layers and multimode optical fibers; in the second situation where the speckle patterns are unknown (blind structured illumination), the so-called memory effect can be harnessed to produce calibration-free photoacoustic images, following the approach initially proposed for fluorescence imaging through thin scattering layers.

Super-resolution reconstruction algorithm for optical-resolution photoacoustic microscopy images based on sparsity and deconvolution.

The lateral resolution of the optical-resolution photoacoustic microscopy (OR-PAM) system depends on the focusing diameter of the probe beam. By increasing the numerical aperture (NA) of optical focusing, the lateral resolution of OR-PAM can be improved. However, the increase in NA results in smaller working distances, and the entire imaging system becomes very sensitive to small optical imperfections. The existing deconvolution-based algorithms are limited by the image signal-to-noise ratio when improving the resolution of OR-PAM images. In this paper, a super-resolution reconstruction algorithm for OR-PAM images based on sparsity and deconvolution is proposed. The OR-PAM image is sparsely reconstructed according to the constructed loss function, which utilizes the sparsity of the image to combat the decrease in the resolution. The gradient accelerated Landweber iterative algorithm is used to deconvolve to obtain high-resolution OR-PAM images. Experimental results show that the proposed algorithm can improve the resolution of mouse retinal images by approximately 1.7 times without increasing the NA of the imaging system. In addition, compared to the Richardson-Lucy algorithm, the proposed algorithm can further improve the image resolution and maintain better imaging quality, which provides a foundation for the development of OR-PAM in clinical research.