A promising approach for quantifying focal stroke modeling and assessing stroke progression:optical resolution photoacoustic microscopy photothrombosis

To investigate the mechanisms underlying the onset and progression of ischemic stroke,some methods have been proposed that can simultaneously monitor and create embolisms in the animal cerebral cortex.However,these methods often require complex systems and the effect of age on cerebral embolism has not been adequately studied,although ischemic stroke is strongly age-related.In this study,we propose an optical-resolution photoacoustic microscopy-based visualized photothrombosis methodology to create and monitor ischemic stroke in mice simultaneously using a 532 nm pulsed laser.We observed the molding process in mice of different ages and presented age-dependent vascular embolism differentiation.Moreover,we integrated optical coherence tomography angiography to investigate age-associated trends in cerebrovascular variability following a stroke.Our imaging data and quantitative analyses underscore the differential cerebrovascular responses to stroke in mice of different ages,thereby highlighting the technique's potential for evaluating cerebrovascular health and unraveling age-related mechanisms involved in ischemic strokes.

Photoacoustic microscopy based on transparent piezoelectric ultrasound transducers

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.

Spatial weight matrix in dimensionality reduction reconstruction for microelectromechanical system-based photoacoustic microscopy

A micro-electromechanical system(MEMS)scanning mirror accelerates the raster scanning of optical-resolution photoacoustic microscopy(OR-PAM).However,the nonlinear tilt angular-voltage characteristic of a MEMS mirror introduces distortion into the maximum back-projection image.Moreover,the size of the airy disk,ultrasonic sensor properties,and thermal effects decrease the resolution.Thus,in this study,we proposed a spatial weight matrix(SWM)with a dimensionality reduction for image reconstruction.The three-layer SWM contains the invariable information of the system,which includes a spatial dependent distortion correction and 3D deconvolution.We employed an ordinal-valued Markov random field and the Harris Stephen algorithm,as well as a modified delay-and-sum method during a time reversal.The results from the experiments and a quantitative analysis demonstrate that images can be effectively reconstructed using an SWM;this is also true for severely distorted images.The index of the mutual information between the reference images and registered images was 70.33 times higher than the initial index,on average.Moreover,the peak signal-to-noise ratio was increased by 17.08%after 3D deconvolution.This accomplishment offers a practical approach to image reconstruction and a promising method to achieve a real-time distortion correction for MEMS-based OR-PAM.

A SIMULTANEOUS MULTI-PROBE DETECTION LABEL-FREE OPTICAL-RESOLUTION PHOTOACOUSTIC MICROSCOPY TECHNIQUE BASED ON MICROCAVITY TRANSDUCER

We demonstrate the feasibility of simultancous multi-probe detection for an optcal-resolution photoacoustic microscopy(OR-PAM)system.OR-P AM has elicited the attention of biomedical imaging researchers because of its optical absorption contrast and high spatial resolution with great imaging depth.OR-PAM allows label-free and noninvasive imaging by maximizing the optical absorption of endogenous biomolecules.However,given the inadequate absoption of some biomolcules,detection sensitivity at the same incident intensity requires improvement.In this study,a modulated continuous wave with power density less than 3mW/cm^(2)(1/4 of the ANSI safety limit)excited the weak photoacoustic(PA)signals of biological cells.A microcavity traneducer is developed based on the bulk modulus of gas five orders of magnitude lower than that of solid;air pressure variation is inversely proportional to cavity volume at the same temperature increase.Considering that a PA wave expands in various directions,detecting PA signals from different positions and adding them together can increase detection sensitivity and signal-to-noise ratio.Therefore,we employ four detectors to acquire tiny PA signals simul-taneously.Experimental results show that the developed OR-PAM system allows the label-free imaging of cells with weak optical absorption.

In vivo evaluation of laser-induced choroidal neovascularization in rats simultaneously using optical coherence tomography and photoacoustic microscopy

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.

Deep-learning-based motion-correction algorithm in optical resolution photoacoustic microscopy

In this study,we propose a deep-learning-based method to correct motion artifacts in optical resolution photoacoustic microscopy(OR-PAM).The method is a convolutional neural network that establishes an end-to-end map from input raw data with motion artifacts to output corrected images.First,we performed simulation studies to evaluate the feasibility and effectiveness of the proposed method.Second,we employed this method to process images of rat brain vessels with multiple motion artifacts to evaluate its performance for in vivo applications.The results demonstrate that this method works well for both large blood vessels and capillary networks.In comparison with traditional methods,the proposed method in this study can be easily modified to satisfy different scenarios of motion corrections in OR-PAM by revising the training sets.

High-speed all-optic optical coherence tomography and photoacoustic microscopy dual-modal system for microcirculation evaluation

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 interfer-ometer 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 ac-quisition 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 sucessfully 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 256×256 pixels is~20s.Using the proposed system,the microvasculature in the mouse auricle is visualized and the blood flow state is accessed.

Fiber laser technologies for photoacoustic microscopy

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.

High-speed high-resolution laser diode-based photoacoustic microscopy for in vivo microvasculature imaging

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 4.8μm 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.

Reflection-mode photoacoustic microscopy using a hollow focused ultrasound transducer for in vivo imaging of blood vessels

A reflection-mode photoacoustic microscope using a hollow focused ultrasound transducer is developed for highresolution in vivo imaging.A confocal structure of the laser and the ultrasound is used to improve the system resolution.The axial and lateral resolutions of the system are measured to be ～ 32 μm and ～ 58 μm,respectively.Ex vivo and in vivo modes are tested to validate the imaging capability of the photoacoustic microscope.The adjacent vein and artery can be seen clearly from the reconstructed photoacoustic images.The results demonstrate that the reflectionmode photoacoustic microscope can be used for high-resolution imaging of micro-blood vessels,which would be of great benefit for monitoring the neovascularization in tumor angiogenesis.

Simulation Platform of Multi-Pulse Photoacoustic Microscopy Based on K-Space Pseudospectral Method

Photoacoustic imaging has emerged in the past decades. Compared with the traditional medical imaging mode, it has better imaging performance and has great development potential in the field of biological imaging. In traditional photoacoustic microscopy, a single laser pulse is generally used to irradiate the sample to produce photoacoustic signal. And sig-nal-to-noise ratio (SNR) is a very important indicator for photoacoustic im-aging. In order to obtain the image with high SNR, multiple acquisition or increasing laser pulse energy is usually adopted. The former will lead to slower imaging speed, and the latter will lead to photobleaching or pho-totoxicity. Here, we propose multi-pulse photoacoustic microscopy, the photoacoustic signals were stimulated sequentially using multiple laser pulses in each A line data acquisition. In order to verify the feasibility of this method, a multi-pulse photoacoustic imaging simulation platform is established using k-Wave toolbox. The performance of multi-pulse photo-acoustic imaging is verified through the three scanning modes of photoa-coustic microscopy A-scan, B-scan, and C-scan. The results indicate that the SNR is proportion to the number of laser pulses used, high SNR can be achieved by low-energy laser pulse. This work will help to expand the ap-plication of photoacoustic imaging.

Flexible depth-of-focus,depth-invariant resolution photoacoustic microscopy with Airy beam

Optical-resolution photoacoustic microscopy(OR-PAM)has rapidly developed and is capable of characterizing optical absorption properties of biological tissue with high contrast and high resolution(micrometer-scale lateral resolution).However,the conventional excitation source of rapidly diverging Gaussian beam imposes limitations on the depth of focus(DOF)in OR-PAM,which in turn affects the depth-resolving ability and detection sensitivity.Here,we proposed a flexible DOF,depth-invariant resolution photoacoustic microscopy(FDIR-PAM)with nondiffraction of Airy beams.The spatial light modulator was incorporated into the optical pathway of the excitation source with matched switching phase patterns,achieving the flexibly adjustable modulation parameters of the Airy beam.We conducted experiments on phantoms and intravital tissue to validate the effectiveness of the proposed approach for high sensitivity and highresolution characterization of variable topology of tissue,offering a promising DOF of 926μm with an invariant lateral resolution of 3.2μm,which is more than 17-fold larger compared to the Gaussian beam.In addition,FDIR-PAM successfully revealed clear individual zebrafish larvae and the pigment pattern of adult zebrafishes,as well as fine morphology of cerebral vasculature in a large depth range with high resolution,which has reached an evident resolving capability improvement of 62%mean value compared with the Gaussian beam.

In vivo spatial-spectral photoacoustic microscopy enabled by optical evanescent wave sensing

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.

Five-wavelength optical-resolution photoacoustic microscopy of blood and lymphatic vessels

Optical-resolution photoacoustic microscopy(OR-PAM)has been developed for anatomical,functional,and molecular imaging but usually requires multiple scanning for different contrasts.We present five-wavelength OR-PAM for simultaneous imaging of hemoglobin concentration,oxygen saturation,blood flow speed,and lymphatic vessels in single raster scanning.We develop a five-wavelength pulsed laser via stimulated Raman scattering.The five pulsed wavelengths,i.e.,532,545,558,570,and 620∕640 nm,are temporally separated by several hundreds of nanoseconds via different optical delays in fiber.Five photoacoustic images at these wavelengths are simultaneously acquired in a single scanning.The 532-and 620∕640-nm wavelengths are used to image the blood vessels and dye-labeled lymphatic vessels.The blood flow speed is measured by a dual-pulse method.The oxygen saturation is calculated and compensated for by the Grüneisen-relaxation effect.In vivo imaging of hemoglobin concentration,oxygen saturation,blood flow speed,and lymphatic vessels is demonstrated in preclinical applications of cancer detection,lymphatic clearance monitoring,and functional brain imaging.

In vivo volumetric monitoring of revascularization of traumatized skin using extended depth-of-field photoacoustic microscopy

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

Recent advances toward clinical applications of photoacoustic microscopy: a review

Photoacoustic imaging(PAI) is an emerging technology that has been dramatically developed in the last decade. PAI, a combination of optical illumination and ultrasound detection, allows us to achieve fine resolution and obtain fruitful information of endogenous and exogenous chromophores. Among PAI imaging techniques, photoacoustic computed tomography(PACT)has been extensively used in human studies due to its deep tissue penetration(several centimeters). Alternatively, photoacoustic microscopy(PAM) offers higher resolution at the expense of penetration depth, which can also be advantageous in clinics.Recently, there has been increasing attention and studies on PAM of human tissues. In this paper, we will review principles of PAM and its applications to human tissues, including the breast tissue, carotid atheroma tissue, eye, gastrointestinal tissue,ovarian tissue and tooth ex vivo as well as the oral cavity and dermatological tissue in vivo. The paper is closed with the outlook regarding the potential applications of PAM in clinics.

Non-invasive and low-artifact in vivo brain imaging by using a scanning acoustic-photoacoustic dual mode microscopy

Photoacoustic imaging is a potential candidate for in vivo brain imaging,whereas,its imaging performance could be degraded by inhomogeneous multi-layered media,consisted of scalp and skull.In this work,we propose a low-artifact photoacoustic microscopy(LAPAM)scheme,which combines conventional acoustic-resolution photoacoustic microscopy with scanning acoustic microscopy to suppress the reflection artifacts induced by multi-layers.Based on similar propagation characteristics of photoacoustic signals and ultrasonic echoes,the ultrasonic echoes can be employed as the filters to suppress the reflection artifacts to obtain low-artifact photoacoustic images.Phantom experiment is used to validate the effectiveness of this method.Furthermore,LAPAM is applied for in-vivo imaging mouse brain without removing the scalp and the skull.Experimental results show that the proposed method successfully achieves the low-artifact brain image,which demonstrates the practical applicability of LAPAM.This work might improve the photoacoustic imaging quality in many biomedical applications which involve tissues with complex acoustic properties,such as brain imaging through scalp and skull.

3D depth-coded photoacoustic microscopy with a large field of view for human skin imaging

Photoacoustic （PA） microscopy comes with high potential for human skin imaging, since it allows noninvasively high-resolution imaging of the natural hemoglobin at depths of several millimeters. Here, we developed a PA microscopy to achieve high-resolution, high-contrast, and large field of view imaging of skin. A three-dimensional （3D） depth-coding technology was used to encode the depth information in PA images, which is very intuitive for identifying the depth of blood vessels in a two-dimensional image, and the vascular structure can be analyzed at different depths. Imaging results demonstrate that the 3D depth-coded PA microscopy should be translated from the bench to the bedside.

Concurrent photoacoustic and ultrasound microscopy with a coaxial dual-element ultrasonic transducer

Simultaneous photoacoustic and ultrasound(PAUS)imaging has attracted increasing attention in biomedical research to probe the optical and mechanical properties of tissue.However,the resolution for majority of the existing PAUS systems is on the order of 1 mm as the majority are designed for clinical use with low-frequency US detection.Here we developed a concurrent PAUS microscopy that consists of optical-resolution photoacoustic microscopy(OR-PAM)and high-frequency US pulse-echo imaging.This dual-modality system utilizes a novel coaxial dual-element ultrasonic transducer(DE-UST)and provides anatomical and functional information with complementary contrast mechanisms,achieving a spatial resolution of 7μm for PA imaging and 106μm for US imaging.We performed phantom studies to validate the system’s performance.The vasculature of a mouse’s hind paw was imaged to demonstrate the potential of this hybrid system for biomedical applications.

Compact long-working-distance laser-diode-based photoacoustic microscopy with a reflective objective

Photoacoustic microscopy(PAM) has quickly developed into a noninvasive biomedical imaging technique to achieve detection, diagnosis, and monitoring.Compared with Q-switched neodymium-doped yttrium aluminum garnet or optical parametric oscillator lasers, a low-cost and small-size laser diode(LD) used as an alternative light source is conducive to achieving the miniaturization and integration for preclinical transformation.However, the LD’s low peak output power needs the high numerical aperture objective to attain tight focus, which limits the working distance(WD) of the system in only2–3 mm, resulting in not achieving the backward coaxial confocal mode.Here, we present a compact visible LD-based PAM system with a reflective objective to achieve a 22 mm long WD and a 10 μm lateral resolution.Different depth subcutaneous microvascular networks in label-free mouse ears have successfully reappeared in vivo with a signal-to-noise ratio up to14 d B by a confocal alignment.It will be a promising tool to develop into a handy tool for subcutaneous blood vessel imaging.