Photoacoustic Microscopy System Research Articles

Multimodal photoacoustic microscopy and optical coherence tomography ocular biomarker imaging in Alzheimer's disease in mice.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by amyloid beta (Aβ)-containing extracellular plaques and tau-containing intracellular neurofibrillary tangles. Reliable and more accessible biomarkers along with associated imaging methods are essential for early diagnosis and to develop effective therapeutic interventions. Described here is an integrated photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality imaging system for multiple ocular biomarker imaging in an AD mouse model. Anti-Aβ-conjugated Au nanochains (AuNCs) were engineered and administered to the mice to provide molecular contrast of Aβ. The retinal vasculature structure and Aβ deposition in AD mice and wild-type (WT) mice were imaged simultaneously by dual-wavelength PAM. OCT distinguished significant differences in retinal layer thickness between AD and WT animals. With the unique ability of imaging the multiple ocular biomarkers via a coaxial multimodality imaging system, the proposed system provides a new tool for investigating the progression of AD in animal models, which could contribute to preclinical studies of AD.

A portable photoacoustic microscopy and ultrasound system for rectal cancer imaging

Photoacoustic microscopy offers functional information regarding tissue vasculature while ultrasound characterizes tissue structure. Combining these two modalities provides novel clinical applications including response assessment among rectal cancer patients undergoing therapy. We have previously demonstrated the capabilities of a co-registered photoacoustic and ultrasound device in vivo, but multiple challenges limited broad adoption. In this paper, we report significant improvements in an acoustic resolution photoacoustic microscopy and ultrasound (ARPAM/US) system characterized by simulation and phantom study, focusing on resolution, optical coupling, and signal characteristics. In turn, higher in-probe optical coupling efficiency, higher signal-to-noise ratio, higher data throughput, and better stability with minimal maintenance requirements were all accomplished. We applied the system to 19 ex vivo resected colorectal cancer samples and found significantly different signals between normal, cancer, and post-treatment tumor tissues. Finally, we report initial results of the first in vivo imaging study.

Deep Learning‐Enhanced Fiber‐Coupled Laser Diode‐Based Photoacoustic Microscopy for High‐Resolution Microvasculature Imaging

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

Enhancing vascular network visualization in 3D photoacoustic imaging: in vivo experiments with a vasculature filter.

Filter-based vessel enhancement algorithms facilitate the extraction of vascular networks from medical images. Traditional filter-based algorithms struggle with high noise levels in images with false vessel extraction, and a low standard deviation (σ) value may introduce gaps at the centers of wide vessels. In this paper, a robust technique with less sensitivity to parameter tuning and better noise suppression than other filter-based methods for two-dimensional and three-dimensional images is implemented. In this study, we propose a filter that employs non-local means (NLM) for denoising, applying the vesselness function to suppress blob-like structures and filling the gaps in wide vessels without compromising edge quality or details. Acoustic resolution photoacoustic microscopy (AR-PAM) systems generate high-resolution volumetric photoacoustic images, but their vascular structure imaging suffers from out-of-focal signal-to-noise ratio (SNR) and lateral resolution loss. Implementing a synthetic aperture focusing technique (SAFT) based on a virtual detector (VD) improves out-of-focal region resolution and SNR. Combining the proposed filter with the SAFT algorithm enhances vascular structural imaging in AR-PAM systems. The proposed method is robust and applicable for animal tissues with less error of vasculature structure extraction in comparison to traditional fliter-based methods like Frangi and Sato filter. Also, the method is faster in terms of processing speed and less tuning parameters. We applied the method to a digital phantom to validate our approach and conducted in vivo experiments to demonstrate its superiority for real volumetric tissue imaging.

Contrast-enhanced near-infrared photoacoustic microscopy and optical coherence tomography imaging of rat fundus.

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.

Miniaturized preamplifier integration in ultrasound transducer design for enhanced photoacoustic imaging.

Photoacoustic imaging (PAI) utilizes the photoacoustic effect to record both vascular and functional characteristics of a biological tissue. Photoacoustic signals have typically low amplitude that cannot be read efficiently by data acquisition systems. This necessitates the use of one or more amplifiers. These amplifiers are somewhat bulky (e.g., the ZFL-500LN+, Mini-Circuits, USA, or 351A-3-50-NI, Analog Modules Inc., USA). Here, we describe the fabrication and development process of a transducer with a built-in low-noise preamplifier that is encased within the transducer housing. This new, to the best of our knowledge, design could be advantageous for applications where a compact transducer + preamplifier is required. We demonstrate the performance of this compact detection unit in a laser scanning photoacoustic microscopy system by imaging a rat ear ex vivo and a rat brain vasculature in vivo.

Efficient Assessment of Tumor Vascular Shutdown by Photodynamic Therapy on Orthotopic Pancreatic Cancer Using High-Speed Wide-Field Waterproof Galvanometer Scanner Photoacoustic Microscopy.

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

Photoacoustic microscopy 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.

Acoustic resolution photoacoustic Doppler flowmetry for assessment of patient rectal cancer blood perfusion.

Photoacoustic Doppler flowmetry offers quantitative blood perfusion information in addition to photoacoustic vascular contrast for rectal cancer assessment. We aim to develop and validate a correlational Doppler flowmetry utilizing an acoustic resolution photoacoustic microscopy (AR-PAM) system for blood perfusion analysis. To extract blood perfusion information, we implemented AR-PAM Doppler flowmetry consisting of signal filtering and conditioning, A-line correlation, and angle compensation. We developed flow phantoms and contrast agent to systemically investigate the flowmetry's efficacy in a series of phantom studies. The developed correlational Doppler flowmetry was applied to images collected during in vivo AR-PAM for post-treatment rectal cancer evaluation. The linearity and accuracy of the Doppler flow measurement system were validated in phantom studies. Imaging rectal cancer patients treated with chemoradiation demonstrated the feasibility of using correlational Doppler flowmetry to assess treatment response and distinguish residual cancer from cancer-free tumor bed tissue and normal rectal tissue. A new correlational Doppler flowmetry was developed and validated through systematic phantom evaluations. The results of its application to in vivo patients suggest it could be a useful addition to photoacoustic endoscopy for post-treatment rectal cancer assessment.

3D imaging of aqueous veins and surrounding sclera using a dual-wavelength photoacoustic microscopy.

Understanding aqueous outflow resistance at the level of aqueous veins has been a challenge to the management of glaucoma. This study investigated resolving the anatomies of aqueous veins and the textures of surrounding sclera using photoacoustic microscopy (PAM). A dual wavelength PAM system was established and validated using imaging phantoms, porcine and human globes perfused with an optical contrast agent ex vivo. The system shows lateral resolution of 8.23 µm and 4.70 µm at 1200 nm and 532 nm, respectively, and an axial resolution of 27.6 µm. The system is able to separately distinguish the aqueous veins and the sclera with high contrast in full circumference of the porcine and human globes.

Review of low-cost light sources and miniaturized designs in photoacoustic microscopy.

Photoacoustic microscopy (PAM) is a promising imaging technique to provide structural, functional, and molecular information for preclinical and clinical studies. However, expensive and bulky lasers and motorized stages have limited the broad applications of conventional PAM systems. A recent trend is to use low-cost light sources and miniaturized designs to develop a compact PAM system and expand its applications from benchtop to bedside. We provide (1)an overview of PAM systems and their limitations, (2)a comprehensive review of PAM systems with low-cost light sources and their applications, (3)a comprehensive review of PAM systems with miniaturized and handheld scanning designs, and (4)perspective applications and a summary of the cost-effective and miniaturized PAM systems. Papers published before July 2023 in the area of using low-cost light sources and miniaturized designs in PAM were reviewed. They were categorized into two main parts: (1)low-cost light sources and (2)miniaturized or handheld designs. The first part was classified into two subtypes: pulsed laser diode and continuous-wave laser diode. The second part was also classified into two subtypes: galvanometer scanner and micro-electro-mechanical system scanner. Significant progress has been made in the development of PAM systems based on low-cost and compact light sources as well as miniaturized and handheld designs. The review highlights the potential of these advancements to revolutionize PAM technology, making it more accessible and practical for various applications in preclinical studies, clinical practice, and long-term monitoring.

An Optically Transparent Focused P(VDF-TrFE) Transducer for Photoacoustic Microscopy (PAM)

This paper reports a new optically-transparent focused P(VDF-TrFE) (poly(vinylidene fluoride-co-trifluoroethylene)) transducer for photoacoustic microscopy (PAM), which is fabricated by a new process based on pre-cutting and direct-lamination. Compared with the previous fabrication process, it is simpler, and makes it possible to achieve a high numerical aperture (NA) without stretching the (brittle) piezoelectric film. For demonstration, a prototype transducer has been fabricated with a 10-μm-thick 70/30 P(VDF-TrFE) film laminated onto a plano-concave glass lens with an acoustic NA of 0.64. Experimental characterizations show that the transducer has an optical transmittance of 88.6% (@ 532 nm), an acoustic center frequency and -3 dB bandwidth of 24 MHz and 29 MHz, respectively. Using the new optically-transparent focused P(VDF-TrFE) transducer, a PAM setup has been built to carry imaging experiments on different targets. Based on the experimental results, it can be concluded that the optically-transparent focused P(VDF-TrFE) transducer could be useful for the development of new PAM systems for different imaging applications.

Photoacoustic microscopy system for biological tissue imaging

A photoacoustic microscopy system was created and examined for biological tissue. Characterization of microphone, laser, and motor stepper was done to get the most appropriate setting for the system. The object table is set in a static state in the scanning process while the radiation sources and detector move in the X-Y direction. The hardware components in this system consist of photoacoustic signal generation components, mechanical components, and photoacoustic signal detection components. The hardware components are integrated and controlled by the computer through the Raspberry-pi microcomputer software. The characterization results of the photoacoustic image system obtained the optimum system setting for detecting biological tissue, namely the frequency of the diode laser in the range of 19 kHz with a duty cycle of 30%, with a minimum motor shift of 0.2 mm. This system can image the same biological tissue as the original sample with good image contrast. The smallest sample that can be scanned is 0.75 mm. The photoacoustic penetration depth is 0.5 mm.

Modeling of Rapid Pam Systems Based on Electrothermal Micromirror for High-Resolution Facial Angiography.

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.

Photoacoustic (532 and 1064 nm) and ultrasonic coscanning microscopy for in vivo imaging on small animals: A productized strategy.

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

Thin-film optical-acoustic combiner enables high-speed wide-field multi-parametric photoacoustic microscopy in reflection mode.

Multi-parametric photoacoustic microscopy (PAM) is uniquely capable of simultaneous high-resolution mapping of blood oxygenation and flow in vivo. However, its speed has been limited by the dense sampling required for blood flow quantification. To overcome this limitation, we have developed a high-speed multi-parametric PAM system, which enables simultaneous acquisition of ∼500 densely sampled B-scans by superposing the rapid optical scanning across the line-shaped focus of a cylindrically focused ultrasonic transducer over the conventional mechanical scan of the optical-acoustic dual foci. A novel, to the best of our knowledge, optical-acoustic combiner (OAC) is designed and implemented to accommodate the short working distance of the transducer, enabling convenient confocal alignment of the dual foci in reflection mode. A resonant galvanometer (GM) provides stabilized high-speed large-angle scanning. This new system can continuously monitor microvascular blood oxygenation (sO2) and flow over a 4.5 × 3 mm2 area in the awake mouse brain with high spatial and temporal resolutions (6.9 µm and 0.3 Hz, respectively).

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.

Front Cover

This study presents a simple, but robust and efficient denoising technique for fast optical resolution photoacoustic microscopy system (OR‐PAM). Impulsive noise and background noise in photoacoustic images are common issues of OR‐PAM system when laser power is limited. Our developed multi‐stage adaptive noise reduction technique efficiently and robustly removes the noises under noisy data acquisition conditions, providing a strong foundation for photoacoustic microscopy with limited laser power.Further details can be found in the article by Nizar Guezzi, Changho Lee, Thanh Dat Le, Hyojin Seong, Kang‐Ho Choi, Jung‐Joon Min, and Jaesok Yu (e202200164). image

Shear‐Force Photoacoustic Microscopy: Toward Super‐resolution Near‐Field Imaging (Laser Photonics Rev. 16(12)/2022)

Super-Resolution Near-Field Imaging In article number 2200296, Byullee Park, Moongyu Han, Hongyoon Kim, Junsuk Rho, and Chulhong Kim developed a lens-free shear-force photoacoustic microscopy system using a tapered fiber and a shear force mechanism. This is the first proof-of-concept study for near-field generation in photoacoustic microscopy toward super-resolution imaging.