Three-dimensional Multistructural Quantitative Photoacoustic and US Imaging of Human Feet in Vivo.

Background Monitoring the microcirculation in human feet is crucial in assessing peripheral vascular diseases, such as diabetic foot. However, conventional imaging modalities are more focused on diagnosis in major arteries, and there are limited methods to provide microvascular information in early stages of the disease. Purpose To investigate a three-dimensional (3D) noncontrast bimodal photoacoustic (PA)/US imaging system that visualizes the human foot morphologically and also reliably quantifies podiatric vascular parameters noninvasively. Materials and Methods A clinically relevant PA/US imaging system was combined with a foot scanner to obtain 3D PA and US images of the human foot in vivo. Healthy participants were recruited from September 2020 to June 2021. The collected 3D PA and US images were postprocessed to present structural information about the foot. The quantitative reliability was evaluated in five repeated scans of 10 healthy feet by calculating the intraclass correlation coefficient and minimal detectable change, and the detectability of microvascular changes was tested by imaging 10 healthy feet intentionally occluded with use of a pressure cuff (160 mm Hg). Statistically significant difference is indicated with P values. Results Ten feet from six healthy male volunteers (mean age ± standard deviation, 27 years ± 3) were included. The foot images clearly visualized the structure of the vasculature, bones, and skin and provided such functional information as the total hemoglobin concentration (HbT), hemoglobin oxygen saturation (SO2), vessel density, and vessel depth. Functional information from five independent measurements of 10 healthy feet was moderately reliable (intraclass correlation coefficient, 0.51-0.74). Significant improvements in HbT (P = .006) and vessel density (P = .046) as well as the retention of SO2 were observed, which accurately described the microvascular change due to venous occlusion. Conclusion Three-dimensional photoacoustic and US imaging was able to visualize morphologic and physiologic features of the human foot, including the peripheral microvasculature, in healthy volunteers. © RSNA, 2022 Online supplemental material is available for this article. See also the editorial by Mezrich in this issue.

Bi-Modal Transfer Learning for Classifying Breast Cancers via Combined B-Mode and Ultrasound Strain Imaging.

Although accurate detection of breast cancer still poses significant challenges, deep learning (DL) can support more accurate image interpretation. In this study, we develop a highly robust DL model based on combined B-mode ultrasound (B-mode) and strain elastography ultrasound (SE) images for classifying benign and malignant breast tumors. This study retrospectively included 85 patients, including 42 with benign lesions and 43 with malignancies, all confirmed by biopsy. Two deep neural network models, AlexNet and ResNet, were separately trained on combined 205 B-mode and 205 SE images (80% for training and 20% for validation) from 67 patients with benign and malignant lesions. These two models were then configured to work as an ensemble using both image-wise and layer-wise and tested on a dataset of 56 images from the remaining 18 patients. The ensemble model captures the diverse features present in the B-mode and SE images and also combines semantic features from AlexNet and ResNet models to classify the benign from the malignant tumors. The experimental results demonstrate that the accuracy of the proposed ensemble model is 90%, which is better than the individual models and the model trained using B-mode or SE images alone. Moreover, some patients that were misclassified by the traditional methods were correctly classified by the proposed ensemble method. The proposed ensemble DL model will enable radiologists to achieve superior detection efficiency owing to enhance classification accuracy for breast cancers in ultrasound (US) images.

A Deep Learning-Based Model That Reduces Speed of Sound Aberrations for Improved In Vivo Photoacoustic Imaging.

Photoacoustic imaging (PAI) has attracted great attention as a medical imaging method. Typically, photoacoustic (PA) images are reconstructed via beamforming, but many factors still hinder the beamforming techniques in reconstructing optimal images in terms of image resolution, imaging depth, or processing speed. Here, we demonstrate a novel deep learning PAI that uses multiple speed of sound (SoS) inputs. With this novel method, we achieved SoS aberration mitigation, streak artifact removal, and temporal resolution improvement all at once in structural and functional in vivo PA images of healthy human limbs and melanoma patients. The presented method produces high-contrast PA images in vivo with reduced distortion, even in adverse conditions where the medium is heterogeneous and/or the data sampling is sparse. Thus, we believe that this new method can achieve high image quality with fast data acquisition and can contribute to the advance of clinical PAI.

Quadruple ultrasound, photoacoustic, optical coherence, and fluorescence fusion imaging with a transparent ultrasound transducer.

Ultrasound and optical imagers are used widely in a variety of biological and medical applications. In particular, multimodal implementations combining light and sound have been actively investigated to improve imaging quality. However, the integration of optical sensors with opaque ultrasound transducers suffers from low signal-to-noise ratios, high complexity, and bulky form factors, significantly limiting its applications. Here, we demonstrate a quadruple fusion imaging system using a spherically focused transparent ultrasound transducer that enables seamless integration of ultrasound imaging with photoacoustic imaging, optical coherence tomography, and fluorescence imaging. As a first application, we comprehensively monitored multiparametric responses to chemical and suture injuries in rats' eyes in vivo, such as corneal neovascularization, structural changes, cataracts, and inflammation. As a second application, we successfully performed multimodal imaging of tumors in vivo, visualizing melanomas without using labels and visualizing 4T1 mammary carcinomas using PEGylated gold nanorods. We strongly believe that the seamlessly integrated multimodal system can be used not only in ophthalmology and oncology but also in other healthcare applications with broad impact and interest.

PAExM: label-free hyper-resolution photoacoustic expansion microscopy.

Reflection-mode ultraviolet photoacoustic microscopy (UV-PAM) is capable of imaging cell nuclei in thick tissue without complex preparation procedures, but it is challenging to distinguish adjacent nuclei due to the limited spatial resolution. Tissue expansion technology has recently been developed to exceed the diffraction-limited fluorescence microscopies, but it is accompanied by limitations including additional staining. Herein, photoacoustic expansion microscopy (PAExM) is presented, which is an advanced histologic imaging strategy combining advantages of fast label-free reflection-mode UV-PAM and the tissue expansion technology. Clustered cell nuclei in an enlarged volume of a mouse brain section can be visually resolved without staining, demonstrating a great potential of the system to be widely used for histologic applications throughout biomedical fields.

Nonlinear pth root spectral magnitude scaling beamforming for clinical photoacoustic and ultrasound imaging.

A recently introduced nonlinear pth root delay-and-sum (NL-p-DAS) beamforming (BF) technique for ultrasound (US) and photoacoustic (PA) imaging, achieving better spatial and contrast resolution compared to a conventional delay and sum (DAS) technique. While the method is advantageous for better resolution, it suffers from grainy speckles and dark areas in the image mainly due to the interference of non-sinusoidal functions. In this Letter, we introduce a modified NL-p-DAS technique called nonlinear pth root spectral magnitude scaling (NL-p-SMS), which performs the pth root on the spectral magnitude instead of the temporal amplitude. We evaluated the US and PA images of NL-p-SMS against those of NL-p-DAS by comparing the axial and lateral line profiles, contrasts, and contrast-to-noise ratios (CNRs) in both phantom and in vivo imaging studies with various p values. As a result, we found that the NL-p-SMS has better axial resolution and CNR than the NL-p-DAS, and reduces the grainy speckles and dark area artifacts. We believe that, with this enhanced performance, our proposed approach could be an advancement compared to the existing nonlinear BF algorithms.

Super-resolution localization photoacoustic microscopy using intrinsic red blood cells as contrast absorbers.

Photoacoustic microscopy (PAM) has become a premier microscopy tool that can provide the anatomical, functional, and molecular information of animals and humans in vivo. However, conventional PAM systems suffer from limited temporal and/or spatial resolution. Here, we present a fast PAM system and an agent-free localization method based on a stable and commercial galvanometer scanner with a custom-made scanning mirror (L-PAM-GS). This novel hardware implementation enhances the temporal resolution significantly while maintaining a high signal-to-noise ratio (SNR). These improvements allow us to photoacoustically and noninvasively observe the microvasculatures of small animals and humans in vivo. Furthermore, the functional hemodynamics, namely, the blood flow rate in the microvasculature, is successfully monitored and quantified in vivo. More importantly, thanks to the high SNR and fast B-mode rate (500 Hz), by localizing photoacoustic signals from captured red blood cells without any contrast agent, unresolved microvessels are clearly distinguished, and the spatial resolution is improved by a factor of 2.5 in vivo. L-PAM-GS has great potential in various fields, such as neurology, oncology, and pathology.

Real-time delay-multiply-and-sum beamforming with coherence factor for in vivo clinical photoacoustic imaging of humans.

In the clinical photoacoustic (PA) imaging, ultrasound (US) array transducers are typically used to provide B-mode images in real-time. To form a B-mode image, delay-and-sum (DAS) beamforming algorithm is the most commonly used algorithm because of its ease of implementation. However, this algorithm suffers from low image resolution and low contrast drawbacks. To address this issue, delay-multiply-and-sum (DMAS) beamforming algorithm has been developed to provide enhanced image quality with higher contrast, and narrower main lobe compared but has limitations on the imaging speed for clinical applications. In this paper, we present an enhanced real-time DMAS algorithm with modified coherence factor (CF) for clinical PA imaging of humans in vivo. Our algorithm improves the lateral resolution and signal-to-noise ratio (SNR) of original DMAS beamformer by suppressing the background noise and side lobes using the coherence of received signals. We optimized the computations of the proposed DMAS with CF (DMAS-CF) to achieve real-time frame rate imaging on a graphics processing unit (GPU). To evaluate the proposed algorithm, we implemented DAS and DMAS with/without CF on a clinical US/PA imaging system and quantitatively assessed their processing speed and image quality. The processing time to reconstruct one B-mode image using DAS, DAS with CF (DAS-CF), DMAS, and DMAS-CF algorithms was 7.5, 7.6, 11.1, and 11.3 ms, respectively, all achieving the real-time imaging frame rate. In terms of the image quality, the proposed DMAS-CF algorithm improved the lateral resolution and SNR by 55.4% and 93.6 dB, respectively, compared to the DAS algorithm in the phantom imaging experiments. We believe the proposed DMAS-CF algorithm and its real-time implementation contributes significantly to the improvement of imaging quality of clinical US/PA imaging system.

Review on practical photoacoustic microscopy.

Photoacoustic imaging (PAI) has many interesting advantages, such as deep imaging depth, high image resolution, and high contrast to intrinsic and extrinsic chromophores, enabling morphological, functional, and molecular imaging of living subjects. Photoacoustic microscopy (PAM) is one form of the PAI inheriting its characteristics and is useful in both preclinical and clinical research. Over the years, PAM systems have been evolved in several forms and each form has its relative advantages and disadvantages. Thus, to maximize the benefits of PAM for a specific application, it is important to configure the PAM system optimally by targeting a specific application. In this review, we provide practical methods for implementing a PAM system to improve the resolution, signal-to-noise ratio (SNR), and imaging speed. In addition, we review the preclinical and the clinical applications of PAM and discuss the current challenges and the scope for future developments.

In vivo label-free functional photoacoustic monitoring of ischemic reperfusion.

Pressure ulcer formation is a common problem among patients confined to bed or restricted to wheelchairs. The ulcer forms when the affected skin and underlying tissues go through repeated cycles of ischemia and reperfusion, leading to inflammation. This theory is evident by intravital imaging studies performed in immune cell-specific, fluorescent reporter mouse skin with induced ischemia-reperfusion (I-R) injuries. However, traditional confocal or multiphoton microscopy cannot accurately monitor the progression of vascular reperfusion by contrast agents, which leaks into the interstitium under inflammatory conditions. Here, we develop a dual-wavelength micro electro mechanical system (MEMS) scanning-based optical resolution photoacoustic microscopy (OR-PAM) system for continuous label-free functional imaging of vascular reperfusion in an IR mouse model. This MEMS-OR-PAM system provides fast scanning speed for concurrent dual-wavelength imaging, which enables continuous monitoring of the reperfusion process. During reperfusion, the revascularization of blood vessels and the oxygen saturation (sO2 ) changes in both arteries and veins are recorded, from which the local oxygen extraction ratios of the ischemic tissue and the unaffected tissue can be quantified. Our MEMS-OR-PAM system provides novel perspectives to understand the I-R injuries. It solves the problem of dynamic label-free functional monitoring of the vascular reperfusion at high spatial resolution.

A Novel 2-D Synthetic Aperture Focusing Technique for Acoustic-Resolution Photoacoustic Microscopy.

Acoustic-resolution photoacoustic microscopy (AR-PAM) is a promising technology for vascular or tumor-targeted molecular imaging. Unique advantages of AR-PM are its non-invasive, non-ionizing real-time, and deeper imaging depth. AR-PAM typically uses an ultrasound transducer with a high acoustic numerical aperture (NA) to enable deeper imaging depth. While high NA achieves good lateral resolution in the focal plane but significantly degrades the lateral resolution in the out-of-focus region. Synthetic aperture focusing technique (SAFT) has been introduced to overcome this out-of-focus degradation by synthesizing the correlated signals. Several 2-D SAFTs have been also reported to improve degraded resolution in all directions. However, the resolution enhancement of the previously reported 2-D SAFTs are suboptimal and are not equivalent to the 1-D SAFT performance under an ideal condition with the sample orientation perpendicular to the synthetic aperture direction. In this paper, we present a new 2-D SAFT called 2-D directional SAFT that improves the lateral resolution significantly and we compare our results against 1-D SAFT under ideal condition. We applied this algorithm to phantom and in vivo images to show the improvement in image quality. We also implement this algorithm in a graphical processing unit to achieve high performance to show the practicality of implementing this new algorithm in a system.

Reflection-mode switchable subwavelength Bessel-beam and Gaussian-beam photoacoustic microscopy in vivo.

We have developed a reflection-mode switchable subwavelength Bessel-beam (BB) and Gaussian-beam (GB) photoacoustic microscopy (PAM) system. To achieve both reflection-mode and high resolution, we tightly attached a very small ultrasound transducer to an optical objective lens with numerical aperture of 1.0 and working distance of 2.5 mm. We used axicon and an achromatic doublet in our system to obtain the extended depth of field (DOF) of the BB. To compare the DOF performance achieved with our BB-PAM system against GB-PAM system, we designed our system so that the GB can be easily generated by simply removing the lenses. Using a 532 nm pulse laser, we achieved the lateral resolutions of 300 and 270 nm for BB-PAM and GB-PAM, respectively. The measured DOF of BB-PAM was approximately 229 µm, which was about 7× better than that of GB-PAM. We imaged the vasculature of a mouse ear using BB-PAM and GB-PAM and confirmed that the DOF of BB-PAM is much better than the DOF of GB-PAM. Thus, we believe that the high resolution achieved at the extended DOF by our system is very practical for wide range of biomedical research including red blood cell (RBC) migration in blood vessels at various depths and observation of cell migration or cell culture.

Optical resolution photoacoustic microscopy based on multimode fibers.

Photoacoustic microscopy (PAM) is a multiscale imaging technique. In optical-resolution photoacoustic microscopy (OR-PAM), a single mode (SM) fiber is normally used as the source of optical excitation to be focused into a diffraction-limited spot. Recent advances in OR-PAM have improved its imaging speed using microelectromechanical systems (MEMS). Here we report for the first time the use of a multimode (MM) fiber as the optical excitation source for high resolution OR-PAM in vivo imaging. A high-speed MEMS scanner based OR-PAM system combined with the mechanical movement to provide wide area imaging was used. The use of multimode fiber for achieving tight optical focus would make the optical alignment easier and high repetition rate light delivery possible for high-speed OR-PAM imaging. A lateral resolution of 3.5 µm and axial resolution of 27 µm with ~1.5 mm imaging depth was successfully demonstrated using the system. The efficacy of multimode fibers for achieving tight focus is beneficial for developing high-resolution photoacoustic endoscopy systems and can be combined with other optical endoscopic imaging modalities as well.

Photoacoustic microscopy for evaluating combretastatin A4 phosphate induced vascular disruption in orthotopic glioma.

The use of an optical resolution photoacoustic microscopy (OR-PAM) system to evaluate the vascular disruptive effect of combretastatin A4 Phosphate (CA4P) on a murine orthotopic glioma with intact skull is described here. Second generation optical-resolution photoacoustic microscopy scanner with a 532 nm pulsed diode-pumped solid-state laser that specifically matches the absorption maximum of hemoglobin in tissues was used to image orthotopic glioma inoculated in mouse brain. Two-dimensional maps of brain vasculature with a lateral resolution of 5 µm and a depth of 700 µm at a field of view 5 × 4 mm were acquired on normal brain and glioma brain. Longitudinal imaging of the brain pre- and post-administration of CA4P, a FDA approved drug for solid tumors, enabled the monitoring of hemodynamic changes in tumor vasculature revealing the well documented vascular shutdown and recovery associated with this drug. Our study marks the beginning of potential prospects of this technology as an imaging tool for preclinical and clinical study of pathologies characterized by changes in the vasculature.

Clinical photoacoustic imaging platforms.

Photoacoustic imaging (PAI) is a new promising medical imaging technology available for diagnosing and assessing various pathologies. PAI complements existing imaging modalities by providing information not currently available for diagnosing, e.g., oxygenation level of the underlying tissue. Currently, researchers are translating PAI from benchside to bedside to make unique clinical advantages of PAI available for patient care. The requirements for a successful clinical PAI system are; deeper imaging depth, wider field of view, and faster scan time than the laboratory-level PAI systems. Currently, many research groups and companies are developing novel technologies for data acquisition/signal processing systems, detector geometry, and an acoustic sensor. In this review, we summarize state-of-the-art clinical PAI systems with three types of the imaging transducers: linear array transducer, curved linear array transducer, and volumetric array transducer. We will also discuss the limitations of the current PAI systems and describe latest techniques being developed to address these for further enhancing the image quality of PAI for successful clinical translation.

Handheld Photoacoustic Microscopy Probe.

Optical resolution photoacoustic microscopy (OR-PAM) is a non-invasive, label-free method of in vivo imaging with microscopic resolution and high optical contrast. Based on intrinsic contrasts, OR-PAM has expanded to include in vivo vessel imaging, flow cytometry, physiological parameter analysis, and single-cell characterization. However, since conventional OR-PAM systems have a fixed tabletop configuration, a large system size, and slow imaging speed, their use in preclinical and clinical studies remains limited. In this study, using microelectromechanical systems (MEMS) technology, we developed a handheld PAM probe with a high signal-to-noise ratio and image rate. To enable broader application of the OR-PAM system, we reduced its size and combined its fast scanning capabilities into a small handheld probe that uses a 2-axis waterproof MEMS scanner (2A-WP-MEMS scanner). All acoustical, optical, and mechanical components are integrated into a single probe with a diameter of 17 mm and a weight of 162 g. This study shows phantom and in vivo images of various samples acquired with the probe, including carbon fibers, electrospun microfibers, and the ear, iris, and brain of a living mouse. In particular, this study investigated the possibility of clinical applications for melanoma diagnosis by imaging the boundaries and morphology of a human mole.

Photothermal strain imaging.

Vulnerable plaques are the major cause of cardiovascular disease, but they are difficult to detect with conventional intravascular imaging techniques. Techniques are needed to identify plaque vulnerability based on the presence of lipids in plaque. Thermal strain imaging (TSI) is an imaging technique based on ultrasound (US) wave propagation speed, which varies with the medium temperature. In TSI, the strain that occurs during tissue temperature change can be used for lipid detection because it has a different tendency depending on the type of tissue. Here, we demonstrate photothermal strain imaging (pTSI) using an intravascular ultrasound catheter. pTSI is performed by slightly and selectively heating lipid using a relatively inexpensive continuous laser source. We applied a speckle-tracking algorithm to US B-mode images for strain calculations. As a result, the strain produced in porcine fat was different from the strain produced in water-bearing gelatin phantom, which made it possible to distinguish the two. This suggests that pTSI could potentially be a way of differentiating lipids in coronary artery.

In Vivo Photoacoustic Imaging of Anterior Ocular Vasculature: A Random Sample Consensus Approach.

Visualizing ocular vasculature is important in clinical ophthalmology because ocular circulation abnormalities are early signs of ocular diseases. Photoacoustic microscopy (PAM) images the ocular vasculature without using exogenous contrast agents, avoiding associated side effects. Moreover, 3D PAM images can be useful in understanding vessel-related eye disease. However, the complex structure of the multi-layered vessels still present challenges in evaluating ocular vasculature. In this study, we demonstrate a new method to evaluate blood circulation in the eye by combining in vivo PAM imaging and an ocular surface estimation method based on a machine learning algorithm: a random sample consensus algorithm. By using the developed estimation method, we were able to visualize the PA ocular vascular image intuitively and demonstrate layer-by-layer analysis of injured ocular vasculature. We believe that our method can provide more accurate evaluations of the eye circulation in ophthalmic applications.

Delay-multiply-and-sum-based synthetic aperture focusing in photoacoustic microscopy.

We propose an improved version of a synthetic aperture focusing technique (SAFT) based on a delay-multiply-and-sum algorithm for acoustic-resolution photoacoustic microscopy (AR-PAM). In this method, the photoacoustic (PA) signals from multiple scan-lines are combinatorially coupled, multiplied, and then summed. This process can be considered a correlation operation of the PA signals in each scan-line, so the spatial coherent information between the PA signals can be efficiently extracted. By applying this method in conventional AR-PAM, lateral resolution and signal-to-noise ratio in out-of-focus regions are much improved compared with those estimated from the previously developed SAFT, respectively, thereby achieving the extension of the imaging focal region. Our phantom and in vivo imaging experiments prove the validity of our proposed method.