Recent Advances in Contrast-Enhanced Photoacoustic Imaging: Overcoming the Physical and Practical Challenges.

For decades now, photoacoustic imaging (PAI) has been investigated to realize its potential as a niche biomedical imaging modality. Despite its highly desirable optical contrast and ultrasonic spatiotemporal resolution, PAI is challenged by such physical limitations as a low signal-to-noise ratio (SNR), diminished image contrast due to strong optical attenuation, and a lower-bound on spatial resolution in deep tissue. In addition, contrast-enhanced PAI has faced practical limitations such as insufficient cell-specific targeting due to low delivery efficiency and difficulties in developing clinically translatable agents. Identifying these limitations is essential to the continuing expansion of the field, and substantial advances in developing contrast-enhancing agents, complemented by high-performance image acquisition systems, have synergistically dealt with the challenges of conventional PAI. This review covers the past four years of research on pushing the physical and practical challenges of PAI in terms of SNR/contrast, spatial resolution, targeted delivery, and clinical application. Promising strategies for dealing with each challenge are reviewed in detail, and future research directions for next generation contrast-enhanced PAI are discussed.

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.

Transcytosis-Inducing Multifunctional Albumin Nanomedicines with Deep Penetration Ability for Image-Guided Solid Tumor Treatment.

Transcytosis is an active transcellular transportation pathway that has garnered interest for overcoming the limited deep penetration of nanomedicines in solid tumors. In this study, a charge-convertible nanomedicine that facilitates deep penetration into solid tumors via transcytosis is designed. It is an albumin-based calcium phosphate nanomedicine loaded with IR820 (mAlb-820@CaP) for high-resolution photoacoustic imaging and enhanced photothermal therapy. Biomineralization on the surface stabilizes the albumin-IR820 complex during circulation and provides calcium ions (Ca2+ ) for tissue penetration on degradation in an acidic environment. pH-triggered transcytosis of the nanomedicine enabled by caveolae-mediated endocytosis and calcium ion-induced exocytosis in 2D cellular, 3D spheroid, and in vivo tumor models is demonstrated. Notably, the extravasation and penetration ability of the nanomedicine is observed in vivo using a high-resolution photoacoustic system, and nanomedicine shows the most potent photothermal antitumor effect in vivo. Overall, the strategy provides a versatile theragnosis platform for both noninvasive photoacoustic imaging and high therapeutic efficiency resulting from deep penetration of nanomedicine.

Wide-field three-dimensional photoacoustic/ultrasound scanner using a two-dimensional matrix transducer array.

Two-dimensional matrix transducer arrays are the most appropriate imaging probes for acquiring dual-modal 3D photoacoustic (PA)/ultrasound (US) images. However, they have small footprints which limit the field-of-view (FOV) to less than 10 mm × 10 mm and degrade the spatial resolution. In this study, we demonstrate a dual-modal PA and US imaging system (using a 2D matrix transducer array and a motorized 2D scanning system) to enlarge the FOV of volumetric images. Multiple PA volumes were merged to form a wide-field image of approximately 45 mm × 45 mm. In vivo imaging was demonstrated using rat sentinel lymph nodes (SLNs) and bladders stained with methylene blue. We believe that this volumetric PA/US imaging technique with a 2D matrix transducer array can be a useful tool for narrow-field real-time monitoring and wide-field imaging of various preclinical and clinical studies.

Review of Three-Dimensional Handheld Photoacoustic and Ultrasound Imaging Systems and Their Applications.

Photoacoustic (PA) imaging is a non-invasive biomedical imaging technique that combines the benefits of optics and acoustics to provide high-resolution structural and functional information. This review highlights the emergence of three-dimensional handheld PA imaging systems as a promising approach for various biomedical applications. These systems are classified into four techniques: direct imaging with 2D ultrasound (US) arrays, mechanical-scanning-based imaging with 1D US arrays, mirror-scanning-based imaging, and freehand-scanning-based imaging. A comprehensive overview of recent research in each imaging technique is provided, and potential solutions for system limitations are discussed. This review will serve as a valuable resource for researchers and practitioners interested in advancements and opportunities in three-dimensional handheld PA imaging technology.

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.

Surfactant-Stripped Semiconducting Polymer Micelles for Tumor Theranostics and Deep Tissue Imaging in the NIR-II Window.

Photoacoustic imaging (PA) in the second near infrared (NIR-II) window presents key advantages for deep tissue imaging owing to reduced light scattering and low background signal from biological structures. Here, a thiadiazoloquinoxaline-based semiconducting polymer (SP) with strong absorption in the NIR-II region is reported. After encapsulation of SP in Pluronic F127 (F127) followed by removal of excess surfactant, a dual functional polymer system named surfactant-stripped semiconductor polymeric micelles (SSS-micelles) are generated with water solubility, storage stability, and high photothermal conversion efficiency, permitting tumor theranostics in a mouse model. SSS-micelles have a wideband absorption in the NIR-II window, allowing for the PA imaging at both 1064 and 1300 nm wavelengths. The PA signal of the SSS-micelles can be detected through 6.5 cm of chicken breast tissue in vitro. In mice or rats, SSS-micelles can be visualized in bladder and intestine overlaid 5 cm (signal to noise ratio, SNR ≈ 17 dB) and 5.8 cm (SNR over 10 dB) chicken breast tissue, respectively. This work demonstrates the SSS-micelles as a nanoplatform for deep tissue theranostics.

In situ x-ray-induced acoustic computed tomography with a contrast agent: a proof of concept.

X-ray-induced acoustic computed tomography (XACT) has shown great potential as a hybrid imaging modality for real-time non-invasive x-ray dosimetry and low-dose three-dimensional (3D) imaging. While promising, one drawback of the XACT system is the underlying low signal-to-noise ratio (SNR), limiting its in vivo clinical use. In this Letter, we propose the first use of a conventional x-ray computed tomography contrast agent, Gastrografin, for improving the SNR of in situ XACT imaging. We obtained 3D volumetric XACT images of a mouse's stomach with orally injected Gastrografin establishing the proposal's feasibility. Thus, we believe, in the future, our proposed technique will allow in vivo imaging and expand or complement conventional x-ray modalities, such as radiotherapy and accelerators.

Bi-modal near-infrared fluorescence and ultrasound imaging via a transparent ultrasound transducer for sentinel lymph node localization.

Sentinel lymph node biopsy with an indocyanine green-based near-infrared fluorescence imaging system avoids the shortcomings of using a radioisotope or a combination of a blue dye and a radioactive tracer. To improve surgical precision, recent research has provided a depth profile of the sentinel lymph node by fusing fluorescence and ultrasound imaging. Here, we present a combined near-infrared fluorescence and ultrasound imaging system based on a transparent ultrasound transducer. The transparent ultrasound transducer enables seamless coaxial alignment of the fluorescence and ultrasound beam paths, allowing bi-modal observation of a single region of interest. Further, we demonstrate that the sentinel lymph node of mice injected with indocyanine green can be successfully localized and dissected based on information from the bi-modal imaging system.

Bi-modal near-infrared fluorescence and ultrasound imaging via a transparent ultrasound transducer for sentinel lymph node localization: publisher's note.

This publisher's note contains a correction to Opt. Lett.47, 393 (2022)10.1364/OL.446041.

Hyaluronate-Black Phosphorus-Upconversion Nanoparticle Complex for Non-invasive Theranosis of Skin Cancer.

Despite the wide investigation on black phosphorus (BP) for biophotonic applications, the finite depth of light penetration has limited further development of BP-based photomedicines. Here, we developed a hyaluronate-BP-upconversion nanoparticle (HA-BP-UCNP) complex for near-infrared (NIR) light-mediated multimodal theranosis of skin cancer with photoacoustic (PA) bioimaging, photodynamic therapy (PDT), and photothermal therapy (PTT). In contrast to the conventional BP-based skin cancer theranosis, the HA-BP-UCNP complex could be non-invasively delivered into the tumor tissue to induce the cancer cell apoptosis upon NIR light irradiation. The PA imaging of BP successfully visualized the non-invasive transdermal delivery of the HA-BP-UCNP complex into the mice skin. HA in the complex facilitated the transdermal delivery of BP into the tumor tissue under the skin. Upon 980 nm NIR light irradiation, the UCNP converted the light to UV-blue light to generate reactive oxygen species by sensitizing BP in the HA-BP-UCNP complex for PDT. Remarkably, 808 nm NIR irradiation with PTT triggered the apoptosis of tumor cells. Taken together, we could confirm the feasibility of the HA-BP-UCNP complex for NIR light-mediated multimodal theranosis of skin cancers.

Whole-Body Photoacoustic Imaging Techniques for Preclinical Small Animal Studies.

Photoacoustic imaging is a hybrid imaging technique that has received considerable attention in biomedical studies. In contrast to pure optical imaging techniques, photoacoustic imaging enables the visualization of optical absorption properties at deeper imaging depths. In preclinical small animal studies, photoacoustic imaging is widely used to visualize biodistribution at the molecular level. Monitoring the whole-body distribution of chromophores in small animals is a key method used in preclinical research, including drug-delivery monitoring, treatment assessment, contrast-enhanced tumor imaging, and gastrointestinal tracking. In this review, photoacoustic systems for the whole-body imaging of small animals are explored and summarized. The configurations of the systems vary with the scanning methods and geometries of the ultrasound transducers. The future direction of research is also discussed with regard to achieving a deeper imaging depth and faster imaging speed, which are the main factors that an imaging system should realize to broaden its application in biomedical studies.

A Four-Channel Low-Noise Readout IC for Air Flow Measurement Using Hot Wire Anemometer in 0.18 µm CMOS Technology.

Air flow measurements provide significant information required for understanding the characteristics of insect movement. This study proposes a four-channel low-noise readout integrated circuit (IC) in order to measure air flow (air velocity), which can be beneficial to insect biomimetic robot systems that have been studied recently. Instrumentation amplifiers (IAs) with low-noise characteristics in readout ICs are essential because the air flow of an insect's movement, which is electrically converted using a microelectromechanical systems (MEMS) sensor, generally produces a small signal. The fundamental architecture employed in the readout IC is a three op amp IA, and it accomplishes low-noise characteristics by chopping. Moreover, the readout IC has a four-channel input structure and implements an automatic offset calibration loop (AOCL) for input offset correction. The AOCL based on the binary search logic adjusts the output offset by controlling the input voltage bias generated by the R-2R digital-to-analog converter (DAC). The electrically converted air flow signal is amplified using a three op amp IA, which is passed through a low-pass filter (LPF) for ripple rejection that is generated by chopping, and converted to a digital code by a 12-bit successive approximation register (SAR) analog-to-digital converter (ADC). Furthermore, the readout IC contains a low-dropout (LDO) regulator that enables the supply voltage to drive digital circuits, and a serial peripheral interface (SPI) for digital communication. The readout IC is designed with a 0.18 µm CMOS process and the current consumption is 1.886 mA at 3.3 V supply voltage. The IC has an active area of 6.78 mm2 and input-referred noise (IRN) characteristics of 95.4 nV/√Hz at 1 Hz.

Pilot Study: Quantitative Photoacoustic Evaluation of Peripheral Vascular Dynamics Induced by Carfilzomib In Vivo.

Carfilzomib is mainly used to treat multiple myeloma. Several side effects have been reported in patients treated with carfilzomib, especially those associated with cardiovascular events, such as hypertension, congestive heart failure, and coronary artery disease. However, the side effects, especially the manifestation of cardiovascular events through capillaries, have not been fully investigated. Here, we performed a pilot experiment to monitor peripheral vascular dynamics in a mouse ear under the effects of carfilzomib using a quantitative photoacoustic vascular evaluation method. Before and after injecting the carfilzomib, bortezomib, and PBS solutions, we acquired high-resolution three-dimensional PAM data of the peripheral vasculature of the mouse ear during each experiment for 10 h. Then, the PAM maximum amplitude projection (MAP) images and five quantitative vascular parameters, i.e., photoacoustic (PA) signal, diameter, density, length fraction, and fractal dimension, were estimated. Quantitative results showed that carfilzomib induces a strong effect on the peripheral vascular system through a significant increase in all vascular parameters up to 50%, especially during the first 30 min after injection. Meanwhile, bortezomib and PBS do not have much impact on the peripheral vascular system. This pilot study verified PAM as a comprehensive method to investigate peripheral vasculature, along with the effects of carfilzomib. Therefore, we expect that PAM may be useful to predict cardiovascular events caused by carfilzomib.

In Vivo Quantitative Vasculature Segmentation and Assessment for Photodynamic Therapy Process Monitoring Using Photoacoustic Microscopy.

Vascular damage is one of the therapeutic mechanisms of photodynamic therapy (PDT). In particular, short-term PDT treatments can effectively destroy malignant lesions while minimizing damage to nonmalignant tissue. In this study, we investigate the feasibility of label-free quantitative photoacoustic microscopy (PAM) for monitoring the vasculature changes under the effect of PDT in mouse ear melanoma tumors. In particular, quantitative vasculature evaluation was conducted based on Hessian filter segmentation. Three-dimensional morphological PAM and depth-resolved images before and after PDT treatment were acquired. In addition, five quantitative vasculature parameters, including the PA signal, vessel diameter, vessel density, perfused vessel density, and vessel complexity, were analyzed to evaluate the influence of PDT on four different areas: Two melanoma tumors, and control and normal vessel areas. The quantitative and qualitative results successfully demonstrated the potential of the proposed PAM-based quantitative approach to evaluate the effectiveness of the PDT method.

Mercury Chloride but Not Lead Acetate Causes Apoptotic Cell Death in Human Lung Fibroblast MRC5 Cells via Regulation of Cell Cycle Progression.

Heavy metals are important for various biological systems, but, in excess, they pose a serious risk to human health. Heavy metals are commonly used in consumer and industrial products. Despite the increasing evidence on the adverse effects of heavy metals, the detailed mechanisms underlying their action on lung cancer progression are still poorly understood. In the present study, we investigated whether heavy metals (mercury chloride and lead acetate) affect cell viability, cell cycle, and apoptotic cell death in human lung fibroblast MRC5 cells. The results showed that mercury chloride arrested the sub-G1 and G2/M phases by inducing cyclin B1 expression. In addition, the exposure to mercury chloride increased apoptosis through the activation of caspase-3. However, lead had no cytotoxic effects on human lung fibroblast MRC5 cells at low concentration. These findings demonstrated that mercury chloride affects the cytotoxicity of MRC5 cells by increasing cell cycle progression and apoptotic cell death.

Transcriptome Analysis Reveals HgCl2 Induces Apoptotic Cell Death in Human Lung Carcinoma H1299 Cells through Caspase-3-Independent Pathway.

Mercury is one of the detrimental toxicants that can be found in the environment and exists naturally in different forms; inorganic and organic. Human exposure to inorganic mercury, such as mercury chloride, occurs through air pollution, absorption of food or water, and personal care products. This study aimed to investigate the effect of HgCl2 on cell viability, cell cycle, apoptotic pathway, and alters of the transcriptome profiles in human non-small cell lung cancer cells, H1299. Our data show that HgCl2 treatment causes inhibition of cell growth via cell cycle arrest at G0/G1- and S-phase. In addition, HgCl2 induces apoptotic cell death through the caspase-3-independent pathway. Comprehensive transcriptome analysis using RNA-seq indicated that cellular nitrogen compound metabolic process, cellular metabolism, and translation for biological processes-related gene sets were significantly up- and downregulated by HgCl2 treatment. Interestingly, comparative gene expression patterns by RNA-seq indicated that mitochondrial ribosomal proteins were markedly altered by low-dose of HgCl2 treatment. Altogether, these data show that HgCl2 induces apoptotic cell death through the dysfunction of mitochondria.

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.

Annual and seasonal patterns in etiologies of pediatric community-acquired pneumonia due to respiratory viruses and Mycoplasma pneumoniae requiring hospitalization in South Korea.

BACKGROUND: Community-acquired pneumonia (CAP) is one of the leading worldwide causes of childhood morbidity and mortality. Its disease burden varies by age and etiology and is time dependent. We aimed to investigate the annual and seasonal patterns in etiologies of pediatric CAP requiring hospitalization. METHODS: We conducted a retrospective study in 30,994 children (aged 0-18 years) with CAP between 2010 and 2015 at 23 nationwide hospitals in South Korea. Mycoplasma pneumoniae (MP) pneumonia was clinically classified as macrolide-sensitive MP, macrolide-less effective MP (MLEP), and macrolide-refractory MP (MRMP) based on fever duration after initiation of macrolide treatment, regardless of the results of in vitro macrolide sensitivity tests. RESULTS: MP and respiratory syncytial virus (RSV) were the two most commonly identified pathogens of CAP. With the two epidemics of MP pneumonia (2011 and 2015), the rates of clinical MLEP and MRMP pneumonia showed increasing trends of 36.4% of the total MP pneumonia. In children < 2 years of age, RSV (34.0%) was the most common cause of CAP, followed by MP (9.4%); however, MP was the most common cause of CAP in children aged 2-18 years of age (45.3%). Systemic corticosteroid was most commonly administered for MP pneumonia. The rate of hospitalization in intensive care units was the highest for RSV pneumonia, and ventilator care was most commonly needed in cases of adenovirus pneumonia. CONCLUSIONS: The present study provides fundamental data to establish public health policies to decrease the disease burden due to CAP and improve pediatric health.