Single-beam Acoustic Tweezers for Cell Biology: Molecular to in vivo level.

Acoustic tweezers have attracted attention in various fields of cell biology, including in vitro single-cell and intercellular mechanics. Compared with other tweezing technologies such as optical and magnetic tweezers, acoustic tweezers possess stronger forces and are safer for use in biological systems. However, due to the limited spatial resolution, or limited size of target objects, acoustic tweezers have primarily been used to manipulate cells in vitro. To extend the advantages of acoustic tweezers to other levels (e.g., molecular and in vivo levels), researchers have recently developed various types of acoustic tweezers such as single-beam acoustic tweezers (SBATs), surface acoustic wave tweezers, and acoustic-streaming tweezers. Among these, SBATs utilize a single-focused beam, making the transducer and system simple, noninvasive, and capable of producing strong forces compared with other types of tweezers. Depending on the acoustic beam pattern, SBATs can be classified into Rayleigh regime, Mie regime, and acoustic vortex with different trapping dynamics and application levels. In this review, we provide an overview of the principles and configuration of each type of SBAT, their applications ranging from molecular to in vivo studies, and their limitations and prospects. Thus, this review demonstrates the significance and potential of SBAT technology in biophysics and biomedical engineering.

Deep learning-based virtual staining, segmentation, and classification in label-free photoacoustic histology of human specimens.

In pathological diagnostics, histological images highlight the oncological features of excised specimens, but they require laborious and costly staining procedures. Despite recent innovations in label-free microscopy that simplify complex staining procedures, technical limitations and inadequate histological visualization are still problems in clinical settings. Here, we demonstrate an interconnected deep learning (DL)-based framework for performing automated virtual staining, segmentation, and classification in label-free photoacoustic histology (PAH) of human specimens. The framework comprises three components: (1) an explainable contrastive unpaired translation (E-CUT) method for virtual H&E (VHE) staining, (2) an U-net architecture for feature segmentation, and (3) a DL-based stepwise feature fusion method (StepFF) for classification. The framework demonstrates promising performance at each step of its application to human liver cancers. In virtual staining, the E-CUT preserves the morphological aspects of the cell nucleus and cytoplasm, making VHE images highly similar to real H&E ones. In segmentation, various features (e.g., the cell area, number of cells, and the distance between cell nuclei) have been successfully segmented in VHE images. Finally, by using deep feature vectors from PAH, VHE, and segmented images, StepFF has achieved a 98.00% classification accuracy, compared to the 94.80% accuracy of conventional PAH classification. In particular, StepFF's classification reached a sensitivity of 100% based on the evaluation of three pathologists, demonstrating its applicability in real clinical settings. This series of DL methods for label-free PAH has great potential as a practical clinical strategy for digital pathology.

Therapeutic Potential of Lactiplantibacillus plantarum FB091 in Alleviating AlcoholInduced Liver Disease through Gut-Liver Axis.

Alcoholic liver disease (ALD) poses a significant global health burden, often requiring liver transplantation and resulting in fatalities. Current treatments, like corticosteroids, effectively reduce inflammation but carry significant immunosuppressive risks. This study evaluates Lactiplantibacillus plantarum FB091, a newly isolated probiotic strain, as a safer alternative for ALD treatment. Using an in vivo mouse model, we assessed the effects of L. plantarum FB091 on alcohol-induced liver damage and gut microbiota composition. Alcohol and probiotics administration did not significantly impact water/feed intake or body weight. Histopathological analysis showed that L. plantarum FB091 reduced hepatocellular ballooning and inflammatory cell infiltration in liver tissues and mitigated structural damage in colon tissues, demonstrating protective effects against alcohol-induced damage. Biomarker analysis indicated that L. plantarum FB091 decreased aspartate aminotransferase levels, suggesting reduced liver damage, and increased alcohol dehydrogenase activity, indicating enhanced alcohol metabolism. Additionally, cytokine assays revealed a reduction in pro-inflammatory TNF-α and an increase in anti-inflammatory IL-10 levels in colon tissues of the L. plantarum FB091 group, suggesting an anti-inflammatory effect. Gut microbiota analysis showed changes in the L. plantarum FB091 group, including a reduction in Cyanobacteria and an increase in beneficial bacteria such as Akkermansia and Lactobacillus. These changes correlated with the recovery and protection of liver and colon health. Overall, L. plantarum FB091 shows potential as a therapeutic probiotic for managing ALD through its protective effects on liver and colon tissues, enhancement of alcohol metabolism, and beneficial modulation of gut microbiota. Further clinical studies are warranted to confirm these findings in humans.

Reorganization of H3K9me heterochromatin leads to neuronal impairment via the cascading destruction of the KDM3B-centered epigenomic network.

Histone H3K9 methylated heterochromatin silences repetitive non-coding sequences and lineage-specific genes during development, but how tissue-specific genes escape from heterochromatin in differentiated cells is unclear. Here, we examine age-dependent transcriptomic profiling of terminally differentiated mouse retina to identify epigenetic regulators involved in heterochromatin reorganization. The single-cell RNA sequencing analysis reveals a gradual downregulation of Kdm3b in cone photoreceptors during aging. Disruption of Kdm3b (Kdm3b +/- ) of 12-month-old mouse retina leads to the decreasing number of cones via apoptosis, and it changes the morphology of cone ribbon synapses. Integration of the transcriptome with epigenomic analysis in Kdm3b +/- retinas demonstrates gains of heterochromatin features in synapse assembly and vesicle transport genes that are downregulated via the accumulation of H3K9me1/2. Contrarily, losses of heterochromatin in apoptotic genes exacerbated retinal neurodegeneration. We propose that the KDM3B-centered epigenomic network is crucial for balancing of cone photoreceptor homeostasis via the modulation of gene set-specific heterochromatin features during aging.

3D Multiparametric Photoacoustic Computed Tomography of Primary and Metastatic Tumors in Living Mice.

Photoacoustic computed tomography (PACT), an emerging imaging modality in preclinical cancer research, can provide multiparametric 3D information about structures, physiological functions, and pharmacokinetics. Here, we demonstrate the use of high-definition 3D multiparametric PACT imaging of both primary and metastatic tumors in living mice to noninvasively monitor angiogenesis, carcinogenesis, hypoxia, and pharmacokinetics. The high-definition PACT system with a 1024-element hemispherical ultrasound transducer array provides an isotropic spatial resolution of 380 µm, an effective volumetric field-of-view of 12.8 mm × 12.8 mm × 12.8 mm without scanning, and an acquisition time of <30 s for a whole mouse body. Initially, we monitor the structural progression of the tumor microenvironment (e.g., angiogenesis and vessel tortuosity) after tumor cell inoculation. Then, we analyze the change in oxygen saturation of the tumor during carcinogenesis, verifying induced hypoxia in the tumor's core region. Finally, the whole-body pharmacokinetics are photoacoustically imaged after intravenous injection of micelle-loaded IR780 dye, and the in vivo PACT results are validated in vivo and ex vivo by fluorescence imaging. By employing the premium PACT system and applying multiparametric analyses to subcutaneous primary tumors and metastatic liver tumors, we demonstrate that this PACT system can provide multiparametric analyses for comprehensive cancer research.

Robust Ensemble of Two Different Multimodal Approaches to Segment 3D Ischemic Stroke Segmentation Using Brain Tumor Representation Among Multiple Center Datasets.

Ischemic stroke segmentation at an acute stage is vital in assessing the severity of patients' impairment and guiding therapeutic decision-making for reperfusion. Although many deep learning studies have shown attractive performance in medical segmentation, it is difficult to use these models trained on public data with private hospitals' datasets. Here, we demonstrate an ensemble model that employs two different multimodal approaches for generalization, a more effective way to perform on external datasets. First, after we jointly train a segmentation model on diffusion-weighted imaging (DWI) and apparent diffusion coefficient (ADC) MR modalities, the model is inferred on the DWI images. Second, a channel-wise segmentation model is trained by concatenating the DWI and ADC images as input, and then is inferred using both MR modalities. Before training with ischemic stroke data, we utilized BraTS 2021, a public brain tumor dataset, for transfer learning. An extensive ablation study evaluates which strategy learns better representations for ischemic stroke segmentation. In our study, nnU-Net well-known for robustness is selected as our baseline model. Our proposed method is evaluated on three different datasets: the Asan Medical Center (AMC) I and II, and the 2022 Ischemic Stroke Lesion Segmentation (ISLES). Our experiments are widely validated over a large, multi-center, and multi-scanner dataset with a huge amount of 846 scans. Not only stroke lesion models can benefit from transfer learning using brain tumor data, but combining the MR modalities using different training schemes also highly improves segmentation performance. The method achieved a top-1 ranking in the ongoing ISLES'22 challenge and performed particularly well on lesion-wise metrics of interest to neuroradiologists, achieving a Dice coefficient of 78.69% and a lesion-wise F1 score of 82.46%. Also, the method was relatively robust on the AMC I (Dice, 60.35%; lesion-wise F1, 68.30%) and II (Dice; 74.12%; lesion-wise F1, 67.53%) datasets in different settings. The high segmentation accuracy of our proposed method could improve radiologists' ability to detect ischemic stroke lesions in MRI images. Our model weights and inference code are available on https://github.com/MDOpx/ISLES22-model-inference .

Nonlinear beamforming for intracardiac echocardiography: a comparative study.

Intracardiac echocardiography (ICE) enables cardiac imaging with a wide field of view, deep imaging depth, and high frame rate during surgery. However, strong sidelobe and grating lobe artifacts created by the ultra-compact transducer degrade its image quality, making diagnosis and monitoring of treatment difficult. Conventionally, aperture apodization algorithms are often used to suppress sidelobe and grating lobe artifacts at the expense of lateral resolution, which is undesirable in ICE. In this study, we present comparative results of the beamforming methods specifically in ICE application. We demonstrate and compare five nonlinear beamforming algorithms in ICE: nonlinear pth root delay and sum (NL-p-DAS), nonlinear pth root spectral magnitude scaling (NL-p-SMS), delay-and-sum with coherence factors (DAS + SCF), delay and sum with apodization (DAS + apodization) and delay and sum (DAS). Phantom and ex-vivo experiment compare the performance of each algorithm in static and dynamic conditions. DAS + SCF shows the best lateral resolution, and all four algorithms improve the image contrast and sidelobe suppression over conventional DAS. NL-p-SMS stands out for the best axial resolution and suppression of grating lobe artifacts. For motion tracking, NL-p-SMS shows better temporal resolution than other methods. Overall, all the beamforming algorithms other than DAS showed improved image quality. Among them, NL-p-SMS, which has a high temporal resolution, showed the potential for providing more accurate information regards movement tracking. Supplementary Information: The online version contains supplementary material available at 10.1007/s13534-024-00352-9.

Video-rate endocavity photoacoustic/harmonic ultrasound imaging with miniaturized light delivery.

Significance: Endocavity ultrasound (US) imaging is a frequently employed diagnostic technique in gynecology and urology for the assessment of male and female genital diseases that present challenges for conventional transabdominal imaging. The integration of photoacoustic (PA) imaging with clinical US imaging has displayed promising outcomes in clinical research. Nonetheless, its application has been constrained due to size limitations, restricting it to spatially confined locations such as vaginal or rectal canals. Aim: This study presents the development of a video-rate (20 Hz) endocavity PA/harmonic US imaging (EPAUSI) system. Approach: The approach incorporates a commercially available endocavity US probe with a miniaturized laser delivery unit, comprised of a single large-core fiber and a line beamshaping engineered diffuser. The system facilitates real-time image display and subsequent processing, including angular energy density correction and spectral unmixing, in offline mode. Results: The spatial resolutions of the concurrently acquired PA and harmonic US images were measured at 318 µm and 291 µm in the radial direction, respectively, and 1.22 deg and 1.50 deg in the angular direction, respectively. Furthermore, the system demonstrated its capability in multispectral PA imaging by successfully distinguishing two clinical dyes in a tissue-mimicking phantom. Its rapid temporal resolution enabled the capture of kinetic dye perfusion into an ex vivo porcine ovary through the depth of porcine uterine tissue. EPAUSI proved its clinical viability by detecting pulsating hemodynamics in the male rat's prostate in vivo and accurately classifying human blood vessels into arteries and veins based on sO2 measurements. Conclusions: Our proposed EPAUSI system holds the potential to unveil previously overlooked indicators of vascular alterations in genital cancers or endometriosis, addressing pressing requirements in the fields of gynecology and urology.

X-ray free-electron laser induced acoustic microscopy (XFELAM).

The X-ray free-electron laser (XFEL) has remarkably advanced X-ray imaging technology and enabled important scientific achievements. The XFEL's extremely high power, short pulse width, low emittance, and high coherence make possible such diverse imaging techniques as absorption/emission spectroscopy, diffraction imaging, and scattering imaging. Here, we demonstrate a novel XFEL-based imaging modality that uses the X-ray induced acoustic (XA) effect, which we call X-ray free-electron laser induced acoustic microscopy (XFELAM). Initially, we verified the XA effect by detecting XA signals from various materials, then we validated the experimental results with simulation outcomes. Next, in resolution experiments, we successfully imaged a patterned tungsten target with drilled various-sized circles at a spatial resolution of 7.8 ± 5.1 µm, which is the first micron-scale resolution achieved by XA imaging. Our results suggest that the novel XFELAM can expand the usability of XFEL in various areas of fundamental scientific research.

An ultrasensitive and broadband transparent ultrasound transducer for ultrasound and photoacoustic imaging in-vivo.

Transparent ultrasound transducers (TUTs) can seamlessly integrate optical and ultrasound components, but acoustic impedance mismatch prohibits existing TUTs from being practical substitutes for conventional opaque ultrasound transducers. Here, we propose a transparent adhesive based on a silicon dioxide-epoxy composite to fabricate matching and backing layers with acoustic impedances of 7.5 and 4-6 MRayl, respectively. By employing these layers, we develop an ultrasensitive, broadband TUT with 63% bandwidth at a single resonance frequency and high optical transparency ( > 80%), comparable to conventional opaque ultrasound transducers. Our TUT maximises both acoustic power and transfer efficiency with maximal spectrum flatness while minimising ringdowns. This enables high contrast and high-definition dual-modal ultrasound and photoacoustic imaging in live animals and humans. Both modalities reach an imaging depth of > 15 mm, with depth-to-resolution ratios exceeding 500 and 370, respectively. This development sets a new standard for TUTs, advancing the possibilities of sensor fusion.

Deep learning-based multimodal fusion network for segmentation and classification of breast cancers using B-mode and elastography ultrasound images.

Ultrasonography is one of the key medical imaging modalities for evaluating breast lesions. For differentiating benign from malignant lesions, computer-aided diagnosis (CAD) systems have greatly assisted radiologists by automatically segmenting and identifying features of lesions. Here, we present deep learning (DL)-based methods to segment the lesions and then classify benign from malignant, utilizing both B-mode and strain elastography (SE-mode) images. We propose a weighted multimodal U-Net (W-MM-U-Net) model for segmenting lesions where optimum weight is assigned on different imaging modalities using a weighted-skip connection method to emphasize its importance. We design a multimodal fusion framework (MFF) on cropped B-mode and SE-mode ultrasound (US) lesion images to classify benign and malignant lesions. The MFF consists of an integrated feature network (IFN) and a decision network (DN). Unlike other recent fusion methods, the proposed MFF method can simultaneously learn complementary information from convolutional neural networks (CNNs) trained using B-mode and SE-mode US images. The features from the CNNs are ensembled using the multimodal EmbraceNet model and DN classifies the images using those features. The experimental results (sensitivity of 100 ± 0.00% and specificity of 94.28 ± 7.00%) on the real-world clinical data showed that the proposed method outperforms the existing single- and multimodal methods. The proposed method predicts seven benign patients as benign three times out of five trials and six malignant patients as malignant five out of five trials. The proposed method would potentially enhance the classification accuracy of radiologists for breast cancer detection in US images.

Contrast Agent-Free 3D Renal Ultrafast Doppler Imaging Reveals Vascular Dysfunction in Acute and Diabetic Kidney Diseases.

To combat the irreversible decline in renal function associated with kidney disease, it is essential to establish non-invasive biomarkers for assessing renal microcirculation. However, the limited resolution and/or vascular sensitivity of existing diagnostic imaging techniques hinders the visualization of complex cortical vessels. Here, a 3D renal ultrafast Doppler (UFD) imaging system that uses a high ultrasound frequency (18 MHz) and ultrahigh frame rate (1 KHz per slice) to scan the entire volume of a rat's kidney in vivo is demonstrated. The system, which can visualize the full 3D renal vascular branching pyramid at a resolution of 167 µm without any contrast agent, is used to chronically and noninvasively monitor kidneys with acute kidney injury (AKI, 3 days) and diabetic kidney disease (DKD, 8 weeks). Multiparametric UFD analyses (e.g., vessel volume occupancy (VVO), fractional moving blood volume (FMBV), vessel number density (VND), and vessel tortuosity (VT)) describe rapid vascular rarefaction from AKI and long-term vascular degeneration from DKD, while the renal pathogeneses are validated by in vitro blood serum testing and stained histopathology. This work demonstrates the potential of 3D renal UFD to offer valuable insights into assessing kidney perfusion levels for future research in diabetes and kidney transplantation.

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.

Immune-Stimulating Potential of Lacticaseibacillus rhamnosus LM1019 in RAW 264.7 Cells and Immunosuppressed Mice Induced by Cyclophosphamide.

Probiotics, including Lacticaseibacillus rhamnosus (L. rhamnosus), have gained recognition for their potential health benefits, such as enhancing immune function, maintaining gut health, and improving nutrient absorption. This study investigated the effectiveness of L. rhamnosus LM1019 (LM1019) in enhancing immune function. In RAW 264.7 cells, LM1019 demonstrated dose-dependent immune stimulation by increasing nitric oxide production, gene expression of proinflammatory cytokines, and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). These effects were mediated through the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) translocation without inducing cytotoxicity. Furthermore, orally administered LM1019 was evaluated in immunosuppressed mice induced by cyclophosphamide (CTX). High-dose administration of LM1019 significantly increased the subpopulations of lymphocytes, specifically helper T cells (CD4+), as well as two subtypes of natural killer (NK) cells, namely, IFN-γ+ and granzyme B+ NK cells. Additionally, LM1019 at a high dose led to elevated levels of proinflammatory cytokines, including IFN-γ and IL-12, compared to CTX-treated mice. These findings highlight the potential of LM1019 in enhancing the immune system. The study contributes to the growing body of research on the beneficial effects of probiotics on immune function.

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.

Review on ultrasound-guided photoacoustic imaging for complementary analyses of biological systems in vivo.

Photoacoustic imaging has been developed as a new biomedical molecular imaging modality. Due to its similarity to conventional ultrasound imaging in terms of signal detection and image generation, dual-modal photoacoustic and ultrasound imaging has been applied to visualize physiological and morphological information in biological systems in vivo. By complementing each other, dual-modal photoacoustic and ultrasound imaging showed synergistic advances in photoacoustic imaging with the guidance of ultrasound images. In this review, we introduce our recent progresses in dual-modal photoacoustic and ultrasound imaging systems at various scales of study, from preclinical small animals to clinical humans. A summary of the works reveals various strategies for combining the structural information of ultrasound images with the molecular information of photoacoustic images.

Functional photoacoustic imaging: from nano- and micro- to macro-scale.

Functional photoacoustic imaging is a promising biological imaging technique that offers such unique benefits as scalable resolution and imaging depth, as well as the ability to provide functional information. At nanoscale, photoacoustic imaging has provided super-resolution images of the surface light absorption characteristics of materials and of single organelles in cells. At the microscopic and macroscopic scales. photoacoustic imaging techniques have precisely measured and quantified various physiological parameters, such as oxygen saturation, vessel morphology, blood flow, and the metabolic rate of oxygen, in both human and animal subjects. This comprehensive review provides an overview of functional photoacoustic imaging across multiple scales, from nano to macro, and highlights recent advances in technology developments and applications. Finally, the review surveys the future prospects of functional photoacoustic imaging in the biomedical field.

Panoramic volumetric clinical handheld photoacoustic and ultrasound imaging.

Photoacoustic (PA) imaging has gained much attention, providing structural and functional information in combination with clinical ultrasound (US) imaging systems. 2D PA and US imaging is easily implemented, but its heavy dependence on operator skills makes 3D imaging preferable. In this study, we propose a panoramic volumetric clinical PA and US imaging system equipping a handheld imaging scanner weighing 600 g and measuring 70 × 62 × 110 mm3. Multiple PA/US scans were performed to cover a large field-of-view (FOV), and the acquired PA/US volumes were mosaic-stitched after manually correcting the positions and rotations in a total of 6 degrees of freedom. PA and US maximum amplitude projection images were visualized online, while spectral unmixed data was quantified offline. The performance of the system was tested via tissue-mimicking phantom experiments. The system's potential was confirmed in vivo by panoramically imaging vascular networks in human arms and necks, with FOVs of 331 × 38 and 129 × 120 mm2, respectively. Further, we quantified hemoglobin oxygen saturation levels in the radial artery, brachial artery, carotid artery, and jugular vein. We hope that this system can be applied for various clinical fields such as cardiovascular imaging, dermatology, vascular surgery, internal medicine, and oncology.