Acoustofluidic scanning fluorescence nanoscopy with a large field of view.

Large-field nanoscale fluorescence imaging is invaluable for many applications, such as imaging subcellular structures, visualizing protein interactions, and high-resolution tissue imaging. Unfortunately, conventional fluorescence microscopy requires a trade-off between resolution and field of view due to the nature of the optics used to form the image. To overcome this barrier, we developed an acoustofluidic scanning fluorescence nanoscope that simultaneously achieves superior resolution, a large field of view, and strong fluorescent signals. The acoustofluidic scanning fluorescence nanoscope utilizes the superresolution capabilities of microspheres that are controlled by a programmable acoustofluidic device for rapid fluorescence enhancement and imaging. The acoustofluidic scanning fluorescence nanoscope resolves structures that cannot be resolved with conventional fluorescence microscopes with the same objective lens and enhances the fluorescent signal by a factor of ~5 without altering the field of view of the image. The improved resolution realized with enhanced fluorescent signals and the large field of view achieved via acoustofluidic scanning fluorescence nanoscopy provides a powerful tool for versatile nanoscale fluorescence imaging for researchers in the fields of medicine, biology, biophysics, and biomedical engineering.

The impact of package selection and versioning on single-cell RNA-seq analysis.

Standard single-cell RNA-sequencing analysis (scRNA-seq) workflows consist of converting raw read data into cell-gene count matrices through sequence alignment, followed by analyses including filtering, highly variable gene selection, dimensionality reduction, clustering, and differential expression analysis. Seurat and Scanpy are the most widely-used packages implementing such workflows, and are generally thought to implement individual steps similarly. We investigate in detail the algorithms and methods underlying Seurat and Scanpy and find that there are, in fact, considerable differences in the outputs of Seurat and Scanpy. The extent of differences between the programs is approximately equivalent to the variability that would be introduced in benchmarking scRNA-seq datasets by sequencing less than 5% of the reads or analyzing less than 20% of the cell population. Additionally, distinct versions of Seurat and Scanpy can produce very different results, especially during parts of differential expression analysis. Our analysis highlights the need for users of scRNA-seq to carefully assess the tools on which they rely, and the importance of developers of scientific software to prioritize transparency, consistency, and reproducibility for their tools.

Acoustothermal transfection for cell therapy.

Transfected stem cells and T cells are promising in personalized cell therapy and immunotherapy against various diseases. However, existing transfection techniques face a fundamental trade-off between transfection efficiency and cell viability; achieving both simultaneously remains a substantial challenge. This study presents an acoustothermal transfection method that leverages acoustic and thermal effects on cells to enhance the permeability of both the cell membrane and nuclear envelope to achieve safe, efficient, and high-throughput transfection of primary T cells and stem cells. With this method, two types of plasmids were simultaneously delivered into the nuclei of mesenchymal stem cells (MSCs) with efficiencies of 89.6 ± 1.2%. CXCR4-transfected MSCs could efficiently target cerebral ischemia sites in vivo and reduce the infarct volume in mice. Our acoustothermal transfection method addresses a key bottleneck in balancing the transfection efficiency and cell viability, which can become a powerful tool in the future for cellular and gene therapies.

Assessing the agreement of chronic lung disease of prematurity diagnosis between radiologists and clinical criteria.

BACKGROUND: Chronic lung disease of prematurity (CLD) is the most prevalent complication of preterm birth and indicates an increased likelihood of long-term pulmonary complications. The accurate diagnosis of this condition is critical for long-term health management. Numerous definitions define CLD with different clinical parameters and radiology findings, making diagnosis of the disease ambiguous and potentially inaccurate. METHODS: 95 patients were identified for this study, as determined by the diagnosis or confirmation of CLD in the impression of the radiologist's report on chest x-ray. Pulmonary function and complications were recorded at multiple benchmark timeframes within each patient's first few months of life and used for determining eligibility under each definition. RESULTS: Each clinical definition of CLD had a high sensitivity for patients identified to have CLD by radiologists, correctly fitting over 90% of patients. Most patients included required invasive mechanical ventilation or positive pressure ventilation at 36 weeks postmenstrual age, indicating patients with radiographically confirmed CLD tended to have more severe disease. Radiologists tended to diagnose CLD before 36 weeks postmenstrual age, a timepoint used by multiple standard clinical definitions, with cases called earlier fitting under a larger percentage of definitions than those called later. CONCLUSIONS: Radiologists tend to diagnose CLD in young patients with severe respiratory compromise, and can accurately diagnose the condition before developmental milestones for clinical definitions are met.

High-yield and rapid isolation of extracellular vesicles by flocculation via orbital acoustic trapping: FLOAT.

Extracellular vesicles (EVs) have been identified as promising biomarkers for the noninvasive diagnosis of various diseases. However, challenges in separating EVs from soluble proteins have resulted in variable EV recovery rates and low purities. Here, we report a high-yield ( > 90%) and rapid ( < 10 min) EV isolation method called FLocculation via Orbital Acoustic Trapping (FLOAT). The FLOAT approach utilizes an acoustofluidic droplet centrifuge to rotate and controllably heat liquid droplets. By adding a thermoresponsive polymer flocculant, nanoparticles as small as 20 nm can be rapidly and selectively concentrated at the center of the droplet. We demonstrate the ability of FLOAT to separate urinary EVs from the highly abundant Tamm-Horsfall protein, addressing a significant obstacle in the development of EV-based liquid biopsies. Due to its high-yield nature, FLOAT reduces biofluid starting volume requirements by a factor of 100 (from 20 mL to 200 µL), demonstrating its promising potential in point-of-care diagnostics.

Aerosol jet printing of surface acoustic wave microfluidic devices.

The addition of surface acoustic wave (SAW) technologies to microfluidics has greatly advanced lab-on-a-chip applications due to their unique and powerful attributes, including high-precision manipulation, versatility, integrability, biocompatibility, contactless nature, and rapid actuation. However, the development of SAW microfluidic devices is limited by complex and time-consuming micro/nanofabrication techniques and access to cleanroom facilities for multistep photolithography and vacuum-based processing. To simplify the fabrication of SAW microfluidic devices with customizable dimensions and functions, we utilized the additive manufacturing technique of aerosol jet printing. We successfully fabricated customized SAW microfluidic devices of varying materials, including silver nanowires, graphene, and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS). To characterize and compare the acoustic actuation performance of these aerosol jet printed SAW microfluidic devices with their cleanroom-fabricated counterparts, the wave displacements and resonant frequencies of the different fabricated devices were directly measured through scanning laser Doppler vibrometry. Finally, to exhibit the capability of the aerosol jet printed devices for lab-on-a-chip applications, we successfully conducted acoustic streaming and particle concentration experiments. Overall, we demonstrated a novel solution-based, direct-write, single-step, cleanroom-free additive manufacturing technique to rapidly develop SAW microfluidic devices that shows viability for applications in the fields of biology, chemistry, engineering, and medicine.

Advances in Microsphere-based Super-resolution Imaging.

Techniques to resolve images beyond the diffraction limit of light with a large field of view (FOV) are necessary to foster progress in various fields such as cell and molecular biology, biophysics, and nanotechnology, where nanoscale resolution is crucial for understanding the intricate details of large-scale molecular interactions. Although several means of achieving super-resolutions exist, they are often hindered by factors such as high costs, significant complexity, lengthy processing times, and the classical tradeoff between image resolution and FOV. Microsphere-based super-resolution imaging has emerged as a promising approach to address these limitations. In this review, we delve into the theoretical underpinnings of microsphere-based imaging and the associated photonic nanojet. This is followed by a comprehensive exploration of various microsphere-based imaging techniques, encompassing static imaging, mechanical scanning, optical scanning, and acoustofluidic scanning methodologies. This review concludes with a forward-looking perspective on the potential applications and future scientific directions of this innovative technology.

Comprehensive Systematic Review of Biomarkers in Metastatic Renal Cell Carcinoma: Predictors, Prognostics, and Therapeutic Monitoring.

BACKGROUND: Challenges remain in determining the most effective treatment strategies and identifying patients who would benefit from adjuvant or neoadjuvant therapy in renal cell carcinoma. The objective of this review is to provide a comprehensive overview of biomarkers in metastatic renal cell carcinoma (mRCC) and their utility in prediction of treatment response, prognosis, and therapeutic monitoring in patients receiving systemic therapy for metastatic disease. METHODS: A systematic literature search was conducted using the PubMed database for relevant studies published between January 2017 and December 2022. The search focused on biomarkers associated with mRCC and their relationship to immune checkpoint inhibitors, targeted therapy, and VEGF inhibitors in the adjuvant, neoadjuvant, and metastatic settings. RESULTS: The review identified various biomarkers with predictive, prognostic, and therapeutic monitoring potential in mRCC. The review also discussed the challenges associated with anti-angiogenic and immune-checkpoint monotherapy trials and highlighted the need for personalized therapy based on molecular signatures. CONCLUSION: This comprehensive review provides valuable insights into the landscape of biomarkers in mRCC and their potential applications in prediction of treatment response, prognosis, and therapeutic monitoring. The findings underscore the importance of incorporating biomarker assessment into clinical practice to guide treatment decisions and improve patient outcomes in mRCC.

Feminizing Adrenocortical Tumor with Multiple Recurrences: A Case Report.

Feminizing adrenocortical tumors (FATs) are exceptionally rare primary adrenal neoplasms that cause high estrogen and low testosterone levels. They are most common in adult males, typically presenting with gynecomastia, hypogonadism, and weight loss. They are almost always malignant, with a poor prognosis and a high recurrence rate. We report a case of a 35-year-old man with an adrenal FAT with high estrogen (181 pg/mL) and low testosterone (37 ng/dL) who presented with gynecomastia, erectile dysfunction, subclinical Cushing syndrome, and pain localizing to different regions of the torso. There was no evidence of metastatic disease initially as seen by visualization of a well-marginated mass on computed tomography scan. Surgical resection of the FAT was performed, and the mass was confirmed to be a low-grade tumor. Clinical symptoms were resolved after surgery. Despite complete resection with negative margins, the patient subsequently had two separate local metastatic recurrences within a few years, treated with a combination of further surgery and medical intervention. This case highlights the unique features of an exceedingly rare adrenal tumor and stresses the importance of early detection and vigilant surveillance following resection due to high recurrence rates.

Localized Multifocal Retroperitoneal Ganglioneuroma with an Infiltrative Appearance on Imaging: A Case Report.

Multifocal ganglioneuromas are characterized by the presence of multiple benign neuroepithelial tumor nodules and are less common than solitary tumors. A small percentage of ganglioneuromas present with a fatty appearance. Only a few cases of multifocal ganglioneuromas have been reported, due to both their rarity and minimal symptomatic presentation; therefore, generalizations about risk factors and predictive markers are very difficult. Here, we report a case of multifocal retroperitoneal ganglioneuroma with an infiltrative appearance on computed tomography (CT). The tumor demonstrated slow growth on multiple imaging studies and was associated with abdominal and flank pain. The aggressive appearance eventually led to surgical resection 18 months after the initial incidental finding on CT. Postsurgical analysis of the tumor on imaging was crucial in revealing its nodularity and infiltration, as well as for clarifying its retroperitoneal location inseparable from the adrenal gland. Histology demonstrated Schwann cells and ganglion cells without atypia or increased cellularity, and with no mitosis or necrosis seen. Our case highlights the consideration of ganglioneuroma with fatty infiltration in the differential diagnosis of a fatty tumor in the mediastinum or retroperitoneum. Additionally, our report differentiates multifocal ganglioneuroma with fatty infiltration from lipomatous ganglioneuroma on radiology and histopathology.

Deep learning image segmentation approaches for malignant bone lesions: a systematic review and meta-analysis.

Introduction: Image segmentation is an important process for quantifying characteristics of malignant bone lesions, but this task is challenging and laborious for radiologists. Deep learning has shown promise in automating image segmentation in radiology, including for malignant bone lesions. The purpose of this review is to investigate deep learning-based image segmentation methods for malignant bone lesions on Computed Tomography (CT), Magnetic Resonance Imaging (MRI), and Positron-Emission Tomography/CT (PET/CT). Method: The literature search of deep learning-based image segmentation of malignant bony lesions on CT and MRI was conducted in PubMed, Embase, Web of Science, and Scopus electronic databases following the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). A total of 41 original articles published between February 2017 and March 2023 were included in the review. Results: The majority of papers studied MRI, followed by CT, PET/CT, and PET/MRI. There was relatively even distribution of papers studying primary vs. secondary malignancies, as well as utilizing 3-dimensional vs. 2-dimensional data. Many papers utilize custom built models as a modification or variation of U-Net. The most common metric for evaluation was the dice similarity coefficient (DSC). Most models achieved a DSC above 0.6, with medians for all imaging modalities between 0.85-0.9. Discussion: Deep learning methods show promising ability to segment malignant osseous lesions on CT, MRI, and PET/CT. Some strategies which are commonly applied to help improve performance include data augmentation, utilization of large public datasets, preprocessing including denoising and cropping, and U-Net architecture modification. Future directions include overcoming dataset and annotation homogeneity and generalizing for clinical applicability.

Acoustic tweezers for high-throughput single-cell analysis.

Acoustic tweezers provide an effective means for manipulating single cells and particles in a high-throughput, precise, selective and contact-free manner. The adoption of acoustic tweezers in next-generation cellular assays may advance our understanding of biological systems. Here we present a comprehensive set of instructions that guide users through device fabrication, instrumentation setup and data acquisition to study single cells with an experimental throughput that surpasses traditional methods, such as atomic force microscopy and micropipette aspiration, by several orders of magnitude. With acoustic tweezers, users can conduct versatile experiments that require the trapping, patterning, pairing and separation of single cells in a myriad of applications ranging across the biological and biomedical sciences. This procedure is widely generalizable and adaptable for investigations in materials and physical sciences, such as the spinning motion of colloids or the development of acoustic-based quantum simulations. Overall, the device fabrication requires ~12 h, the experimental setup of the acoustic tweezers requires 1-2 h and the cell manipulation experiment requires ~30 min to complete. Our protocol is suitable for use by interdisciplinary researchers in biology, medicine, engineering and physics.

Acoustofluidic scanning fluorescence nanoscopy with large field of view.

Nanoscale fluorescence imaging with a large-field view is invaluable for many applications such as imaging of subcellular structures, visualizing protein interaction, and high-resolution tissue imaging. Unfortunately, conventional fluorescence microscopy has to make a trade-off between resolution and field of view due to the nature of the optics used to form an image. To overcome this barrier, we have developed an acoustofluidic scanning fluorescence nanoscope that can simultaneously achieve superior resolution, a large field of view, and enhanced fluorescent signal. The acoustofluidic scanning fluorescence nanoscope utilizes the super-resolution capability of microspheres that are controlled by a programable acoustofluidic device for rapid fluorescent enhancement and imaging. The acoustofluidic scanning fluorescence nanoscope can resolve structures that cannot be achieved with a conventional fluorescent microscope with the same objective lens and enhances the fluorescent signal by a factor of ~5 without altering the field of view of the image. The improved resolution with enhanced fluorescent signal and large field of view via the acoustofluidic scanning fluorescence nanoscope provides a powerful tool for versatile nanoscale fluorescence imaging for researchers in the fields of medicine, biology, biophysics, and biomedical engineering.

Acousto-dielectric tweezers for size-insensitive manipulation and biophysical characterization of single cells.

The intrinsic biophysical properties of cells, such as mechanical, acoustic, and electrical properties, are valuable indicators of a cell's function and state. However, traditional single-cell biophysical characterization methods are hindered by limited measurable properties, time-consuming procedures, and complex system setups. This study presents acousto-dielectric tweezers that leverage the balance between controllable acoustophoretic and dielectrophoretic forces applied on cells through surface acoustic waves and alternating current electric fields, respectively. Particularly, the balanced acoustophoretic and dielectrophoretic forces can trap cells at equilibrium positions independent of the cell size to differentiate between various cell-intrinsic mechanical, acoustic, and electrical properties. Experimental results show our mechanism has the potential for applications in single-cell analysis, size-insensitive cell separation, and cell phenotyping, which are all primarily based on cells' intrinsic biophysical properties. Our results also show the measured equilibrium position of a cell can inversely determine multiple biophysical properties, including membrane capacitance, cytoplasm conductivity, and acoustic contrast factor. With these features, our acousto-dielectric tweezing mechanism is a valuable addition to the resources available for biophysical property-based biological and medical research.

Modification of Deoxynivalenol by a Fungal Laccase Paired with Redox Mediator TEMPO.

Mycotoxins such as deoxynivalenol introduce a health risk to the food supply and are costly to manage or avoid. Technologies for reducing or eliminating the toxicity of deoxynivalenol could be useful in a variety of processes, such as in preserving the value as animal feed of byproducts of ethanol production. We characterized transformation products of deoxynivalenol that were formed by the combination of a fungal laccase paired with the chemical mediator 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), using chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. Alcohol groups at the C3 and C15 positions of deoxynivalenol were oxidized to ketones, and the chemical mediator became covalently linked to the C4 position. Conditions experienced during gas chromatography led to the dissociation of TEMPO, forming 3,15-diketodeoxynivalenol. Understanding the range of possible modifications to deoxynivalenol and other trichothecenes is a necessary step toward effective remediation of contaminated grain.

An acoustofluidic scanning nanoscope using enhanced image stacking and processing.

Nanoscale optical resolution with a large field of view is a critical feature for many research and industry areas, such as semiconductor fabrication, biomedical imaging, and nanoscale material identification. Several scanning microscopes have been developed to resolve the inverse relationship between the resolution and field of view; however, those scanning microscopes still rely upon fluorescence labeling and complex optical systems. To overcome these limitations, we developed a dual-camera acoustofluidic nanoscope with a seamless image merging algorithm (alpha-blending process). This design allows us to precisely image both the sample and the microspheres simultaneously and accurately track the particle path and location. Therefore, the number of images required to capture the entire field of view (200 × 200 µm) by using our acoustofluidic scanning nanoscope is reduced by 55-fold compared with previous designs. Moreover, the image quality is also greatly improved by applying an alpha-blending imaging technique, which is critical for accurately depicting and identifying nanoscale objects or processes. This dual-camera acoustofluidic nanoscope paves the way for enhanced nanoimaging with high resolution and a large field of view.

Intelligent nanoscope for rapid nanomaterial identification and classification.

Machine learning image recognition and classification of particles and materials is a rapidly expanding field. However, nanomaterial identification and classification are dependent on the image resolution, the image field of view, and the processing time. Optical microscopes are one of the most widely utilized technologies in laboratories across the world, due to their nondestructive abilities to identify and classify critical micro-sized objects and processes, but identifying and classifying critical nano-sized objects and processes with a conventional microscope are outside of its capabilities, due to the diffraction limit of the optics and small field of view. To overcome these challenges of nanomaterial identification and classification, we developed an intelligent nanoscope that combines machine learning and microsphere array-based imaging to: (1) surpass the diffraction limit of the microscope objective with microsphere imaging to provide high-resolution images; (2) provide large field-of-view imaging without the sacrifice of resolution by utilizing a microsphere array; and (3) rapidly classify nanomaterials using a deep convolution neural network. The intelligent nanoscope delivers more than 46 magnified images from a single image frame so that we collected more than 1000 images within 2 seconds. Moreover, the intelligent nanoscope achieves a 95% nanomaterial classification accuracy using 1000 images of training sets, which is 45% more accurate than without the microsphere array. The intelligent nanoscope also achieves a 92% bacteria classification accuracy using 50 000 images of training sets, which is 35% more accurate than without the microsphere array. This platform accomplished rapid, accurate detection and classification of nanomaterials with miniscule size differences. The capabilities of this device wield the potential to further detect and classify smaller biological nanomaterial, such as viruses or extracellular vesicles.

Acoustofluidics for simultaneous nanoparticle-based drug loading and exosome encapsulation.

Nanocarrier and exosome encapsulation has been found to significantly increase the efficacy of targeted drug delivery while also minimizing unwanted side effects. However, the development of exosome-encapsulated drug nanocarriers is limited by low drug loading efficiencies and/or complex, time-consuming drug loading processes. Herein, we have developed an acoustofluidic device that simultaneously performs both drug loading and exosome encapsulation. By synergistically leveraging the acoustic radiation force, acoustic microstreaming, and shear stresses in a rotating droplet, the concentration, and fusion of exosomes, drugs, and porous silica nanoparticles is achieved. The final product consists of drug-loaded silica nanocarriers that are encased within an exosomal membrane. The drug loading efficiency is significantly improved, with nearly 30% of the free drug (e.g., doxorubicin) molecules loaded into the nanocarriers. Furthermore, this acoustofluidic drug loading system circumvents the need for complex chemical modification, allowing drug loading and encapsulation to be completed within a matter of minutes. These exosome-encapsulated nanocarriers exhibit excellent efficiency in intracellular transport and are capable of significantly inhibiting tumor cell proliferation. By utilizing physical forces to rapidly generate hybrid nanocarriers, this acoustofluidic drug loading platform wields the potential to significantly impact innovation in both drug delivery research and applications.

Sonoporation: Past, Present, and Future.

A surge of research in intracellular delivery technologies is underway with the increased innovations in cell-based therapies and cell reprogramming. Particularly, physical cell membrane permeabilization techniques are highlighted as the leading technologies because of their unique features, including versatility, independence of cargo properties, and high-throughput delivery that is critical for providing the desired cell quantity for cell-based therapies. Amongst the physical permeabilization methods, sonoporation holds great promise and has been demonstrated for delivering a variety of functional cargos, such as biomolecular drugs, proteins, and plasmids, to various cells including cancer, immune, and stem cells. However, traditional bubble-based sonoporation methods usually require special contrast agents. Bubble-based sonoporation methods also have high chances of inducing irreversible damage to critical cell components, lowering the cell viability, and reducing the effectiveness of delivered cargos. To overcome these limitations, several novel non-bubble-based sonoporation mechanisms are under development. This review will cover both the bubble-based and non-bubble-based sonoporation mechanisms being employed for intracellular delivery, the technologies being investigated to overcome the limitations of traditional platforms, as well as perspectives on the future sonoporation mechanisms, technologies, and applications.

Adherence, Sexual Behavior and Sexually Transmitted Infections in a New Zealand Prospective PrEP Cohort: 12 Months Follow-up and Ethnic Disparities.

Inequities in pre-exposure prophylaxis (PrEP) experiences will impede HIV epidemic elimination among gay and bisexual men (GBM). Ethnicity is a strong marker of inequity in the United States, but evidence from other countries is lacking. We investigated experiences on-PrEP to 12 months follow-up in a prospective cohort of 150 GBM in Auckland, New Zealand with an equity quota of 50% non-Europeans. Retention at 12 months was 85.9%, lower among Maori/Pacific (75.6%) than non-Maori/Pacific participants (90.1%). Missed pills increased over time and were higher among Maori/Pacific. PrEP breaks increased, by 12 months 35.7% of Maori/Pacific and 15.7% of non-Maori/Pacific participants had done so. Condomless receptive anal intercourse partners were stable over time. STIs were common but chlamydia declined; 12-month incidence was 8.7% for syphilis, 36.0% gonorrhoea, 46.0% chlamydia, 44.7% rectal STI, 64.0% any STI. Structural interventions and delivery innovations are needed to ensure ethnic minority GBM gain equal benefit from PrEP.Clinical trial number ACTRN12616001387415.