High-resolution Cell Imaging Research Articles

Real-Time Resolution Enhancement of Confocal Laser Scanning Microscopy via Deep Learning

Confocal laser scanning microscopy is one of the most widely used tools for high-resolution imaging of biological cells. However, the imaging resolution of conventional confocal technology is limited by diffraction, and more complex optical principles and expensive optical-mechanical structures are usually required to improve the resolution. This study proposed a deep residual neural network algorithm that can effectively improve the imaging resolution of the confocal microscopy in real time. The reliability and real-time performance of the algorithm were verified through imaging experiments on different biological structures, and an imaging resolution of less than 120 nm was achieved in a more cost-effective manner. This study contributes to the real-time improvement of the imaging resolution of confocal microscopy and expands the application scenarios of confocal microscopy in biological imaging.

Unpaired data training enables super-resolution confocal microscopy from low-resolution acquisitions.

Supervised deep-learning models have enabled super-resolution imaging in several microscopic imaging modalities, increasing the spatial lateral bandwidth of the original input images beyond the diffraction limit. Despite their success, their practical application poses several challenges in terms of the amount of training data and its quality, requiring the experimental acquisition of large, paired databases to generate an accurate generalized model whose performance remains invariant to unseen data. Cycle-consistent generative adversarial networks (cycleGANs) are unsupervised models for image-to-image translation tasks that are trained on unpaired datasets. This paper introduces a cycleGAN framework specifically designed to increase the lateral resolution limit in confocal microscopy by training a cycleGAN model using low- and high-resolution unpaired confocal images of human glioblastoma cells. Training and testing performances of the cycleGAN model have been assessed by measuring specific metrics such as background standard deviation, peak-to-noise ratio, and a customized frequency content measure. Our cycleGAN model has been evaluated in terms of image fidelity and resolution improvement using a paired dataset, showing superior performance than other reported methods. This work highlights the efficacy and promise of cycleGAN models in tackling super-resolution microscopic imaging without paired training, paving the path for turning home-built low-resolution microscopic systems into low-cost super-resolution instruments by means of unsupervised deep learning.

Beyond neurons: computer vision methods for analysis of morphologically complex astrocytes

The study of the geometric organization of biological tissues has a rich history in the literature. However, the geometry and architecture of individual cells within tissues has traditionally relied upon manual or indirect measures of shape. Such rudimentary measures are largely a result of challenges associated with acquiring high resolution images of cells and cellular components, as well as a lack of computational approaches to analyze large volumes of high-resolution data. This is especially true with brain tissue, which is composed of a complex array of cells. Here we review computational tools that have been applied to unravel the cellular nanoarchitecture of astrocytes, a type of brain cell that is increasingly being shown to be essential for brain function. Astrocytes are among the most structurally complex and functionally diverse cells in the mammalian body and are essential partner cells of neurons. Light microscopy does not allow adequate resolution of astrocyte morphology, however, large-scale serial electron microscopy data, which provides nanometer resolution 3D models, is enabling the visualization of the fine, convoluted structure of astrocytes. Application of computer vision methods to the resulting nanoscale 3D models is helping reveal the geometry and organizing principles of astrocytes, but a complete understanding of astrocyte structure and its functional implications will require further adaptation of existing computational tools, as well as development of new approaches.

Altered Endoplasmic Reticulum Integrity and Organelle Interactions in Living Cells Expressing INF2 Variants.

The cytoskeleton mediates fundamental cellular processes by organizing inter-organelle interactions. Pathogenic variants of inverted formin 2 (INF2) CAAX isoform, an actin assembly factor that is predominantly expressed in the endoplasmic reticulum (ER), are linked to focal segmental glomerulosclerosis (FSGS) and Charcot-Marie-Tooth (CMT) neuropathy. To investigate how pathogenic INF2 variants alter ER integrity, we used high-resolution live imaging of HeLa cells. Cells expressing wild-type (WT) INF2 showed a predominant tubular ER with perinuclear clustering. Cells expressing INF2 FSGS variants that cause mild and intermediate disease induced more sheet-like ER, a pattern similar to that seen for cells expressing WT-INF2 that were treated with actin and microtubule (MT) inhibitors. Dual CMT-FSGS INF2 variants led to more severe ER dysmorphism, with a diffuse, fragmented ER and coarse INF2 aggregates. Proper organization of both F-actin and MT was needed to modulate the tubule vs. sheet conformation balance, while MT arrays regulated spatial expansion of tubular ER in the cell periphery. Pathogenic INF2 variants also induced mitochondria fragmentation and dysregulated mitochondria distribution. Such mitochondrial abnormalities were more prominent for cells expressing CMT-FSGS compared to those with FSGS variants, indicating that the severity of the dysfunction is linked to the degree of cytoskeletal disorganization. Our observations suggest that pathogenic INF2 variants disrupt ER continuity by altering interactions between the ER and the cytoskeleton that in turn impairs inter-organelle communication, especially at ER-mitochondria contact sites. ER continuity defects may be a common disease mechanism involved in both peripheral neuropathy and glomerulopathy.

Japanese encephalitis virus hijacks ER-associated degradation regulators for its replication.

Flaviviruses target their replication on membranous structures derived from the ER, where both viral and host proteins play crucial structural and functional roles. Here, we have characterized the involvement of the ER-associated degradation (ERAD) pathway core E3 ligase complex (SEL1L-HRD1) regulator proteins in the replication of Japanese encephalitis virus (JEV). Through high-resolution immunofluorescence imaging of JEV-infected HeLa cells, we observe that the virus replication complexes marked by NS1 strongly colocalize with the ERAD adapter SEL1L, lectin OS9, ER-membrane shuttle factor HERPUD1, E3 ubiquitin ligase HRD1 and rhomboid superfamily member DERLIN1. NS5 positive structures also show strong overlap with SEL1L. While these effectors show significant transcriptional upregulation, their protein levels remain largely stable in infected cells. siRNA mediated depletion of OS9, SEL1L, HERPUD1 and HRD1 significantly inhibit viral RNA replication and titres, with SEL1L depletion showing the maximum attenuation of replication. By performing protein translation arrest experiments, we show that SEL1L, and OS9 are stabilised upon JEV infection. Overall results from this study suggest that these ERAD effector proteins are crucial host-factors for JEV replication.

Ouabain modulates collective cell migration via a Na,K-ATPase activated Ca2+ signal pathway

Collective cell migration is important for many physiological and pathological processes. Ouabain, a cardiotonic steroid and specific ligand to Na,K-ATPase, has been reported to modulate collective cell migration, inhibiting it in cancer cells, and increasing it in normal cells. However, it is not clear what the mechanism is behind this difference in response. It is well-documented that ouabain blocks the pump function of Na,K-ATPase. At low concentrations this activates on the cellular level a Ca2+ mediated signaling chain with multiple downstream effects, including adhesion and migration pathways. At higher concentrations the inhibition of Na+ and K+ transport dominates the effect on the cellular level. We hypothesize that the difference in migration response of cancer and normal cells to ouabain is due to a difference in the Ca2+ signaling response. We further hypothesize that the spread and amplification of Ca2+signals between cells via gap junctions is upregulated by ouabain. To test the hypothesis, we made scratch assays in cultures of MDCK cells and studied migration rate and Ca2+signaling. 10 nM ouabain increases the migration rate, an effect that is inhibited by 2-APB, that blocks the IP3R Ca2+ release from ER. The effect on migration is also blocked by KN-93 that inhibits Ca2+ activated CAMKII. Interestingly, ouabain at 200 nM concentration decreased the migration rate compared to untreated cells. To analyze the strength of intercellular communication we analyzed the spread and synchronicity of spontaneous Ca2+ signals between neighboring cells. For this we used MDCK cells stable transfected with the Ca2+ sensitive fluorescent protein GCamp6f. Cells were treated with vehicle, ouabain 10 nM, or heptanol (blocks gap junctions). Timelapse Ca2+ imaging for 10-30 minutes was done. Machine learning based image analysis with Cellpose was then used to segment cells, followed by cross correlation of the integrated Ca2+signals from neighboring cells. Despite findings from previous phosphoproteomic studies indicating that there should be an upregulation of gap junctions, no conclusive differences in intercellular signaling could be identified for the different treatments. Finally, we made high resolution imaging of cells immunolabeled for vinculin and FAK, two proteins that are involved in migration and adhesion. We found ouabain correlated effects on the localization of vinculin and FAK with a polarized and concentrated localization after high dose ouabain, compared to a diffuse and unpolarized localization after low dose ouabain. Taken together we suggest that low dose ouabain via Na,K-ATPase triggers a Ca2+ signal that destabilize cell adhesions and that this is an important mechanism contributing to the observed upregulation of cell migration. Marta and Gunnar V Philipson's foundation, and VR 2020-05347 (Swedish Research Council). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Two-Photon-Responsive "TICT + AIE" Active Naphthyridine-BF2 Photoremovable Protecting Group: Application for Specific Staining and Killing of Cancer Cells.

The combined effects of twisted intramolecular charge transfer (TICT) and aggregation-induced emission (AIE) phenomena have demonstrated a significant influence on excited-state chemistry. These combined TICT and AIE features have been extensively utilized to enhance photodynamic and photothermal therapy. Herein, we demonstrated the synergistic capabilities of TICT and AIE phenomena in the design of the photoremovable protecting group (PRPG), namely, NMe2-Napy-BF2. This innovative PRPG incorporates TICT and AIE characteristics, resulting in four remarkable properties: (i) red-shifted absorption wavelength, (ii) strong near-infrared (NIR) emission, (iii) viscosity-sensitive emission property, and (iv) accelerated photorelease rate. Inspired by these intriguing attributes, we developed a nanodrug delivery system (nano-DDS) using our PRPG for cancer treatment. In vitro studies showed that our nano-DDS manifested effective cellular internalization, specific staining of cancer cells, high-resolution confocal imaging of cancerous cells in the NIR region, and controlled release of the anticancer drug chlorambucil upon exposure to light, leading to cancer cell eradication. Most notably, our nano-DDS exhibited a substantially increased two-photon (TP) absorption cross section (435 GM), exhibiting its potential for in vivo applications. This development holds promise for significant advancements in cancer treatment strategies.

High-resolution cell imaging using white light phase shifting interferometry and iterative phase deconvolution.

An optimization algorithm is presented for the deconvolution of a complex field to improve the resolution and accuracy of quantitative phase imaging (QPI). A high-resolution phase map can be recovered by solving a constrained optimization problem of deconvolution using a complex gradient operator. The method is demonstrated on phase measurements of samples using a white light based phase shifting interferometry (WLPSI) method. The application of the algorithm on real and simulated objects shows a significant resolution and contrast improvement. Experiments performed on Escherichia coli bacterium have revealed its sub-cellular structures that were not visible in the raw WLPSI images obtained using a five phase shifting method. These features can give valuable insights into the structures and functioning of biological cells. The algorithm is simple in implementation and can be incorporated into other QPI modalities .

Recent advances in SERS-based bioanalytical applications: live cell imaging

Abstract Raman scattering can provide information on molecular fingerprints, which have been widely applied in various fields of material science and nanobiotechnology. Notably, low interference with water molecules in obtaining the Raman spectra between 500 and 2000 cm−1 made it a powerful spectroscopic tool in biology, such as imaging and signaling for a living cell. To be a robust tool for cell biology, the performance of obtaining molecular-specific information with high sensitivity, high resolution in real time, and without inducing cell damage is strongly required. The conventional fluorescence-based method has been suffered from the rapid photobleaching of organic fluorophores and the lack of molecular information. In contrast, Raman scattering is a promising spectroscopic tool to acquire cellular information, and the extremely low signal intensity of Raman scattering could be amplified by incorporating the plasmonic nanomaterials. Along with the fundamental research focus on surface-enhanced Raman scattering (SERS), the practical approaches of SERS for cellular imaging as a new tool for drug screening and monitoring cellular signals have been extensively explored based on new optical setups and new designing strategies for the nanostructures. Diverse nanostructure and surface chemistry for targeting or sensing have been played pivotal roles in acquiring cellular information and high resolution cell imaging. In this regard, this review focused on the recent advances of SERS-based technologies for a live cell imaging investigated such as potential drug screening, signaling for chemicals or biomolecules in cell, in situ sensing, and high spatiotemporal resolution.

Comparative analysis of human, rodent and snake deltavirus replication.

The recent discovery of Hepatitis D (HDV)-like viruses across a wide range of taxa led to the establishment of the Kolmioviridae family. Recent studies suggest that kolmiovirids can be satellites of viruses other than Hepatitis B virus (HBV), challenging the strict HBV/HDV-association dogma. Studying whether kolmiovirids are able to replicate in any animal cell they enter is essential to assess their zoonotic potential. Here, we compared replication of three kolmiovirids: HDV, rodent (RDeV) and snake (SDeV) deltavirus in vitro and in vivo. We show that SDeV has the narrowest and RDeV the broadest host cell range. High resolution imaging of cells persistently replicating these viruses revealed nuclear viral hubs with a peculiar RNA-protein organization. Finally, in vivo hydrodynamic delivery of viral replicons showed that both HDV and RDeV, but not SDeV, efficiently replicate in mouse liver, forming massive nuclear viral hubs. Our comparative analysis lays the foundation for the discovery of specific host factors controlling Kolmioviridae host-shifting.

Carlos Gutiérrez-Merino: Synergy of Theory and Experimentation in Biological Membrane Research.

Professor Carlos Gutiérrez-Merino, a prominent scientist working in the complex realm of biological membranes, has made significant theoretical and experimental contributions to the field. Contemporaneous with the development of the fluid-mosaic model of Singer and Nicolson, the Förster resonance energy transfer (FRET) approach has become an invaluable tool for studying molecular interactions in membranes, providing structural insights on a scale of 1-10 nm and remaining important alongside evolving perspectives on membrane structures. In the last few decades, Gutiérrez-Merino's work has covered multiple facets in the field of FRET, with his contributions producing significant advances in quantitative membrane biology. His more recent experimental work expanded the ground concepts of FRET to high-resolution cell imaging. Commencing in the late 1980s, a series of collaborations between Gutiérrez-Merino and the authors involved research visits and joint investigations focused on the nicotinic acetylcholine receptor and its relation to membrane lipids, fostering a lasting friendship.

Applications of scanning probe microscopy in neuroscience research

Abstract Scanning probe microscopy techniques allow for label-free high-resolution imaging of cells, tissues, and biomolecules in physiologically relevant conditions. These techniques include atomic force microscopy (AFM), atomic force spectroscopy, and Kelvin probe force microscopy, which enable high resolution imaging, nanomanipulation and measurement of the mechanoelastic properties of neuronal cells, as well as scanning ion conductance microscopy, which combines electrophysiology and imaging in living cells. The combination of scanning probe techniques with optical spectroscopy, such as with AFM-IR and tip-enhanced Raman spectroscopy, allows for the measurement of topographical maps along with chemical identity, enabled by spectroscopy. In this work, we review applications of these techniques to neuroscience research, where they have been used to study the morphology and mechanoelastic properties of neuronal cells and brain tissues, and to study changes in these as a result of chemical or physical stimuli. Cellular membrane models are widely used to investigate the interaction of the neuronal cell membrane with proteins associated with various neurological disorders, where scanning probe microscopy and associated techniques provide significant improvement in the understanding of these processes on a cellular and molecular level.

In Vivo Optical Interrogation of Neuronal Responses to Genetic, Cell Type-Specific Silencing.

We established a low background, Cre-dependent version of the inducible Tet-On system for fast, cell type-specific transgene expression in vivo Coexpression of a constitutive, Cre-dependent fluorescent marker selectively allowed single-cell analyses before and after inducible, Tet-dependent transgene expression. Here, we used this method for precise, acute manipulation of neuronal activity in the living brain. The goal was to study neuronal network homeostasis at cellular resolution. Single induction of the potassium channel Kir2.1 produced cell type-specific silencing within hours that lasted for at least 3 d. Longitudinal in vivo imaging of spontaneous calcium transients and neuronal morphology demonstrated that prolonged silencing did not alter spine densities or synaptic input strength. Furthermore, selective induction of Kir2.1 in parvalbumin interneurons increased the activity of surrounding neurons in a distance-dependent manner. This high-resolution, inducible interference and interval imaging of individual cells (high I5, HighFive) method thus allows visualizing temporally precise, genetic perturbations of defined cells.SIGNIFICANCE STATEMENT Gene function is studied by KO or overexpression of a specific gene followed by analyses of phenotypic changes. However, being able to predict and analyze exactly those cells in which genetic manipulation will occur is not possible. We combined two prominent transgene overexpression methods to fluorescently highlight the targeted cells appropriately before cell type-specific transgene induction. By inducing a potassium channel that decreases neuronal firing, we investigated how neuronal networks in the living mouse brain possibly compensate swift changes in cellular activities. Unlike in vitro, known compensatory homeostatic mechanisms, such as changes in synapses, were not observed in vivo Overall, we demonstrated with our method rapid genetic manipulation and analysis of neuronal activities as well as precision transgene expression.

Segmentation in large-scale cellular electron microscopy with deep learning: A literature survey.

Electron microscopy (EM) enables high-resolution imaging of tissues and cells based on 2D and 3D imaging techniques. Due to the laborious and time-consuming nature of manual segmentation of large-scale EM datasets, automated segmentation approaches are crucial. This review focuses on the progress of deep learning-based segmentation techniques in large-scale cellular EM throughout the last six years, during which significant progress has been made in both semantic and instance segmentation. A detailed account is given for the key datasets that contributed to the proliferation of deep learning in 2D and 3D EM segmentation. The review covers supervised, unsupervised, and self-supervised learning methods and examines how these algorithms were adapted to the task of segmenting cellular and sub-cellular structures in EM images. The special challenges posed by such images, like heterogeneity and spatial complexity, and the network architectures that overcame some of them are described. Moreover, an overview of the evaluation measures used to benchmark EM datasets in various segmentation tasks is provided. Finally, an outlook of current trends and future prospects of EM segmentation is given, especially with large-scale models and unlabeled images to learn generic features across EM datasets.

A-143 Imaging Flow Cytometry: A Novel Method Using ADVIA 2120i System Reagents and Microscopic Image Analysis

Abstract Background Flow cytometry based ADVIA 2120i Hematology System reagents are used to modify blood cells. Laser-scattering flow cytometry replaced with digital microscopic image analysis significantly simplifies the complex system to a compact, low-cost microscope. A novel method for imaging flow cytometry is proposed here that simultaneously generates complete blood count (CBC) and high-resolution images of blood cells. Methods Image processing algorithms are implemented to process the pixels containing blood cells. RBCs are made isovolumetrically spherical using the RBC reagent (Fig. 1A) and imaged. RBC volumes are easily determined from the diameter of the isovolumetrically spherical RBCs. By illuminating the spherical RBCs with approximately400 nm light, the increasing grayscale values of the pixels corresponds to increasing haemoglobin concentration due to absorption. WBC nuclei are stained using the Perox reagent (Fig. 1B). The grayscale values of the nuclei pixels can be used to determine the peroxidase stain component, thereby obtaining cell size-to-peroxidase activity scatter plots similar to those on the ADVIA 2120i system. Using the Baso reagent, the cytoplasm of all WBCs are stripped off except for basophils, as shown in Fig. 1C. All the microscopic images have good contrast and high resolution. Image processing techniques can thus provide a 5-part differential using the nuclei/lobular density of WBCs. Results Images were obtained under a 20×x Nikon microscope with a Thorlabs DCC1545M monochrome camera. ADVIA reagents were mixed with venous blood and <5 µL of reagent treated sample was spotted between a microscopic slide and coverslip. Image analysis was performed using ImageJ particle analysis tools. Conclusion Analysis of digital microscopic images of blood cells acted upon by ADVIA 2120i system reagents can offer a novel, simplified, cost-effective method of hematology analysis. Simultaneous CBC scatter plots and high-resolution images of the blood cells can be obtained using this new approach.Fig. 1.(a) Isovolumetricallly spherical RBCs, (b) peroxidase stain of WBC nuclei, and (c) cytoplasm of WBCs stripped off (except for basophils). Images taken under a 20× Nikon microscope. *Under development and not available for commercial sale.

Flat mount preparation for whole-mount fluorescent imaging of zebrafish embryos.

The zebrafish is a widely used model organism for biomedical research due to its ease of maintenance, external fertilization of embryos, rapid embryonic development, and availability of established genetic tools. One notable advantage of using zebrafish is the transparency of the embryos, which enables high-resolution imaging of specific cells, tissues, and structures through the use of transgenic and knock-in lines. However, as the embryo develops, multiple layers of tissue wrap around the lipid-enriched yolk, which can create a challenge to image tissues located deep within the embryo. While various methods are available, such as two-photon imaging, cryosectioning, vibratome sectioning, and micro-surgery, each of these has limitations. In this study, we present a novel deyolking method that allows for high-quality imaging of tissues that are obscured by other tissues and the yolk. Embryos are lightly fixed in 1% PFA to remove the yolk without damaging embryonic tissues and are then refixed in 4% PFA and mounted on custom-made bridged slides. This method offers a simple way to prepare imaging samples that can be subjected to further preparation, such as immunostaining. Furthermore, the bridged slides described in this study can be used for imaging tissue and organ preparations from various model organisms.

Novel image analysis tool for rapid screening of cell morphology in preclinical animal models of disease

The field of cell biology has seen major advances in both cellular imaging modalities and the development of automated image analysis platforms that increase rigor, reproducibility, and throughput for large imaging data sets. However, there remains a need for tools that provide accurate morphometric analysis of single cells with complex, dynamic cytoarchitecture in a high-throughput and unbiased manner. We developed a fully automated image-analysis algorithm to rapidly detect and quantify changes in cellular morphology using microglia cells, an innate immune cell within the central nervous system, as representative of cells that exhibit dynamic and complex cytoarchitectural changes. We used two preclinical animal models that exhibit robust changes in microglia morphology: (1) a rat model of acute organophosphate intoxication, which was used to generate fluorescently labeled images for algorithm development; and (2) a rat model of traumatic brain injury, which was used to validate the algorithm using cells labeled using chromogenic detection methods. All ex vivo brain sections were immunolabeled for IBA-1 using fluorescence or diaminobenzidine (DAB) labeling, images were acquired using a high content imaging system and analyzed using a custom-built algorithm. The exploratory data set revealed eight statistically significant and quantitative morphometric parameters that distinguished between phenotypically distinct groups of microglia. Manual validation of single-cell morphology was strongly correlated with the automated analysis and was further supported by a comparison with traditional stereology methods. Existing image analysis pipelines rely on high-resolution images of individual cells, which limits sample size and is subject to selection bias. However, our fully automated method integrates quantification of morphology and fluorescent/chromogenic signals in images from multiple brain regions acquired using high-content imaging. In summary, our free, customizable image analysis tool provides a high-throughput, unbiased method for accurately detecting and quantifying morphological changes in cells with complex morphologies.

Optical photothermal infrared spectroscopy and discrete wavenumber imaging for high content screening of single cells.

Major drawbacks of direct mid-infrared spectroscopic imaging of single cells in an aqueous buffer are strong water absorption, low resolution typically above 10 μm, and Mie scattering effects. This study demonstrates how an indirect detection principle can overcome these drawbacks using the optical photothermal infrared (O-PTIR) technique for high-resolution discrete wavenumber imaging and fingerprint spectroscopy of cultivated cells as a model system in a simple liquid sample chamber. The O-PTIR spectra of six leukemia- and cancer-derived cell lines showed main IR bands near 1648, 1547, 1447, 1400, 1220, and 1088 cm-1. Five spectra of approximately 260 single cells per cell type were averaged, the O-PTIR data set was divided into leukemia-derived cells (THP-1, HL 60, Jurkat, and Raji) and cancer cells (HeLa and HepaRG), and partial least squares linear discriminant analysis (PLS-LDA) was applied in the spectral range 800-1800 cm-1 to train three classification models. A leukemia versus cancer cell model showed an accuracy of 90.0%, the HeLa versus HepaRG cell model had an accuracy of 95.4%, and the model for the distinction of leukemia cells had an accuracy of 75.4%. IR bands in linear discriminants (LDs) of the models were correlated with second derivative spectra that resolved more than 25 subbands. The IR and second derivative spectra of proteins, DNA, RNA and lipids were collected as references to confirm band assignments. O-PTIR images of single cells at a 200 nm step size were acquired at 1086, 1548, and 1746 cm-1 to visualize the nucleic acid, protein, and lipid distribution, respectively. Variations in subcellular features and in the lipid-to-protein and nucleic acid-to-protein ratios were identified that were consistent with biomolecular information in LDs. In conclusion, O-PTIR can provide high-quality spectra and images with submicron resolution of single cells in aqueous buffers that offer prospects in high-content screening applications.

Receptor-independent fluid-phase macropinocytosis promotes arterial foam cell formation and atherosclerosis.

Accumulation of lipid-laden foam cells in the arterial wall plays a central role in atherosclerotic lesion development, plaque progression, and late-stage complications of atherosclerosis. However, there are still fundamental gaps in our knowledge of the underlying mechanisms leading to foam cell formation in atherosclerotic arteries. Here, we investigated the role of receptor-independent macropinocytosis in arterial lipid accumulation and pathogenesis of atherosclerosis. Genetic inhibition of fluid-phase macropinocytosis in myeloid cells (LysMCre+ Nhe1fl/fl) and repurposing of a Food and Drug Administration (FDA)-approved drug that inhibits macrophage macropinocytosis substantially decreased atherosclerotic lesion development in low-density lipoprotein (LDL) receptor-deficient and Apoe-/- mice. Stimulation of macropinocytosis using genetic (H-RASG12V) and physiologically relevant approaches promoted internalization of unmodified native (nLDL) and modified [e.g., acetylated (ac) and oxidized (ox) LDL] lipoproteins in both wild-type and scavenger receptor (SR) knockout (Cd36-/-/Sra-/-) macrophages. Pharmacological inhibition of macropinocytosis in hypercholesterolemic wild-type and Cd36-/-/Sra-/- mice identified an important role of macropinocytosis in LDL uptake by lesional macrophages and development of atherosclerosis. Furthermore, serial section high-resolution imaging, LDL immunolabeling, and three-dimensional (3D) reconstruction of subendothelial foam cells provide visual evidence of lipid macropinocytosis in both human and murine atherosclerotic arteries. Our findings complement the SR paradigm of atherosclerosis and identify a therapeutic strategy to counter the development of atherosclerosis and cardiovascular disease.

Partnered Excited-State Intermolecular Proton Transfer Fluorescence (P-ESIPT) Signaling for Nitrate Sensing and High-Resolution Cell-Imaging.

Nitrite (NO2−) is a common pollutant and is widely present in the environment and in human bodies. The development of a rapid and accurate method for NO2− detection is always a very important task. Herein, we synthesized a partnered excited-state intermolecular proton transfer (ESIPT) fluorophore using the “multi-component one pot” method, and used this as a probe (ESIPT-F) for sensing NO2−. ESIPT-F exhibited bimodal emission in different solvents because of the solvent-mediated ESIPT reaction. The addition of NO2− caused an obvious change in colors and tautomeric fluorescence due to the graft of NO2− into the ESIPT-F molecules. From this basis, highly sensitive and selective analysis of NO2− was developed using tautomeric emission signaling, achieving sensitive detection of NO2− in the concentration range of 0~45 mM with a detection limit of 12.5 nM. More importantly, ESIPT-F showed the ability to anchor proteins and resulted in a recognition-driven “on-off” ESIPT process, enabling it to become a powerful tool for fluorescence imaging of proteins or protein-based subcellular organelles. MTT experimental results revealed that ESIPT-F is low cytotoxic and has good membrane permeability to cells. Thus, ESIPT-F was further employed to image the tunneling nanotube in vitro HEC-1A cells, displaying high-resolution performance.