Search Results (1,359)

Neutrophils constitute the largest fraction of total circulating leukocytes in humans and mediate early innate immune responses. Although they are often considered a uniform population of short-lived immune cells, emerging evidence from single-cell RNA sequencing and high-dimensional flow cytometry has revealed that neutrophils are functionally and phenotypically heterogeneous in both healthy and pathological conditions. However, a critical gap is how molecularly defined neutrophil states translate into distinct spatiotemporal behaviors in vivo. This review summarizes our current understanding of the molecular signatures underlying neutrophil heterogeneity and explores the functional in vivo behaviors in various diseases, including cancer, sepsis, and ischemic stroke. We also discuss the potential of intravital imaging to bridge the gap between static molecular profiling and dynamic cellular behavior, offering a comprehensive view of the functional heterogeneity of neutrophils. Full article

Machine learning (ML) is transforming the analysis of biomolecular data, holding significant promise for improving the efficiency and accuracy of microscopy image analysis and for studying the dynamics of molecules in live cells. As data-driven approaches continue to evolve, they may eventually replace traditional statistical methods that rely on conventional analytical methods. This review examines and critically analyses the state of the art of ML techniques as applied to various levels of data supervision in the analysis of dynamic single-molecule datasets obtained using superresolution optical microscopy. Collectively encompassed under the umbrella of “nanoscopy”, these methods currently comprise targeted techniques such as stimulated emission depletion (STED) microscopy and stochastic techniques like single-molecule localization microscopies (SMLMs), comprising photoactivated localization microscopy (PALM), DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy, and minimal fluorescence photon flux (MINFLUX) microscopy. These techniques all enable the imaging of subcellular components and molecules beyond the diffraction limit, and some are additionally capable of studying their dynamics in real time, as reviewed here, using several ML techniques that facilitate motion analysis in two or three dimensions with qualitative and quantitative characterisation in the live cell. It is expected that the growing use of learning-based approaches in biological microscopy data processing will dramatically increase throughput and accelerate progress in this rapidly developing field. Full article

As critical regulators of gene expression, microRNAs (miRNAs) are key biomarkers and therapeutic targets in cancer. However, current methods for intracellular miRNA imaging are often limited by poor sensitivity and operational complexity. In this study, we identified a site-specifically modified DNAzyme variant, 11Bn, which exhibits up to 7-fold higher catalytic activity than the wild-type 8-17 through systematic screening. Using this variant, we constructed a DNAzyme-based sensor for miRNA-21 imaging in living cells. The sensor achieves a limit of detection of 7.89 nM, threefold lower than that of the wild-type sensor, and enables sensitive visualization of intracellular miRNA-21 without signal amplification. Moreover, it can capture dynamic changes in miRNA levels within cells, providing a versatile molecular tool for miRNA imaging and related biomedical applications. Full article

Surface-enhanced Raman spectroscopy (SERS) amplifies Raman scattering by placing molecules in the near-field of plasmonic nanostructures, enabling label-free molecular fingerprinting. While attractive for living cell phenotyping, many cellular SERS works rely on internalized colloidal nanoparticles, leading to variable uptake/localization, aggregation-driven hotspot fluctuations, and potential cellular perturbation. Here, we report a chip-like Au/SiO₂ nanolaminate SERS substrate that supports direct culture and label-free measurements of living cells on spatially defined hotspots without nanoparticle uptake. The periodic nanolaminate forms dense nanogaps and is engineered for 785 nm excitation, providing uniform enhancement over a large, culture-compatible area with high hotspot uniformity. By engineering the cell–substrate nano–bio interface, the platform enables reproducible acquisition of intrinsic cellular vibrational fingerprints under physiological conditions without Raman tags. Using MCF-7 and MDA-MB-231 breast cancer cells, we collected hundreds of spectra per line, and MDA-MB-231 exhibited broader spectral variations, indicating greater heterogeneity. Principal component analysis and linear discriminant analysis achieved 99% classification accuracy for MCF-7 and MDA-MB-231, and bright-field imaging confirmed preserved adhesion and canonical morphologies. This chip-based, label-free living cell SERS platform enables scalable, nonperturbative phenotyping and may support rapid malignancy classification and treatment response screening across subtle cancer states. Full article

Natural products from plants have played an important role in cancer and neurodegenerative diseases. In this context, the root bark of Maytenus chiapensis (Celastraceae) was investigated to examine its chemical constituents and potential biological activities. Chromatographic separation of the root bark extract yielded a new Diels–Alder adduct (morenine) formed by a triterpenophenolic moiety derived from tingenone and a bicyclic guaiane-type sesquiterpene linked through a 1,4-dioxane bridge. In addition, eight previously reported Diels–Alder adducts—retusonine and cheiloclines A–D and F–H—were isolated, together with their biosynthetic precursors, the quinone-methide triterpenoids (QMTs) pristimerin and tingenone. Structural elucidation was achieved through detailed 1D and 2D NMR spectroscopic analyses. The adducts were tested for cytotoxicity against six cancer cell lines (A549, SW1573, MIA PaCa-2, T-47D, HeLa, and WiDr cell lines), showing moderate-to-low activity compared with their precursors. Continuous live cell imaging identified apoptosis and vacuole formation as the main modes of action of pristimerin in SW1573 cells. Moreover, acetylcholinesterase inhibition assays revealed that cheiloclines B–D, F, and H exhibited up to 50% inhibition. These findings reinforce the potential of Celastraceae species as a source of unique and complex compounds and enhance our understanding of their therapeutic potential. Full article

Serotonin is a critical morphogen in early development, yet the mechanisms regulating its homeostasis in the preimplantation embryo remain unclear, particularly under conditions of maternal antidepressant exposure. Here, we investigated embryonic serotonergic autonomy using mouse models of pharmacological transport blockade (maternal fluoxetine treatment) and in vitro treatment with the monoamine oxidase inhibitor pargyline. We employed immunofluorescence, RT-qPCR, and live-cell imaging to assess metabolic flux, gene expression, and physiological health. We demonstrate that monoamine oxidase functions as a metabolic firewall, progressively maturing from zygote to blastocyst to degrade excess amines. Paradoxically, maternal serotonin transporter blockade triggered significant intracellular serotonin hyper-accumulation in blastocysts, associated with a trend toward a compensatory upregulation of the biosynthetic gene Ddc. While this serotonin overload did not compromise morphology, mitochondrial function, or pluripotency marker expression, it induced a robust epigenetic response. Excess serotonin promoted elevated H3Q5ser immunoreactivity in both nuclear and cytoplasmic compartments via a transglutaminase-dependent mechanism. These findings reveal that the preimplantation embryo possesses a resilient, autonomous serotonergic system capable of compensatory synthesis. However, environmental fluctuations are chemically recorded via transglutaminase-mediated serotonylation, representing an epigenetic mark that warrants further long-term study within the Developmental Origins of Health and Disease (DOHaD) framework. Full article

Background: Biodegradable magnesium-based alloys are increasingly explored as emerging biomaterials for dental and maxillofacial applications due to their osteoconductive properties and potential to reduce long-term implant-related complications. However, early-stage evaluation requires predictive diagnostic screening methods capable of assessing cytocompatibility and cellular response under clinically relevant extract conditions. Objectives: In this study, Mg–0.5Ca alloys modified with increasing strontium concentrations (0.5–3 wt.%) were investigated through an in vitro diagnostic framework using MG-63 osteoblast-like cells. Methods: Cell viability was quantitatively assessed via MTT assays after 24 and 72 h of exposure, while fluorescence-based live-cell imaging provided complementary morphological insights. Results: demonstrated a composition-associated cytocompatibility profile, with Sr-enriched compositions showing improved cellular metabolic activity and adhesion patterns compared to lower-Sr compositions. Conclusions: These findings support the role of strontium as a functional alloying element and highlight the importance of standardized diagnostic screening workflows for emerging dental biomaterials. Overall, this study proposes a simplified predictive platform for early biocompatibility diagnostics, contributing to the integration of biomaterial evaluation into future digitalized dental regeneration workflows. Full article

Cellular senescence is a stress-induced state characterised by durable proliferative arrest and extensive transcriptional and secretory reprogramming. In cancer, senescence can suppress early tumour outgrowth, yet persistence of senescent cells and their senescence-associated secretory phenotype (SASP) may drive maladaptive inflammation, immune dysfunction, vascular perturbation and extracellular matrix (ECM) remodelling. Head and neck squamous cell carcinoma (HNSCC) provides a clinically informative context because tumours arise in injury-prone mucosa and standard therapies (radiotherapy and platinum-based chemotherapy) can induce long-lived senescent phenotypes across stromal and vascular compartments. Here, we synthesise the evidence through a signal → matrix → function framework, in which the therapy-induced SASP modules reshape collagen density, alignment, confinement and crosslinking, thereby influencing invasion, immune access, perfusion and post-treatment fibrosis. We emphasise that senescence detection in head and neck tissues is highly context-dependent and readily confounded by inflammageing, chronic mucosal injury and HPV-associated biology, necessitating a cell-type-resolved, spatially anchored, multi-axis definition that integrates growth-arrest context, nuclear/DNA damage response hallmarks and functional outputs. We highlight oral submucous fibrosis (OSF) as a matrix-primed precursor state that exemplifies convergence between chronic injury, fibrosis and senescence-adjacent programmes. Finally, we propose an integrated translational roadmap combining multiplex spatial pathology with quantitative collagen imaging to map therapy-induced senescence–ECM niches and support biomarker-guided testing of senomorphic, senolytic and matrix-normalising strategies in HNSCC. Full article

Cervical cancer is one of the most prevalent and easily contracted diseases among women, significantly impacting their daily lives. Computer vision-based cervical cell morphology diagnosis technology can offer robust support for cervical cell analysis at a lower cost. However, the presence of a substantial number of overlapping cells in cervical images renders existing cell segmentation methods less accurate, thereby complicating the guidance of medical diagnosis. In this paper, we introduce a tristage Progressive Refinement method (PRefiner) for overlapping cell segmentation that decouples the traditional end-to-end pipeline, with the final stage specifically correcting anomalous results to enhance precision. We achieve separable overlapping cervical cell segmentation results through a cell nucleus locator, a single-cell segmenter, and a Segmentation Result Mask Refiner. Specifically, we employ a hybrid U-Net as the primary network for the cell nucleus locator and single-cell segmenter, which determines the position of the cell nucleus and procures the initial coarse segmentation result. In the mask refiner, we incorporate a conditional generation framework to address the perception decision problem and design a local–global dual-scale discriminator to ensure that the segmentation result aligns with the prior of a single-cell mask. Experimental results on CCEDD and ISBI2015 demonstrate that PRefiner achieves optimal performance by effectively resolving abnormal segmentations. Notably, our method improves the Dice coefficient of abnormal results from five different models by an average of 2.62% (ranging from 1.0% to 5.1%). Full article

Cell-based immuno-biosensors are novel platforms for studying immune responses of biological cells, with real-time insights more similar to physiological and pathological conditions. These systems utilize living immune cells as their main components, enabling them to detect disease-related biomarkers and cellular traits in a way that is often highly sensitive and label-free. Integration with microfluidics and organ-on-chip technologies has facilitated precise manipulational control over the cellular microenvironment. Not only has this resulted in high-throughput screening, but it also enabled smaller, more portable systems which can be used at the point of care. In this work, we review the recent advance in microfluidic cell-based immuno-biosensing associated with immune cells such as neutrophils, macrophages, T cell and dendrite cells. Some of the exciting developments include fusion with methods such as advanced imaging, electrical impedance sensing and application of machine learning to phenotyping. We will also elaborate on the issues related to the standardization of these systems, cell heterogeneity, and the challenges for translating these technologies for clinical application. Taken together, such integrated platforms have potential to fill the gap left in-between cellular immunology with biosensor engineering. Full article

Background/Objectives: This article develops theory and methods for 3D tomographic imaging of absorption coefficient distributions using axial scanning with EUV microscopes at 46× and 345× magnification. Unlike conventional CT that requires sample rotation, axial scanning moves cells through the microscope focus. The aim is tomographic reconstruction of living cell fine structure without the organelle staining used in optical fluorescence microscopy or ultra-thin cell slicing as in electron microscopy. Methods: By generalizing the geometric-optical approximation for small absorption coefficient inhomogeneities in absorbing media, we derived a new explicit tomography equation and solution algorithm validated through numerical simulation. The approach was applied to Convallaria cell analysis using the ×46 microscope. For the ×345 microscope, we developed an alternative method where the kernel of the tomography integral equation was determined experimentally using gold nanospheres with known absorption coefficient, shape, and position. This method was tested through modeling and applied to diagnostics of Convallaria and mouse cerebellar granule cells. Results: The developed methods resolve subcellular features down to 140 nm using the ×46 microscope and 50 nm using the ×345 microscope. Thin low-contrast intracellular structures and individual 50–100 nm organelles were detected. Conclusions: Methods for retrieving absorption coefficient distributions in cone-beam geometry based on geometric-optical theory generalization and on calibration by gold nanoparticles have been developed and validated through numerical simulation and cell analysis. These methods demonstrate for the first time the effectiveness of axial nanotomography using multilayer mirror microscopes for cell diagnostics. Full article

Fluorogenic glycosides are widely used substrates for assaying lysosomal glycosidase activities in vitro, but they do not provide subcellular information in living cells. In this study, we used glucosylceramide (GlcCer) liposomes as carriers to deliver fluorogenic substrates into live PC12 cells for confocal imaging. The α-4-methylumbelliferyl glucoside (α-4MUG) and β-glucosidase substrate β-4-(trifluoromethyl)umbelliferyl glucoside (β-4FMUG) were co-encapsulated in liposomes. The liposomes (approximately 100 nm in diameter) were taken up by PC12 cells after pulse exposure. Punctate fluorescence signals from both hydrolyzed substrates were observed. The relative intensity of two signals varied among puncta, as assessed by dual-channel imaging and line-scan analysis. These results show that GlcCer liposomes provide a practical platform for long-term and differential analyses of relative α- and β-glucosidase activities in living cells. Full article

Molecular interactions underpin the functioning of the living cell. Molecules exist in distinct quaternary structural forms, associate with molecular partners in signaling cascades, form transient quinary interactions, localize in membrane domains, and cluster in membrane-less condensates. Measuring the concentration, size, and dynamics of these molecular assemblies remains an enduring biophysical challenge, particularly in cells, where heterogeneity is the rule rather than the exception. Orthogonal signals derived from fluorescence lifetime, fluorescence fluctuations, and fluorescence polarization provide valuable metrics for probing interactions and environments, concentration and size, and rotational dynamics, respectively. This paper combines fluorescence lifetime imaging microscopy with image correlation analysis and polarization to determine the concentrations, brightness, lifetime, and rotational correlation time of different fluorescent states. A two-population model is examined as a prototypical example of a heterogeneous system. The analysis is illustrated on a simple fluorescence model system, where cluster densities, relative brightnesses, lifetimes, and rotational correlation times are extracted. Full article

Neural regeneration requires an optimal environment, including structural, chemical, mechanical, and electrical properties. Alginate (Alg) and graphene oxide (GO) are promising biomaterials for nerve tissue engineering, as Alg provides biocompatibility and hydrogel formation, while GO enhances mechanical strength and conductivity. For this study, GO was synthesized using the modified Hummer’s method, and Alg–GO scaffolds with varying GO concentrations were developed. FTIR spectroscopy confirmed the successful incorporation of GO into the Alg matrix, while UV–Vis and photoluminescence analyses demonstrated GO-induced modifications of the optical properties. Thermal analysis revealed improved stability with increasing GO content, whereas swelling tests showed enhanced water uptake and retention. Conductivity measurements indicated a clear improvement in electrical conductivity, particularly at moderate GO concentrations. SEM imaging confirmed a homogeneous distribution of GO within the Alg matrix, with structural uniformity across all samples. Cytocompatibility was assessed using SH–SY5Y neuroblastoma cells through MTT, LDH, and LIVE/DEAD assays. All composites supported cell attachment, viability, and proliferation, with GO concentrations up to 6% promoting optimal cell growth without inducing cytotoxicity. In contrast, excessive GO content (9%) resulted in reduced proliferation, although biocompatibility was maintained. These results highlight the potential of Alg–GO scaffolds as promising candidates for neural tissue engineering. The findings demonstrate the potential of Alg–GO scaffolds as advanced biomaterials for regenerative medicine. Future research should focus on in vivo evaluations to confirm their therapeutic applicability. Full article

Astrocytomas are among the most common primary tumors of the central nervous system, arising from astrocytic glial cells and encompassing a wide spectrum of histopathological grades and clinical behaviors. Human immunodeficiency virus (HIV) infection is characterized by chronic immune dysregulation, neuroinflammation, and increased susceptibility to both opportunistic infections and malignancies. The management of astrocytomas in patients living with HIV presents unique clinical challenges but should, whenever feasible, follow standard neuro-oncological principles. We report the case of a 34-year-old man with well-controlled HIV infection who presented in February 2025 with progressive neurological symptoms. Brain imaging revealed a left temporo-insular lesion, and the diagnosis was confirmed by neuronavigation-guided biopsy performed on 31 March 2025. Histopathological and immunohistochemical evaluation established the diagnosis of an isocitrate dehydrogenase (IDH) wild-type astrocytoma, central nervous system (CNS) World Health Organization (WHO) grade 2, according to the 2021 classification of central nervous system tumors. Full article