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At least 19 recordsLinked to original sources
PubMed2026

Cumulative microscopy reveals cellular states in fibroblasts from patients with genetic disorders.

Analysis of cellular states and signaling trajectories can provide insights into causes of disease. We developed cumulative microscopy, a method to perform cyclical imaging without elution or quenching steps. Cumulative microscopy computationally extracts individual signals from accumulating fluorescence during sequential imaging. We use cumulative microscopy to quantitatively assess cell cycle and stress markers in individual primary fibroblasts from patients with rare genetic proliferative disorders with increased cancer risk. Neural network-based analysis of cumulative microscopy data suggests that cells from patients with Cartilage-hair hypoplasia (CHH), but not Mulibrey Nanism (MUL), show replication stress. We analyze cell states and cell trajectories and find that a subset of cells from patients with CHH show spontaneous replication stress, followed by cell cycle exit in both G1 and G2 phases. We note that replication stress potentially could underlie both proliferative defects and increased cancer risk in CHH patients and conclude that cumulative microscopy is an efficient, quantitative, and generalizable approach to multiplex microscopy.

Humans↗
PubMed2026

Advanced High-Resolution and Super-Resolution Microscopy Techniques for Investigating Chromatin Structure and Epigenetics.

Fluorescence microscopy has emerged as an indispensable tool for investigating chromatin structure and epigenetic states. This technique not only enables super-resolution imaging to unveil detailed localizations and structures but also offers precise quantitative capabilities for comparing variations across multiple conditions. In this chapter, we present a comprehensive guide to immunofluorescence methods, detailing two primary fixation protocols. Additionally, we describe the application of super-resolution spinning disc microscopy, emphasizing its utility in detecting nuclear and epigenetic markers.

Chromatin↗
PubMed2025

Monitoring Influenza A Virus Entry Using Quantitative Fluorescence Microscopy.

Influenza A virus (IAV) is a major threat to global human health and is a topic of intense research. With the continuous problem of seasonal influenza and the threat of potential pandemics due to frequent emergence of new viral strains, development of new, broad-spectrum antivirals is an urgent priority. In antiviral development against influenza, the process of host cell entry of IAV is of particular interest as inhibiting the virus at the entry step should stop infection early on, blocking the downstream infection processes including viral replication and transcription. Therefore, a detailed understanding of the IAV entry processes is essential to illuminate virus-assisting host factors that can serve as potentially valuable targets for therapeutic interventions. To accelerate the identification of novel antivirals or host-directed targets that play essential role in IAV entry, quantitative assays that can be used to monitor the virus at sequential entry steps would be important for performing high-content genetic or inhibitor screens. In this chapter, we describe how IAV entry can be monitored at the sequential entry steps, spanning from the initial attachment of the virus particle to the cell surface to the transmission of the viral genome to the nucleus, by fluorescence microscopy. Further, we provide the methods to quantify the images acquired with high-content microscope for each of the major IAV entry steps. The fluorescence microscopy-based IAV entry assays and the image quantification methods described here can be used to boost our understanding of the virus-host cell interactions and can lead to the discovery of novel host-directed prophylactic or therapeutic interventions.

Humans↗
PubMed2026

Volumetric DNA microscopy for mapping spatial transcriptomes in three dimensions.

The architecture and function of biological systems are inherently three-dimensional, yet most existing spatial transcriptomic technologies remain restricted to thin tissue sections, limiting their capacity to resolve cellular organization and microenvironments within intact tissue volumes. To address this limitation, we developed volumetric DNA microscopy, a scalable, optics-free approach for spatial transcriptome profiling directly within intact biological specimens. The method encodes spatial information into DNA molecules that form a dense intermolecular network in situ, enabling the reconstruction of three-dimensional spatial relationships through short-read sequencing and computational analysis. Here we detail the complete workflow including in situ cDNA synthesis, spatial encoding through DNA nanoball formation, dual-scale proximity bridging between neighboring nanoballs and spatial reconstruction via geodesic spectral embedding. Sequencing libraries can be generated within 7-8 d by a competent graduate-level molecular biologist, followed by standardized downstream computational analysis. Because the workflow requires only routine molecular biology reagents and a benchtop sequencer, volumetric DNA microscopy provides a versatile platform for exploring genetic and morphological features in intact tissues.

Spatial Transcriptomics↗
PubMed2005

The Open Microscopy Environment (OME) Data Model and XML file: open tools for informatics and quantitative analysis in biological imaging.

The Open Microscopy Environment (OME) defines a data model and a software implementation to serve as an informatics framework for imaging in biological microscopy experiments, including representation of acquisition parameters, annotations and image analysis results. OME is designed to support high-content cell-based screening as well as traditional image analysis applications. The OME Data Model, expressed in Extensible Markup Language (XML) and realized in a traditional database, is both extensible and self-describing, allowing it to meet emerging imaging and analysis needs.

Computational Biology↗
PubMed2025

Label-free structural imaging of plant roots and microbes using third-harmonic generation microscopy.

Root biology is pivotal in addressing global challenges including sustainable agriculture and climate change. However, roots have been relatively understudied among plant organs, partly due to the difficulties in imaging root structures in their natural environment. Here we used microfabricated ecosystems (EcoFABs) to establish growing environments with optical access and employed nonlinear multimodal microscopy of third-harmonic generation (THG) and three-photon fluorescence (3PF) to achieve label-free, in situ imaging of live roots and microbes at high spatiotemporal resolution. THG enabled us to observe key plant root structures including the vasculature, Casparian strips, dividing meristematic cells, and root cap cells, as well as subcellular features including nuclear envelopes, nucleoli, starch granules, and putative stress granules. THG from the cell walls of bacteria and fungi also provides label-free contrast for visualizing these microbes in the root rhizosphere. With simultaneously recorded 3PF signal, we demonstrated our ability to investigate root-microbe interactions by achieving single-bacterium tracking and subcellular imaging of fungal spores and hyphae in the rhizosphere.

Plant Roots↗
PubMed2023

EMPIAR: the Electron Microscopy Public Image Archive.

Public archiving in structural biology is well established with the Protein Data Bank (PDB; wwPDB.org) catering for atomic models and the Electron Microscopy Data Bank (EMDB; emdb-empiar.org) for 3D reconstructions from cryo-EM experiments. Even before the recent rapid growth in cryo-EM, there was an expressed community need for a public archive of image data from cryo-EM experiments for validation, software development, testing and training. Concomitantly, the proliferation of 3D imaging techniques for cells, tissues and organisms using volume EM (vEM) and X-ray tomography (XT) led to calls from these communities to publicly archive such data as well. EMPIAR (empiar.org) was developed as a public archive for raw cryo-EM image data and for 3D reconstructions from vEM and XT experiments and now comprises over a thousand entries totalling over 2 petabytes of data. EMPIAR resources include a deposition system, entry pages, facilities to search, visualize and download datasets, and a REST API for programmatic access to entry metadata. The success of EMPIAR also poses significant challenges for the future in dealing with the very fast growth in the volume of data and in enhancing its reusability.

Imaging, Three-Dimensional↗
PubMed2022

Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination.

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of chromatin, show most DNA repair enzymes remove DNA damage at reduced rates as compared to free DNA. The molecular details on how base excision repair (BER) enzymes recognize and remove DNA damage in nucleosomes have not been elucidated. However, biochemical BER data of nucleosomal substrates suggest the nucleosome presents different structural barriers dependent on the location of the DNA lesion and the enzyme. This indicates the mechanisms employed by these enzymes to remove DNA damage in free DNA may be different than those employed in nucleosomes. Given that the majority of genomic DNA is assembled into nucleosomes, structural information of these complexes is needed. To date, the scientific community lacks detailed protocols to perform technically feasible structural studies of these complexes. Here, we provide two methods to prepare a complex of two genetically fused BER enzymes (Polymerase β and AP Endonuclease1) bound to a single-nucleotide gap near the entry-exit of the nucleosome for cryo-electron microscopy (cryo-EM) structural determination. Both methods of sample preparation are compatible for vitrifying quality grids via plunge freezing. This protocol can be used as a starting point to prepare other nucleosomal complexes with different BER factors, pioneer transcription factors, and chromatin-modifying enzymes.

Chromatin↗
PubMed2025

Unique growth and morphology properties of Clade 5 Clostridioides difficile strains revealed by single-cell time-lapse microscopy.

Clostridioides difficile is a gastrointestinal pathogen of both humans and agricultural animals and thus a major One Health threat. The C. difficile species consists of five main clades, with Clade 5 currently undergoing speciation from Clades 1-4. Clade 5 strains are highly prevalent in agricultural animals and can cause zoonotic infections, suggesting that these strains have evolved phenotypes that distinguish them from Clade 1-4 strains. Here, we compare the growth properties of Clade 5 strains to those of Clade 1-4 strains using anaerobic time-lapse microscopy coupled with automated image analysis. Our analyses indicate that Clade 5 strains grow faster and are more likely to form long chains of cells than Clade 1-4 strains. Using comparative genomic and CRISPRi analyses, we show that the chaining phenotype of Clade 5 strains is driven by the orientation of the invertible cmr switch sequence, with chaining strains exhibiting a bias to the cmr-ON state. Interestingly, Clade 5 strains with a bias towards the cmr-ON state shifted to a largely cmr-OFF state during murine infection, suggesting that the cmr-OFF state is under positive selection during infection. Collectively, our data reveal that Clade 5 strains have distinct growth properties, which may allow them to inhabit diverse ecological niches.

Journal Article↗
PubMed2025

Unique growth and morphology properties of Clade 5 Clostridioides difficile strains revealed by single-cell time-lapse microscopy.

Clostridioides difficile is a gastrointestinal pathogen of both humans and agricultural animals and thus a major One Health threat. The C. difficile species consists of five main clades, with Clade 5 currently undergoing speciation from Clades 1-4. Since Clade 5 strains are highly prevalent in agricultural animals and a frequent cause of zoonotic infections, these strains may have evolved phenotypes that distinguish them from Clade 1-4 strains. Here, we compare the growth properties of Clade 5 strains to those of Clade 1-4 strains using anaerobic time-lapse microscopy coupled with automated image analysis. Our analyses indicate that Clade 5 strains grow faster and are more likely to form long chains of cells than Clade 1-4 strains. Using comparative genomic and CRISPRi analyses, we show that the chaining phenotype of Clade 5 strains is driven by the orientation of the invertible cmr switch sequence, with chaining strains exhibiting a bias to the cmr-ON state. Interestingly, Clade 5 strains with a bias towards the cmr-ON state shifted to a largely cmr-OFF state during murine infection, suggesting that the cmr-OFF state is under positive selection during infection. Collectively, our data reveal that Clade 5 strains have distinct growth properties, which may allow them to inhabit diverse ecological niches.

Clostridioides difficile↗
PubMed2025

4D Microscopy and Tracking of Chromosomes and the Spindle in C. elegans Early Embryos.

Maintaining genomic integrity throughout successive cell divisions is essential for the proper development and functioning of organisms. Chromosome alignment and segregation occur on a microtubule-based spindle originating from centrosomes. The molecular and cellular mechanisms involved in accurate chromosome segregation during early embryonic divisions are highly conserved between worms and humans. Therefore, C. elegans serves as a robust model for investigating mitotic cell divisions within a metazoan system. Throughout early embryonic development, filming and tracking successive cell divisions becomes progressively more challenging as the number of cells increases and cell size decreases. To address this challenge, we describe a method for preparing live samples, performing 4D time-lapse imaging, and semi-automated tracking of chromosomes and spindle poles during early mitotic divisions in C. elegans embryos.

Caenorhabditis elegans↗
PubMed2026

Towards time-resolved MicroED grid preparation using mix-and-inject gas dynamic virtual nozzles.

Recent progress in gas dynamic virtual nozzle (GDVN) technologies in combination with high-brilliance synchrotron and X-ray free-electron lasers (XFELs) has allowed the visualization of protein dynamics in crystallo by mixing macromolecular protein crystals with a substrate using tunable mixing times on the order of milliseconds to seconds prior to serial X-ray diffraction data collection. This has become the method of choice for high-resolution structure determination of intermediate states. However, such experiments require large counts of crystals of proper sizes for high-resolution data collection, and premium beam times for screening efforts. Cryogenic microcrystal electron diffraction (MicroED) represents a complementary technique that may be a more accessible avenue for time-resolved nanocrystallography compared with serial X-ray diffraction experiments. MicroED can produce full diffraction datasets from just a few submicrometre-thick crystals, and the approach is more readily accessible, requiring standard cryogenic transmission electron microscopy (TEM) equipment available at many universities and institutes. Cryogenic MicroED, like other forms of cryo-EM, begins with rapidly freezing biological material on electron microscopy grids. In the case of MicroED, micro- to nano-crystals (<500&#x2005;nm thick) are deposited onto electron microscopy grids and plunge-frozen for subsequent electron diffraction data collection. Here, we have incorporated GDVN technology developed originally for XFEL experiments into the freezing process as a first step towards time-resolved studies. We describe the limited deposition efficiency of the model MicroED protein proteinase K on TEM grids using GDVNs, preceding sample vitrification and successful MicroED data collection. We discuss both the initial results from such experiments and the methodological challenges in developing this approach into a reliable workflow for millisecond-to-second time-resolved structural studies of macromolecules. Our results promise a strategy to deposit crystals on grids using GDVNs and determine high-resolution structures by MicroED, constituting a first step towards development of time-resolved MicroED experiments.

MicroED↗
PubMed2026

Digital and computational morphology in hematology: current platforms, clinical evidence, and future requirements.

INTRODUCTION: Morphologic examination of peripheral blood and bone marrow remains central to the diagnosis and classification of hematologic disorders. Conventional optical microscopy, however, is labor-intensive, dependent on operator expertise, and affected by interobserver variability. Digital morphology has developed from automated image acquisition and cell pre-classification into a broader field that includes whole-slide imaging, remote review, quantitative morphometry, and artificial intelligence-based analysis. CONTENT: This review examines current applications of digital morphology in peripheral blood, bone marrow aspirates, malaria detection, and body-fluid analysis. Commercial platforms are evaluated with particular attention to the distinction between raw automated pre-classification, expert digital post-classification, and comparison with independent optical microscopy. Digital systems generally perform well for common mature leukocyte populations but remain less reliable for rare or diagnostically critical cells, including blasts, abnormal lymphoid cells, plasma cells, and intermediate maturation stages. Research systems increasingly extend analysis from individual-cell classification to whole-slide, specimen-level, and patient-level assessment. SUMMARY: Digital morphology can improve standardization, image traceability, remote consultation, education, proficiency testing, quality assurance, and selected aspects of laboratory workflow. Its clinical value depends on appropriate validation, transparent reporting of reference methods, recognition of algorithm-specific failure modes, and clearly defined criteria for expert review and conventional microscopy. Human expertise remains essential not only for validating results but also for adapting cell taxonomies and interpretive rules to evolving classifications of hematologic diseases. OUTLOOK: Future progress will require representative multicenter datasets, harmonized morphologic terminology, external validation, interoperability with laboratory information systems, and continuous monitoring after software or hardware updates. Integration of morphology with quantitative hematology, flow cytometry, cytogenetics, genomics, and clinical data may support more comprehensive computational diagnosis. Digital platforms may also broaden access to specialist expertise, training, and quality programs in resource-limited institutions and regions, provided that infrastructure, governance, and professional competency are adequately supported.

artificial intelligence↗
PubMed2026

HoT auto-blinking probes enable real-time, super-resolution chromatin imaging in live cells and tissues.

Single-molecule localization microscopy (SMLM) enables visualization of chromatin architecture at nanoscale resolution. However, high-performance DNA probes suitable for SMLM in both live cells and tissues remain limited. We developed Hoechst-6-Carboxytetramethylrhodamine (6-TAMRA) derivative (HoT) probes-rhodamine-based derivatives conjugated to a Hoechst moiety-through structural fine-tuning of rhodamine spirocyclization. HoTs are self-assembling, auto-blinking probes with excellent photostability and high temporal resolution. They permeate live cells, enabling long-term, real-time nanoscopic chromatin imaging in live and fixed cells and in tissue sections. In live cells, we identified nanoscale features in the 3D organization of chromatin and quantified DNA fiber kinetics at high resolution. We quantified DNA compaction in single cells within retinal and colon cancer sections. OligoSTORM (stochastic optical reconstruction microscopy)-labeled gene loci can be visualized and measured within their HoT-labeled chromatin footprints. Our work provides powerful tools for investigating chromatin structure and functions in living cells and tissues, with applications ranging from cancer diagnosis to retinal regeneration.

Chromatin↗
PubMed2025

3D STED Imaging of Isolated Arabidopsis thaliana Nuclei.

Microscopy imaging of chromatin offers valuable insights into its spatial organization in the nucleus, a novel epigenetic dimension influencing the genome's functions. Particularly, visualization at the nanoscale in single cells is uniquely complementary to molecular profiling methods averaging chromatin configuration and composition over thousands of cells. How are chromatin and chromosomal domains distributed in relation to gene expression? How variable are these configurations? How do chromatin domains evolve in structure, composition, and distribution during cellular differentiation or cellular responses to environmental stimuli? Super-resolution microscopy techniques, like stimulated emission depletion (STED), are key in answering such questions. However, such imaging techniques are not often used in the field of plant cell biology compared to mammalian counterparts, which has greatly advanced our understanding of the 3D principles in genome organization. In an effort to bridge this gap, we provide a clear guide for isolating, embedding, immunostaining, and STED imaging intact leaf nuclei from Arabidopsis thaliana in 3D.

Arabidopsis↗
PubMed2026

CONCR lncRNA organizes a 3'-end structural domain that engages DDX11 for DNA replication and sister chromatid cohesion.

CONCR (DDX11-AS1) is a long noncoding RNA (lncRNA) necessary for the establishment of sister chromatid cohesion. Despite its activity, whether it contains structural elements essential for its function remains unknown. We determined CONCR structural organization and its functional relevance by integrating selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), atomic force microscopy (AFM), evolutionary analyses, cryo-electron microscopy (cryo-EM), and cellular genetic studies. We found that CONCR molecular topology is modular, with highly structured domains connected by flexible linkers. A large 3'-end domain is responsible for binding to DDX11 helicase, can trigger DDX11 ATPase activity, and is essential for proper DNA replication and sister chromatid cohesion. This 3' end comprises two helical arms connecting two multiway junctions with structural motifs conserved among all primate groups and required for DDX11 binding and sister chromatid cohesion. Our results highlight the critical role of RNA structure in CONCR function, with a highly structured 3'-end domain acting as a loading and activation platform for DDX11 helicase.

DEAD-box RNA Helicases↗
PubMed2026

Subcellular interactions of neuropeptide Y and corticotropin-releasing factor in the central nucleus of the amygdala in the mouse.

Neuropeptide Y (NPY) is ubiquitously distributed throughout the central nervous system. Recognized as a mediator of stress resilience, NPY has been shown to counteract the excitatory effects of the neuropeptide corticotropin-releasing factor (CRF), that orchestrates the stress response. In the mouse, while NPY and CRF exhibit a high degree of neuroanatomical association in the central nucleus of the amygdala (CeA) indicating potential significant interactions, the synaptic organizations of these neuropeptides have not been elucidated. In the present study, we determined the anatomical interactions between NPY and CRF in the CeA. Immunofluorescence microscopy presented that NPY-immunoreactive varicose processes were distributed throughout the CeA and appeared to be closely apposed to CRF-containing neurons. Using electron microscopy, immunoperoxidase labeling for NPY and gold-silver labeling for CRF showed that NPY-labeled axon terminals (NPY-t) form synapses with CRF-labeled dendrites (CRF-d). Semi-quantitative analysis revealed that 247 of NPY-t directly target CRF-d. In addition, approximately 80% of NPY-t form symmetric synapses with CRF-d while approximately 1% form asymmetric synapses. These findings provide the first ultrastructural evidence that NPY-containing axon terminals make direct contact with CRF-containing dendrites in the CeA. This suggests that the CRF-containing neurons in the CeA may be a key site for NPY action, potentially influencing brain regions involved in stress responses and stress-related psychiatric disorders, and alcohol use disorders.

Animals↗
PubMed2024

Dynamic Pathology of Enteric Neural Network Using Curcumin-assisted Multiphoton Laser Imaging in Hirschsprung Disease.

BACKGROUND: In living tissue, it has been difficult to make microscopic-level observations without damaging the tissue. We have invented a novel intravital fluorescent observation method (IFOM) for real-time tissue observation, combining multiphoton laser scanning microscopy with curcumin vital staining (CVS-IFOM). The aim of this study was to use CVS-IFOM to analyze the enteric nervous system (ENS) in mice and human patients with hypoganglionosis and Hirschsprung disease (HSCR). METHODS: In an initial viability study, we compared live ENS images from nonfluorescent C57BL6 mice stained with curcumin (n = 5) and green fluorescent protein mice (n = 5) using multiphoton laser scanning microscopy. We then explored CVS-IFOM for the live examination of resected colon tissues from 1 patient with hypoganglionosis and 3 patients with HSCR. RESULTS: In the viability study, detailed ENS histologic features were only observed in the curcumin-stained mice. In the patient with hypoganglionosis, CVS-IFOM provided ENS details that were not visualized under hematoxylin and eosin staining or calretinin immunohistochemistry, allowing the analysis of ENS size, neural bundle number, and neural cell number per plexus. In patients with HSCR, CVS-IFOM showed a gradual hypoplastic change in the ENS from the oral edge to the anal edge, detecting disproportionate changes in the ENS within the same intestinal level, supporting a circumferentially uneven distribution of the intestinal ENS. CONCLUSIONS: CVS-IFOM may be supportive for intraoperative pathologic diagnosis during surgeries for HSCR.

Hirschsprung Disease↗