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جستوجوی دقیقمهندسی پزشکی و فناوری سلامت
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ورود به زیرشاخهفناوری تصویربرداری پزشکی
مقالهها، منابع و پژوهشهای تازه حوزه فناوری تصویربرداری پزشکی
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شواهد مهندسی پزشکی و فناوری سلامت
PubMed2027
Towards a justice-based reimagination of digital health research and innovation: actionable considerations for learning health systems.
The need to embed decolonising methodologies in health-care practice has been recognised globally. The term decolonising health, which has become a topic of increasing public and academic discourse since 2021, refers to the dismantling of colonial systems of dominance and oppression ingrained within health systems and structures. Despite the growing recognition of the need for decolonisation in broader health research and practice, the application of a decolonial lens to the rapidly evolving field of digital health remains largely underexplored. An active commitment to decolonisation should be driven by a quest for social justice. However, a key concern for those committed to decolonisation in any field of inquiry is the continuing hegemony of western Eurocentric knowledge and knowledge systems that dominate much of the global research machinery. Hence, we suggest that decolonisation of digital health should start at the ontological level of coloniality. In this Viewpoint, we present a justice-based reimagination of digital health using the established research paradigm framing of ontology, epistemology, axiology, and methodology. We also map decolonial recommendations across the learning health system model and provide a translational reflexivity checklist to support digital health project teams in decolonising research and innovation activities.
PubMed2027
A Microscopy Toolkit for Root Studies in B. distachyon.
The study of plant organogenesis presents significant challenges due to the small number of cells involved in its initial stages. Observing the initial cell divisions becomes increasingly challenging when transitioning from Arabidopsis thaliana (Arabidopsis) to other species. Lateral root (LR) initiation is an essential process for enhancing a plant's ability to access water and nutrients. In most cases, LR formation starts in the pericycle of the parent root, giving rise to a new meristem. Understanding the mechanisms coordinating LR development is important for improving plant resilience to biotic and abiotic stresses. The prediction of the precise timing and location of LR initiation along the root axis remains challenging, even in Arabidopsis. This is magnified when attempting to observe LR development in crops. Brachypodium distachyon (Brachypodium) has emerged as a versatile model for cereal crops. However, studying Brachypodium LR development requires a revision of protocols and methodologies to be applied from seedling growth to root imaging. Here, we present protocols for seed preparation, in vitro growth, and tissue clearing for Brachypodium. Whereas ClearSee appeared to be unsuitable for rendering Brachypodium root tissues transparent after a reasonable incubation period, our modified DEEP-Clear method resulted in improved efficiency in tissue clearing and is compatible with major fluorescent proteins and dyes. Finally, we introduce a simple and straightforward approach to locally synchronize LR development. These tools and methodologies are crucial for advancing our knowledge of plant root system architecture, transitioning from a model plant to agronomically important species.
PubMed2027
A New Combined Technique for Neophallus Elongation and Girth Enhancement for Metoidioplasty (Barroso Procedure): Total Corpora Mobilization with Martius Flap.
PURPOSE: Metoidioplasty is used in transmasculine gender-affirming surgery; however, limited neophallus length and girth remain significant challenges (1-4). We describe a combined technique using Total Corpora Mobilization (TCM) for elongation and a Martius flap for girth enhancement (5-9). MATERIALS AND METHODS: Fifteen transgender men underwent first-stage metoidioplasty with TCM and Martius flap between 2023 and 2026. The vestibular plate and urethra were mobilized, followed by circumferential clitoral degloving and suspensory ligament division. A pubic periosteal incision was made, and subperiosteal dissection was performed using bipolar cautery while preserving a portion of the periosteum attached to the corpora cavernosa. The urethra was further mobilized and the triangular ligament was divided, followed by lateral dissection of both corpora cavernosa and complete release from the crura. An oral mucosa graft was used to create the urethral plate. The preserved periosteum was fixed to the dorsal tunica albuginea, and a vascularized Martius flap harvested from the labia majora was secured over the corpora for girth augmentation. Scrotal flaps provided wound coverage. RESULTS: The index patient achieved a neophallus length of 6.4 cm, representing a 1.9 cm gain over the preoperative stretched clitoral length. Median length gain was 3.7 cm, and mean circumference increase was 1.6 cm. Glans perfusion and tactile sensitivity were preserved in all patients, with no vascular or sensory complications recorded. CONCLUSIONS: Combined TCM and Martius flap metoidioplasty is feasible and reproducible. By achieving corporal release beyond previously described techniques, TCM may meaningfully enhance neophallus projection while preserving perfusion and sensation. Larger cohorts and longer follow-up are required to confirm the durability and functional outcomes of this approach.
PubMed2027
Advanced Image Generation for Cancer and Stem Cell Biology Using Diffusion Models.
Deep learning has transformed medical image analysis, but progress in cancer and stem cell applications is often constrained by limited access to large, diverse, well-annotated imaging datasets. This bottleneck is especially acute for studies of tumor heterogeneity and cancer stem cell (CSC) biology, where rare phenotypes and dynamic cell-state transitions-frequently linked to stemness-associated transcriptional programs (e.g., OCT4, SOX2, NANOG)-benefit from high-quality imaging across many samples and conditions. At the same time, regulatory and practical barriers (patient privacy, acquisition cost, and uneven institutional data sharing) restrict dataset scale and reuse. Diffusion models offer a practical route to synthetic data expansion by generating high-fidelity synthetic images that retain salient radiologic and pathologic features. In this chapter, we present an end-to-end protocol for adapting latent diffusion (Stable Diffusion) to oncology imaging using DreamBooth fine-tuning with small numbers of representative images, coupled with text-to-image and image-to-image workflows to generate controlled variations across modalities and disease presentations (e.g., brain tumor MRI, breast cancer mammography/CESM). We also describe quantitative and qualitative evaluation strategies, including Fréchet Inception Distance (FID) benchmarking and expert review considerations, to assess realism and diversity. These methods enable cancer and stem cell biologists to augment training data for segmentation and classification, build shareable educational resources, and prototype analyses for rare tumors or stemness-enriched subtypes while potentially reducing reliance on direct sharing of patient images.
PubMed2027
An Integrated Workflow for Culturing and AI-Based Profiling of Mouse Intestinal Epithelium.
Two-dimensional (2D) culture systems are powerful in vitro tools that have revolutionized the study of intestinal stem cell biology. These culture systems derived from mouse intestinal and colonic epithelia enable the modeling of tissue architecture, barrier function, and disease mechanisms under physiologically relevant conditions. This protocol details methods for isolating and maintaining mouse intestinal crypts as 2D monolayers. Monolayers are established via enzymatic dissociation and seeding onto ECM-functionalized hydrogels. This protocol also describes a deep learning pipeline that performs virtual fluorescent staining of label-free phase-contrast images of live organoids. This protocol enables the reproducible generation and computational analysis of murine intestinal epithelial cultures for studies in epithelial biology, drug testing, and disease modeling. This integrated system is designed for facilitating high-throughput imaging, immunolabeling, and functional analysis, expanding its use in regenerative medicine, tissue engineering, and gastrointestinal research.
PubMed2027
Automated Quantification and Morphometric Analysis of Pluripotent Stem Cell Colonies Using ColonyQuant.
Alkaline phosphatase staining is routinely used to evaluate the undifferentiated state of embryonic stem cells, yet quantitative interpretation of colony assays frequently depends on manual inspection and categorical scoring that introduce subjectivity and limit scalability. ColonyQuant provides a standardized, automated framework for objective analysis of alkaline phosphatase-stained colonies from conventional bright-field images. The software performs adaptive colony detection, per-colony intensity measurement, and extraction of eight geometric descriptors that collectively characterize colony size, compactness, symmetry, and boundary complexity. Feature tables generated by the workflow are structured to support statistical comparison across replicates and experimental conditions. Integrated analysis modules enable dimensionality reduction, supervised classification, and feature ranking, facilitating interpretation of phenotypic differences without requiring custom scripting. Visualization routines generate distribution plots, contour-density maps, multivariate embeddings, and representative shape mosaics to summarize population heterogeneity and morphological organization. Applied to pluripotent stem cell cultures subjected to chromatin perturbation, the platform detects coordinated changes in colony growth behavior and structural architecture that may not be evident through visual scoring alone. The protocol describes installation, configuration, batch image processing, quality control, hierarchical data aggregation, and downstream statistical analysis, providing a reproducible approach for transforming qualitative colony assays into quantitative, high-content phenotypic datasets suitable for stem cell research and screening applications.
PubMed2027
Cell Biology of Botrytis: Live-Cell Imaging Techniques and Protein Localization Protocols.
Botrytis cinerea is a widely studied model organism for plant-fungal interactions. This chapter provides simple and easy protocols for live-cell imaging techniques and subcellular protein localization studies in B. cinerea in vitro. Using a simple microscopy setup, we describe methods for imaging spore germlings and hyphae and for studying protein dynamics in response to the antifungal compound α-tomatine. The protocols highlight the application of fluorescence microscopy to visualize sterol distribution and protein localization patterns, enabling deeper insights into fungal cell biology.
PubMed2027
Characteristic Analysis of the Resting-State fMRI Global Signal in Schizophrenia.
The global signal (GS) represents the broad variations in neural activity throughout the brain. Recent research has identified changes in the GS of functional magnetic resonance imaging (fMRI) in schizophrenia, challenging the traditional view of GS as mere noise that is typically discarded during data preprocessing. However, there has been no comprehensive characteristic analysis of GS in schizophrenia. This study recruited 100 patients with schizophrenia and 113 healthy subjects to perform resting-state fMRI. The averaged gray matter fMRI signal is denoted as GS. The GS topography was constructed by calculating Pearson correlation (i.e. GSCORR) between the GS and time series of each gray matter voxel. Furthermore, the relevance between GS topography and the function network features was constructed according to graph theory. Finally, we implemented an integrated analytical framework combining independent component analysis and multiple linear regression to quantify the contributions of resting-state brain networks to the spatiotemporal characteristics of the GS. The GSCORR of schizophrenia decreased in the bilateral insula and exhibited a significant negative association with disease duration. The increased GSCORR in the thalamus and default mode network (DMN) showed a positive correlation with the scale scores. We found that global properties are represented by GS topography in schizophrenia. In addition, the linear characterization results of GS showed that the component contribution decreased at the primary sensory network but increased at the high-order associated network. Our results further demonstrated that GS contains brain features associated with schizophrenia, which would help to understand the neural mechanisms underlying the psychopathological symptoms of schizophrenia.
PubMed2027
Chlamydia trachomatis Infection in a Microphysiologic Cell Culture Model.
Chlamydia trachomatis is an obligate intracellular bacterium that infects the columnar epithelium of the human endocervix. While conventional two-dimensional cell cultures and animal models have been instrumental in advancing our understanding of C. trachomatis biology, they are limited in capturing the multicellularity, architecture, and physiological microenvironment of the human cervix. This chapter describes the use of a three-dimensional (3D) microphysiologic model to study Chlamydia trachomatis infection. The model is inexpensively made without specialized equipment and is designed to recreate the epithelial-stromal interface. We outline procedures for coculturing cervical epithelial cells and fibroblasts, infecting epithelial cells with fluorescently labeled C. trachomatis, monitoring infection progression via fluorescent microscopy, and quantifying infectious progeny. The complete developmental cycle of C. trachomatis within this model provides a robust and accessible platform to investigate C. trachomatis-specific host-pathogen interactions, immune responses, and the influence of diverse physiological and environmental stimuli within a relevant cervical context.
PubMed2027
Culture and Maintenance Methods for Botrytis cinerea.
Culturing and maintaining Botrytis cinerea in vitro is essential for advancing research on fungal biology, host-pathogen interactions, and plant disease management. Reliable culture methods provide the basis for reproducible experiments, ensuring stable growth and long-term preservation of isolates. This chapter details standardized procedures for establishing fungal colonies on agar and broth media, including Universal Beer Agar, Potato Dextrose Agar, and Czapek-Dox Agar, as well as specialized formulations such as Carrot Agar and Honey Peptone Agar. Protocols for short-term maintenance on agar slants and long-term storage in glycerol at -80°C are described to ensure strain viability over time. Critical variables affecting fungal growth, such as temperature, light, pH, and medium composition, are also discussed, providing guidance for optimizing culture conditions. Together, these methods serve as practical tools for laboratories aiming to maintain pure B. cinerea cultures for physiological, genetic, and pathological studies.
PubMed2027
Deep Insights Without Clearing: An Optimized Protocol for Patient-Derived Whole-Organoid Immunofluorescence Imaging.
Here, we present an optimized whole-organoid immunofluorescence staining protocol that preserves morphology, achieves uniform antibody penetration, and enables deep-tissue imaging without the need for tissue clearing. Organoids are three-dimensional, self-organizing structures that replicate many of the cellular and architectural features of their tissue of origin, making them powerful preclinical models for studying development, disease, and therapeutic responses. Most organoid research is performed on formalin-fixed, paraffin-embedded (FFPE) samples, in which organoids are sectioned across the Z-planes. While this method facilitates conventional histological analysis, it limits spatially resolved visualization of protein expression and tissue organization. The inherent density and abundant extracellular matrix of organoids present substantial barriers to reagent penetration and imaging depth, further constraining the ability to capture their three-dimensional architecture in full. Our new workflow includes steps for fixation, permeabilization, and antibody incubation and has been validated on intestinal organoids using markers for different membrane-associated proteins. By providing high-quality staining through the full organoid depth without clearing, this protocol streamlines sample preparation, reduces processing artifacts, and allows more rapid, accurate 3D mapping of cell types and structures in organoid-based research.
PubMed2027
EdU-Based Quantitative Analysis of Cell Proliferation in Arabidopsis Root Meristems.
The division of a mother cell into two daughter cells is a fundamental process in biology, involving the transfer of genomic information to the next generation of cells. In plant roots, cell divisions occur in the root apical meristem, a specialized tissue located at the plant's root tip. Cell proliferation rates in the root apical meristem shape root growth, thereby contributing to general development as well as the acclimatization to stressful environmental conditions through phenotypic plasticity. Here, we present a protocol for the microscopical analysis of cell proliferation in Arabidopsis thaliana root meristems by 5-ethynyl-2'-deoxyuridine (EdU) staining. EdU, a thymine-analog, is incorporated into newly synthesized DNA of proliferative cells during the S-phase of the cell cycle. Subsequently, EdU-containing nuclei can be labeled with a fluorophore and detected by confocal laser scanning microscopy. The combination of the EdU assay with cell wall staining enables the quantification of root meristem architecture parameters and cell division activity from microscopic images. Together, this can elucidate how root meristems respond to changes in environmental conditions or address fundamental questions of developmental plant biology.
PubMed2027
Fluorescence and Ultrastructural Microscopy for Botrytis cinerea.
Botrytis cinerea, a notorious pathogenic fungus, leads to postharvest decay in horticultural crops and substantial economic losses, while the pathogenicity of B. cinerea is closely related to its structural characteristics and cellular homeostasis. Among the currently available techniques, fluorescence and ultrastructural observations serve as crucial tools for investigating subcellular structures and cellular events during B. cinerea growth. These techniques enable the monitoring of cell structural integrity, such as variations in the cell membrane and cell wall, as well as intracellular changes, such as reactive oxygen species bursts and cell apoptosis. The results at cytological levels may not only facilitate close-up dissection of B. cinerea cells, but also provide important cues to uncover the inhibitory mechanism of exogenous treatments. This chapter introduces fluorescence staining and observation, as well as the preparation of ultrathin sections of B. cinerea conidia, aiming to provide methodological guidance for cytological examinations of B. cinerea.
PubMed2027
Global Genomic Surveillance.
Global genomic surveillance has emerged as a foundational pillar of public health in the twenty-first century, enabling real-time tracking of pathogen evolution and informing outbreak response. This chapter examines the strategic architecture of global genomic surveillance, focusing on its application to arboviruses such as chikungunya virus (CHIKV). It explores the integration of genomic data with epidemiological, clinical, and environmental information within a One Health framework, while addressing critical challenges in governance, equity, and interoperability. The discussion covers the entire genomic surveillance workflow, from sample collection and sequencing to bioinformatic analysis and phylogenetic inference, and highlights the transformative role of artificial intelligence (AI) in predictive surveillance. By analyzing global initiatives, operational barriers, and emerging technologies, this chapter underscores the necessity of sustainable, equitable, and interoperable genomic systems to proactively address current and future infectious disease threats.
PubMed2027
High-Resolution Detection of Chlamydia Proteins by Expansion Microscopy.
The pathogenic bacterium Chlamydia replicates via an unusual developmental cycle that takes place within a membrane-bound inclusion inside an infected host cell. Immunofluorescence microscopy has revealed many details about the two specialized developmental forms and the recruitment of host organelles to the inclusion, but lacks the high resolution of electron microscopy (EM). In this chapter, we describe the use of an innovative microscopy method, called Tenfold Robust Expansion (TREx) microscopy, to visualize proteins in Chlamydia-infected cells at nanoscale resolution. We present a detailed protocol describing the steps of this expansion microscopy method, including immunostaining, anchoring, gelation, homogenization, expansion, and visualization with a standard confocal microscope.
PubMed2027
High-Resolution Detection of Sphingolipids During Chlamydia trachomatis Infection via Expansion Microscopy.
The obligate intracellular bacterial pathogen Chlamydia trachomatis establishes a membrane-bound compartment called the inclusion, which serves as a replicative niche, within host cells. Sphingolipids, a major class of eukaryotic membrane lipids, are acquired from the host and incorporated into chlamydial membranes. Visualization of individual bacteria within inclusions or sub-bacterial structures by fluorescence microscopy remains challenging due to the limited resolution of conventional light microscopy. Expansion microscopy (ExM), a super-resolution imaging technique, enables nanoscale resolution on standard confocal microscopes and is an ideal tool to study Chlamydia infections. However, current ExM protocols are not compatible with sphingolipid visualization. Here, we present a strategy that enables ExM-based imaging of sphingolipids during C. trachomatis infection.
PubMed2027
High-Throughput Root Phenotyping of Maize Seedlings.
Imaging has revolutionized phenotyping analysis by providing accurate documentation and records of observed phenotypes. Analyzing imaging data remains a major bottleneck in root phenotyping analysis. Advances in technology offer a plethora of analysis options at varying automation levels and price points, providing a reliable open-source pipeline for image analysis that delivers stable, reproducible results. Here, we discuss a maize seedling image analysis pipeline powered by open-source tools that enables rapid analysis of seedling root phenotypes. This image analysis pipeline uses Root Painter, an optional AI cleanup step, and RhizoVision Explorer to extract phenotypic data from maize seedlings grown on rolled towel assays, which are commonly used, enabling rapid analysis of these images.
PubMed2027
Imaging of Ceratopteris richardii Root Anatomy.
Here, we present detailed protocols for histological staining and imaging of root cross sections of the fern Ceratopteris richardii, providing high-resolution visualization of cellular and tissue structures essential for understanding root development, nutrient and water uptake, and responses to environmental cues. This chapter describes the preparation of root samples, fixation, embedding, sectioning, and the application of specific histological stains that highlight key anatomical features, including the epidermis, cortex, endodermis, vascular tissues, and root cap. The protocol also covers imaging techniques using confocal microscopy to obtain clear, reproducible images suitable for quantitative and qualitative analyses. This standardized approach facilitates comparative studies of root anatomy and development, offering a valuable tool for plant developmental biology research while ensuring the preservation of tissue morphology.
PubMed2027
Improving Image Quality in 10× Visium Spatial Transcriptomics Using Vispro.
10× Visium is a widely used spatial transcriptomics platform that enables joint profiling of gene expression and the spatial locations of cells. However, the histology images generated by the 10× Visium platform often contain technical artifacts, including fiducial markers and background noise, which degrade image quality. Here, we describe how a computational method, Vispro, can be applied to process and enhance these images. The resulting high-quality images lead to improved performance across a range of downstream analyses.
PubMed2027
Labeling and Detection of Peptidoglycan in Chlamydia and Chlamydia-Related Organisms Utilizing D-Alanine Analogs.
The incorporation and visualization of modified D-amino acids into the peptidoglycan (PG) of actively replicating microbes have made a tremendous impact on the field of bacterial physiology. Here, we describe the optimization of this technique for labeling PG in obligate, intracellular Chlamydia species as well as Chlamydia-like organisms. Fluorescent D-amino acids (FDAAs) are D-alanine analogs that readily incorporate into the stem peptides of living bacteria and can be visualized rapidly with a fluorescence microscope without the need for laborious postfixation procedures. By comparison, D-amino acid analogs (DAAAs) and D-amino acid dipeptides (DAADs) similarly incorporate into newly synthesized peptidoglycan but require postfixation steps to effectively visualize PG. These probes were foundational in demonstrating the presence of PG in Protochlamydia and Chlamydia species, and PG labeling is a convenient tool that can augment other fluorescence imaging approaches. Here, we provide a protocol for the incorporation of D-alanine analogs into the PG of intracellular bacteria and the subsequent steps enabling these amino acids to be visualized via fluorescence microscopy. We also describe the use of an analysis tool that enables the mapping of PG-labeled objects in three-dimensional space and a protocol for confirming that modified amino acids are incorporating into bacterial PG.
PubMed2027
Lipase-Catalyzed Synthesis of Nutritional Structured Lipids at Laboratory-Scale Using Crude or Refined Oils.
In this chapter, some examples of laboratory protocols to produce nutritional structured lipids, namely human milk fat substitutes, dietetic triacylglycerols, and interesterified fat blends with improved functional and rheological properties, catalyzed by either immobilized commercial or noncommercial lipase preparations, are presented. The use of crude oils instead of refined counterparts to reduce oil purification costs is also addressed. In addition to batch synthesis, the continuous production in packed- or fluidized-bed bioreactors is addressed, as well as the evaluation of operational stability of the biocatalysts used (either in batch reuses or in continuous mode).
PubMed2027
Live Cell Imaging of Chlamydia Inclusion Membrane Proteins Using Genetically Encoded Antibodies.
Chlamydia subverts host cell pathways by secreting effector proteins into the membrane of its inclusion. These bacterial membrane proteins, called Incs, mediate interactions between the inclusion and the host cell. To study Inc protein dynamics in live cells, we have developed a methodology that relies on host cell expression of antibodies fused to fluorescent proteins, known as frankenbodies, which detect epitopes exposed to the cytosol. HeLa cells are first stably transfected with a plasmid encoding an mScarlet3-tagged anti-FLAG frankenbody. These cells are then infected with Chlamydia expressing a FLAG-tagged Inc protein, which is secreted into the inclusion membrane with the FLAG tag facing the cytosol. Recruitment of the fluorescent anti-FLAG frankenbody to the inclusion is monitored in real time using time-lapse confocal microscopy. This approach has been successfully employed to elucidate the dynamics of IncA on the inclusion membrane and can be expanded to simultaneously investigate multiple Incs, as well as Inc:Inc interactions, by using different tag-frankenbody combinations.
PubMed2027
Live Fluorescence Lifetime Imaging Microscopy (FLIM) of the Epithelial Polarity and Cell-Specific Segmentation in the Small Intestinal Organoids.
Apical-basal polarity is essential for gastrointestinal epithelium function, where the apical membrane mediates nutrient absorption, host-microbiota interactions, and pathogen defense. However, conventional intestinal organoid culture displays an "apical-in" topology, limiting functional research with respect to the apical membrane. F-actin labeling with fluorescent phalloidin, a well-established apical marker, is also incompatible with live imaging. To address these limitations, in this chapter we describe an extracellular matrix (ECM) removal protocol to achieve "apical-out" topology in porcine intestinal organoid model and further validated WGA and Nile Red live imaging tracers enabling discrimination between apical-out (AO) and basal-out (BO) organoids. Subsequently, using integrated computational pipelines (automatic nuclei segmentation and single-nucleus phasor-based lifetime analysis), we can quantify organoid proliferative states at single-nucleus resolution following BrdU and Hoechst 33342 staining. Using the analysis of Hoechst 33342 fluorescence lifetime, we demonstrate that both organoid topologies exhibit intrinsic proliferative heterogeneity, but overall proliferative capacity is topology-dependent: apical-out organoids displayed decreased proliferation, whereas basal-out organoids remain highly proliferative.
PubMed2027
Live Imaging of Grass Roots Growth Dynamics Using Microfluidics.
Microfluidic tools enable manipulation of microliter-scale volumes within a stable microenvironment. Microfluidics has been widely adopted in various biological fields for, among other applications, controlled delivery of sensory, chemical, and mechanical stimuli to the observed organisms. In this chapter, we describe the design and usage of a custom-made double-layered PDMS microfluidic device, named BUGchip, developed for the purpose of studying plant root growth and metabolic responses in rapidly changing conditions with a high time-scale resolution. We demonstrate the utility of this device by a showcase experiment with a hyperosmotic treatment of Brachypodium distachyon roots, in which changes in growth rate and relative pH across the root surface in response to the treatment can be observed within minutes.
PubMed2027
Live Imaging of Ions in Cells Infected with Chlamydia trachomatis.
This chapter describes methodological approaches for studying intracellular ion dynamics during Chlamydia trachomatis infection. It emphasizes the requirement for live-cell imaging to capture active and compartmentalized ion fluxes in infected cells precluded by fixed-cell techniques. Specifically, we present step-by-step protocols for labeling Chlamydia-infected cells with a fluorescent potassium ion (K+)-sensitive probe for performing live imaging with confocal microscopy or spinning disc confocal microscopy for higher resolution and procedures for image processing and quantification. We also describe time-lapse microscopy to monitor K+ dynamics over the extended time course of the infection. These advanced microscopy methods have the potential to visualize ion transport in real time at the host-pathogen interface.
PubMed2027
Mapping Labile Fe2+ and Fe3+ in Living Arabidopsis thaliana Roots: Imaging and Quantification.
Iron (Fe) is involved in numerous key physiological processes due to its transfer or gain electron capacity. However, in excess, Fe is toxic to cells. The understanding of mechanisms governing its homeostasis requires imaging techniques capable of localizing and quantifying Fe and its redox state in situ. The Perls-DAB histochemical staining is a widely used method for visualizing Fe at a cellular level. Other advanced approaches, such as micro X-ray fluorescence and X-ray absorption near-edge structure spectroscopy, also provide information on the spatial distribution and oxidation state of Fe. However, all of these methods require sample fixation before observing Fe localization, which can introduce biases in the results. Recent development in synthetic fluorescent probes now enables the real-time visualization of ferrous (Fe2+) and ferric (Fe3+) forms in living roots, offering a robust and user-friendly approach to study Fe redox distribution in planta. This live imaging approach provides access to Fe dynamics and enables semi-quantitative analyses, thereby opening new avenues for Fe homeostasis research. This chapter provides detailed protocols for applying these methods in Arabidopsis roots.
PubMed2027
Measuring Membrane Protein Dynamics in Plant Roots Using Fluorescence Correlation Spectroscopy.
Quantitative methods using imaging and spectroscopy offer valuable insights into cell biology and molecular biophysics by elucidating complex mechanisms across various cellular environments. Fluorescence correlation spectroscopy (FCS) is particularly advantageous for quantifying the dynamics of fluorescent particles in low-concentration samples. As a high-resolution spectroscopic method with single-molecule sensitivity, FCS is well-suited to investigating membrane proteins, molecular interactions, aggregation, and conformational changes in living systems, including plant cells. However, challenges such as background fluorescence and artifacts arising from biological sample properties require careful experimental design, thereby limiting the routine application of FCS in plant tissues. Recent advancements in technology, such as confocal optics and highly efficient photon detectors, have substantially enhanced FCS's sensitivity and expanded its capacity for single-molecule detection. These advancements now empower plant biologists to explore the dynamics of plant proteins under various conditions. This chapter focuses on methodologies in fluorescence autocorrelation spectroscopy (FACS/FCS) and their application to plant research, with a particular emphasis on plasma membrane proteins in Arabidopsis root cells.
PubMed2027
Monitoring Calcium Dynamics in Pig Intestinal Organoids with FLIM and FRET.
Intracellular calcium signaling plays a crucial role in intestinal epithelial function. In this chapter, we describe a method to monitor calcium dynamics in live pig small intestinal organoids using stably expressed genetically encoded FRET-based biosensor in combination with intensity-based ratiometric and fluorescence lifetime imaging (FLIM) microscopy readouts. The calcium biosensor Twitch-2B was introduced into organoids by electroporation. Despite the modest lifetime contrast, the biosensor provides a highly reliable ratiometric signal and is compatible with both conventional fluorescence and FLIM-based imaging platform. Following electroporation, organoids are allowed to form and subsequently were selected to establish stable biosensor-expressing lines. This is followed by the live microscopy of heterogeneous and cell-specific calcium responses upon pharmacological stimulation. The presented protocol offers a versatile approach for studies of calcium signaling in relevant 3D cell models, such as organoids and tissues-on-a-chip.
PubMed2027
Robot-Assisted Partial Nephrectomy in a High-Complexity Renal Tumor in a Horseshoe Kidney.
INTRODUCTION: Horseshoe kidney is an uncommon congenital fusion anomaly that can make renal tumor surgery especially challenging because of altered rotation, limited mobility, variable vascular supply, and an unpredictable collecting system (1-7). This video presents a robot-assisted partial nephrectomy for a high-complexity renal tumor in this setting. CASE PRESENTATION: A 33-year-old man, with ECOG 0 and no relevant comorbidities, was diagnosed with a 7.5-cm solid renal mass in the central posterior portion of the left moiety of a horseshoe kidney. The lesion had a RENAL score of 10p. Contrast-enhanced computed tomography and three-dimensional reconstruction were used to understand the relationship between the tumor, aberrant vessels, renal hilum, and collecting system, supporting the decision to attempt nephron-sparing surgery (5, 8). Surgical technique and results: The procedure was performed through a transperitoneal robotic approach with the patient in right lateral decubitus using the Da Vinci Si platform. Port placement followed a standard renal robotic configuration, with a paramedian supraumbilical camera port, three robotic working ports along a craniocaudal lateral axis, a caudal fourth-arm port, and two medial assistant ports for suction, exposure, and support during renorrhaphy. After exposure of the horseshoe kidney and left hilar dissection, two arterial branches and one renal vein were identified. Tumor excision was performed under vascular control, with 20 minutes of warm ischemia and no collecting system opening, followed by two-layer absorbable renorrhaphy with adjunctive hemostatic agents. The operative time was 150 minutes. No transfusion, conversion, drain placement, or relevant immediate complication occurred. The urinary catheter was removed after 24 hours, and the patient was discharged 72 hours after surgery. Pathology showed clear cell renal cell carcinoma, Fuhrman grade 3, pT2N0M0, with negative surgical margins. During 12 months of oncologic follow-up, renal function remained stable and semiannual imaging showed no evidence of recurrence. Contemporary video reports have also emphasized the feasibility of advanced robotic renal surgery and complex partial nephrectomy strategies in selected patients (9, 10). CONCLUSION: In a carefully selected patient, robot-assisted partial nephrectomy supported by three-dimensional planning was feasible for a complex renal tumor in a horseshoe kidney, with negative surgical margins, preserved renal function, and no recurrence during 12 months of follow-up.
PubMed2027
RootHairML: A Simple Machine-Learning Tool for Quantifying Root Hairs.
Root hairs play pivotal roles in nutrient and water acquisition and in plant-microbe interactions. Consequently, understanding the mechanisms underlying root hair development and their regulatory pathways is an important aspect of plant physiology research. Quantifying root hairs and root hair length is often essential in such studies, but is labor-intensive and prone to subjectivity. The availability of straightforward tools for automated root hair measurements is limited, and existing options are often tailored for specific images or do not measure individual root hairs. To address this, we developed RootHairML, a flexible and simple Python-based machine learning tool designed for efficient quantification of root hair lengths. Based on labeled images and pixel features, RootHairML trains a Random Forest model to enable the detection of root hairs in new images. It provides measurements of individual root hair lengths per image and generates annotated images showing all detected root hairs, allowing for manual verification and adjustments. Here, we describe and showcase the use of RootHairML. Overall, RootHairML offers a valuable tool for root hair analysis, enabling researchers to increase data collection and enhance the reproducibility of root hair studies.