Methods最新文献

{"title":"Multifunctional nanophotonic photoacoustic biosensors: a new era in molecular imaging–guided deep-tissue cancer monitoring","authors":"Bakr Ahmed Taha ,&nbsp;Ghassan M. Sulaiman ,&nbsp;Ali J. Addie ,&nbsp;Khalil A.A. Khalil ,&nbsp;Elsadig M. Ahmed ,&nbsp;Vishal Chaudhary ,&nbsp;Norhana Arsad","doi":"10.1016/j.ymeth.2025.06.005","DOIUrl":"10.1016/j.ymeth.2025.06.005","url":null,"abstract":"<div><div>Monitoring cancer therapy is difficult because of restricted imaging depth, inadequate molecular specificity, and delayed response evaluation. Moreover, conventional imaging techniques fail to provide high-resolution, real-time views of the dynamic tumor microenvironment during therapy. Among emerging technologies, nanophotonic photoacoustic biosensors have gained prominence as multifunctional platforms that enable real-time, non-invasive imaging and dynamic monitoring of cancer therapy. This review discusses advances in nanophotonic engineering, including plasmonic nanostructures, NIR-II fluorophore-integrated systems, SERS-active materials, fiber-optic probes, and hybrid nanosystems, all tailored to enhance molecular targeting and signal specificity. In addition, biomimetic and biologically inspired nanosystems with enhanced tissue penetration and reduced autofluorescence in the NIR-II spectrum can be specifically highlighted. The key aspects of clinical translation are examined including biosafety, molecular specificity, and scalability. Furthermore, further explore the convergence of these biosensors with artificial intelligence and Internet of Things (IoT) frameworks to support adaptive, patient-specific decision-making in oncology. As a result of these multifunctional systems that combine nanophotonics, machine learning, and molecular diagnostics, oncology could shift towards precision-guided treatment. Finally, it proposes strategic avenues for clinical adoption, placing PAS at the vanguard of the next generation of cancer diagnostics.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"242 ","pages":"Pages 1-23"},"PeriodicalIF":4.2,"publicationDate":"2025-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144293159","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Inside the cell: Approaches to evaluating mRNA internalization and trafficking","authors":"Claudia Del Toro Runzer ,&nbsp;Christian Plank ,&nbsp;Martijn van Griensven ,&nbsp;Elizabeth R. Balmayor","doi":"10.1016/j.ymeth.2025.06.006","DOIUrl":"10.1016/j.ymeth.2025.06.006","url":null,"abstract":"<div><div>With the growing prominence of mRNA-based therapeutics and vaccines, accurately assessing the cellular uptake of mRNA complexes is a critical first step in evaluating both the efficiency of delivery systems and their downstream therapeutic potential. This is especially important when working with novel mRNA constructs, comparing different delivery vectors, or targeting diverse cell types. In this study, we present a suite of methods to quantify and visualize mRNA internalization following transfection of three types of human primary cells: mesenchymal stromal cells, fibroblasts, and osteoblasts. We highlight the utility of fluorescent probes for both qualitative and quantitative assessment of mRNA uptake and intracellular trafficking. To dissect the pathways involved in uptake, we employed three distinct endocytic inhibitors—chlorpromazine, wortmannin, and genistein—each targeting specific endocytic mechanisms. Additionally, we provide protocols for the lipid-based transfection agents Lipofectamine 3000 and 3DFect, which can be adapted for use with similar vectors. Key methodologies such as flow cytometry and correlative light and electron microscopy, known as CLEM, are described in detail for their effectiveness in analyzing mRNA internalization. A deeper understanding of the internalization and intracellular fate of mRNA is essential for the advancement of more efficient and safer mRNA-based delivery platforms.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 173-183"},"PeriodicalIF":4.2,"publicationDate":"2025-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144280431","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterization of targeted knock-in achieved via tandem paired nicking mediated by CRISPR/Cas9 nickases","authors":"Md Towhid Ahmed Shihan ,&nbsp;Toshinori Hyodo ,&nbsp;Takeshi Fujino ,&nbsp;Md. Lutfur Rahman ,&nbsp;Muhammad Nazmul Hasan ,&nbsp;Mrityunjoy Biswas ,&nbsp;Lam Quang Vu ,&nbsp;Nushrat Jahan ,&nbsp;Yuko Mihara ,&nbsp;Sivasundaram Karnan ,&nbsp;Akinobu Ota ,&nbsp;Shinobu Tsuzuki ,&nbsp;Atsushi Toyoda ,&nbsp;Yoshitaka Hosokawa ,&nbsp;Masahiro Kasahara ,&nbsp;Hiroyuki Konishi","doi":"10.1016/j.ymeth.2025.06.004","DOIUrl":"10.1016/j.ymeth.2025.06.004","url":null,"abstract":"<div><div>Targeted knock-in of specific DNA sequences using CRISPR/Cas9 is an advanced technology that enables programmed genome alterations including insertions, deletions, and base substitutions exactly as designed. Despite its utility in life sciences and promise for medical and industrial applications, it remains critical to establish a methodology for highly precise and efficient targeted knock-in to facilitate the practical use of this technology. Tandem paired nicking (TPN) is a genome editing methodology leveraging nicking variants of CRISPR/Cas9 nucleases (Cas9 nickases) to create site-specific nicks within the homologous region of the genome and donor DNA. Such nicking configuration promotes precise and efficient targeted knock-in while repressing the formation of unintended insertions and deletions and p53-mediated DNA damage response. In this study, we conducted a detailed characterization of TPN-based targeted knock-in by performing genome editing assays with various nicking configurations modified from TPN. Our results demonstrated that genomic nicks remarkably contribute to TPN-based targeted knock-in, whereas donor nicks play a less critical role. The introduction of additional nicks beyond the standard two-nick configuration did not further improve the efficiency of TPN-based targeted knock-in. Comparison with other Cas9 nickase-based methodologies for targeted knock-in demonstrated largely equivalent knock-in efficiencies achieved by these methodologies. High-throughput long-read sequencing confirmed a lower incidence of undesired insertions and deletions of various lengths by TPN, in comparison with a conventional Cas9 nuclease-based approach. These findings underscore TPN as a methodology for precise and efficient targeted knock-in, and highlight the broad potential of Cas9 nickase-based targeted knock-in for clinical and industrial applications.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 184-195"},"PeriodicalIF":4.2,"publicationDate":"2025-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144281923","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhancing biliary tract cancer diagnosis using AI-driven 3D optical diffraction tomography","authors":"Se Woo Park ,&nbsp;Hee Chan Moon ,&nbsp;Seok Jin Hong ,&nbsp;Anna Choi ,&nbsp;Seung-Lee Lee ,&nbsp;Da Hae Park ,&nbsp;Eun Shin ,&nbsp;Jung Hyun Jo ,&nbsp;Dong Hee Koh ,&nbsp;Jin Lee ,&nbsp;Jong-Uk Hou ,&nbsp;Kyong Joo Lee","doi":"10.1016/j.ymeth.2025.06.003","DOIUrl":"10.1016/j.ymeth.2025.06.003","url":null,"abstract":"<div><div>Biliary tract cancer is associated with distinct metabolic alterations, particularly in lipid metabolism. This study aimed to classify biliary tract cancer cells automatically based on lipid droplet (LD) characteristics using three-dimensional (3D) optical diffraction tomography (ODT) combined with convolutional neural networks (CNNs). Human biliary tract cancer cell lines (SNU1196, SNU308, and SNU478) and a normal cholangiocyte cell line (H69) were cultured to evaluate the LD volume, mass, and count. We generated 3D refractive index tomograms and developed a CNN-based diagnostic system for automated classification. The biliary tract cancer cells exhibited significantly increased LD volume, mass, and count compared with those of normal cholangiocytes, reflecting distinct metabolic profiles. The EfficientNet-b3 model achieved an area under the curve (AUC) of 0.982 and an accuracy of 93.79%. Incorporating LD metadata, such as volume and dry mass, improved performance, yielding an AUC of 0.997 and an accuracy of 97.94%. Combining LD metadata with multi-view score fusion enhanced diagnostic performance (AUC: 0.999, accuracy: 98.61%). Further, LayerCAM analysis revealed that the model focused on LD-rich cytoplasmic regions, thereby aligning with known metabolic phenotypes. Overall, our findings demonstrate the diagnostic potential of LD characteristics and support the clinical utility of 3D ODT combined with deep learning for early detection of biliary tract cancer and future multimodal applications.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 196-203"},"PeriodicalIF":4.2,"publicationDate":"2025-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144245524","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comprehensive account of exosome isolation from rat substantia nigra for mass spectrometry-based proteomics study","authors":"Satyajit Ghosh ,&nbsp;Surojit Ghosh ,&nbsp;Aniket Jana ,&nbsp;Rajsekhar Roy ,&nbsp;Surajit Ghosh","doi":"10.1016/j.ymeth.2025.06.002","DOIUrl":"10.1016/j.ymeth.2025.06.002","url":null,"abstract":"<div><div>Exosomes, small extracellular vesicles originating from endocytic processes, have garnered increasing attention due to their roles in both physiological functions and pathological conditions. Initially identified in the 1980 s, exosomes are formed within multivesicular bodies (MVBs) through the invagination of the endosomal membrane, leading to the creation of intraluminal vesicles (ILVs). These ILVs can either be degraded by lysosomes or released into the extracellular space as exosomes, facilitating intercellular communication. In the nervous system, exosomes are implicated in various functions, including neural development and the progression of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. This study presents a novel protocol for the isolation and proteomic analysis of exosomes derived from the substantia nigra (SN) of rat brains. By employing a combination of differential centrifugation and immunocapture techniques, we achieved a purer exosome fraction and higher exosome yield compared to traditional ultracentrifugation methods. Our proteomics analysis identified 51, 48, and 70 proteins from three distinct exosome samples (SN-EV-1, SN-EV-2, and SN-EV-4), with Gene Ontology annotation revealing their involvement in diverse biological functions. This research not only establishes a reliable method for isolating brain-derived exosomes but also sets the stage for comparative studies between healthy and neurodegenerative conditions. Ultimately, our findings aim to enhance the understanding of exosomal roles in disease mechanisms and contribute to the identification of potential biomarkers and therapeutic targets for neurodegenerative disorders.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 150-162"},"PeriodicalIF":4.2,"publicationDate":"2025-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144241615","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"cfDNAFE: Comprehensively extracting multi-omics features of cell-free DNA for noninvasive diagnosis","authors":"Wanxin Cui ,&nbsp;Junjie You ,&nbsp;Wenlong Jie ,&nbsp;Zihao Li ,&nbsp;Xiaoqing Peng","doi":"10.1016/j.ymeth.2025.05.013","DOIUrl":"10.1016/j.ymeth.2025.05.013","url":null,"abstract":"<div><div>The tissues-of-origin of circulating cell-free DNA (cfDNA) holds great promise for non-invasive diagnosing cancers, monitoring allograft rejection, and prenatal testing. Many features for inferring the tissues-of-origin of cfDNAs are being revealed from different angles, including genetics, epigenetics, and fragmentomics, with whole-genome sequencing (WGS) and whole-genome bisulfite sequencing (WGBS) data of cfDNA. However, it lacks integrative toolkits for automatically extracting the revealed features from the WGS and WGBS data of cfDNA samples. Here, we propose cfDNAFE, a comprehensive and easy-to-use python package for extracting multi-omics features from the aligned cfDNA sequencing data. It covers three aspects: cfDNA genetic features, cfDNA methylation features, and cfDNA fragmentation features, including 13 types of feature profiles. The genetic features include substitution mutations, mutation signatures and copy number variations. The methylation features are the proportions of methylated fragments, unmethylated fragments, and mixed methylated fragments on cell-type-specific markers. The fragmentation features related to the fragment sizes, end/breakpoint motifs, and nucleosome positions are also integrated. To verify the functions of cfDNAFE, we perform analysis on the WGS/WGBS data of cfDNA samples based on the feature profiles extracted by cfDNAFE. The comparison between the cfDNA samples of hepatocellular carcinoma (HCC) patients and normal controls suggests HCC cfDNA samples exhibit significant difference in fragment size related features and breakpoint/end motif patterns, and obtain significant higher OCF values in the liver-specific open regions than the health controls. Conclusively, cfDNAFE is a most comprehensive toolkit which covers the most features for inferring the tissues-of-origin of cfDNAs in existing studies up to date. It will facilitate researchers to build machine learning models for auxiliary diagnosis based on these features. Availability and implementation: <span><span>https://github.com/Cuiwanxin1998/cfDNAFE</span><svg><path></path></svg></span>.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 163-172"},"PeriodicalIF":4.2,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144232867","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Label-free spectral confocal reflectance microscopy for ex vivo neuroimaging and neural structure visualization","authors":"Reinher Pimentel-Domínguez ,&nbsp;Rainald Pablo Ordaz ,&nbsp;Abraham J. Cisneros-Mejorado ,&nbsp;Rogelio O. Arellano ,&nbsp;Remy Avila","doi":"10.1016/j.ymeth.2025.06.001","DOIUrl":"10.1016/j.ymeth.2025.06.001","url":null,"abstract":"<div><div>Confocal microscopy is an essential technique in the field of life sciences, commonly used to study molecules in a variety of preparations, ranging from cells and tissues to entire organisms. In the field of neuroscience, it is a widely utilized tool for both anatomical-structural and functional studies. However, a lesser-known application of this microscopy method is confocal reflectance microscopy, which involves image acquisition based on reflected light from the sample. In this study, we present the use of spectral confocal reflectance microscopy (SCoRe) to explore multiple structures in the rat brain, without the use of any dyes or immunolabeling. Our results demonstrate that this technique allows for the distinction between different brain structures with high spatial resolution, enabling the observation of fibers in the cerebral cortex, corpus callosum, hippocampus, cerebellum, and other regions. These findings highlight the ability of SCoRe to provide detailed anatomical insights that are comparable to those obtained through conventional methods. Additionally, we have shown that SCoRe is compatible with samples prepared using traditional techniques, such as histochemical staining and immunofluorescence. This research emphasizes the value of SCoRe as a cost-effective and label-free method for high-resolution brain imaging, which can improve neuroscience studies and reduce long-term expenses.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 140-149"},"PeriodicalIF":4.2,"publicationDate":"2025-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144223914","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development and interpretation of a pathomics-based model for the prediction of immune therapy response in colorectal cancer","authors":"Yan Luo ,&nbsp;Qiang Tian ,&nbsp;Lei Xu ,&nbsp;Dongmei Zeng ,&nbsp;Hua Zhang ,&nbsp;Tianyu Zeng ,&nbsp;Hua Tang ,&nbsp;Chao Wang ,&nbsp;Yihua Chen","doi":"10.1016/j.ymeth.2025.05.012","DOIUrl":"10.1016/j.ymeth.2025.05.012","url":null,"abstract":"<div><div>Colorectal cancer (CRC) is the third most common malignancy and the second leading cause of cancer-related deaths worldwide, with a 5-year survival rate below 20 %. Immunotherapy, particularly immune checkpoint blockade (ICB)-based therapies, has become an important approach for CRC treatment. However, only specific patient subsets demonstrate significant clinical benefits. Although the TIDE algorithm can predict immunotherapy responses, the reliance on transcriptome sequencing data limits its clinical applicability. Recent advances in artificial intelligence and computational pathology provide new avenues for medical image analysis. In this study, we classified TCGA-CRC samples into immunotherapy responder and non-responder groups using the TIDE algorithm. Further, a pathomics model based on convolutional neural networks was constructed to directly predict immunotherapy responses from histopathological images. Single-cell analysis revealed that fibroblasts may induce immunotherapy resistance in CRC through collagen-CD44 and ITGA1 + ITGB1 signaling axes. The developed pathomics model demonstrated excellent classification performance in the test set, with an AUC of 0.88 at the patch level and 0.85 at the patient level. Moreover, key pathomics features were identified through SHAP analysis. This innovative predictive tool provides a novel method for clinical decision-making in CRC immunotherapy, with potential to optimize treatment strategies and advance precision medicine.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 128-139"},"PeriodicalIF":4.2,"publicationDate":"2025-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144207316","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metabolic impacts of long-chain fatty acids on cardiomyocyte maturation in neonatal mammalian hearts","authors":"Seong-Eung Cha ,&nbsp;Mi Nam Lee ,&nbsp;Eung-Sam Kim","doi":"10.1016/j.ymeth.2025.05.010","DOIUrl":"10.1016/j.ymeth.2025.05.010","url":null,"abstract":"<div><div>Cardiomyocytes are essential models for cardiac disease modeling, drug development, and regenerative therapies. Specifically, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as widely used cellular models with high reproducibility. However, cardiomyocytes generated <em>in vitro</em> tend to remain immature and insufficient in replicating the electrophysiological and mechanical functions of adult cardiomyocytes, limiting the clinical and experimental applications of these models. Thus, various biochemical and biophysical strategies have been explored to promote the maturation of cardiomyocytes, to address these limitations, and more accurately mimic the characteristics of mature cardiomyocytes. This review summarizes recent studies on multiple methodologies employed to induce cardiomyocyte maturation, with a particular emphasis on the role of long-chain fatty acids (LCFAs). The evidence summarized in this review is derived from studies utilizing cardiomyocytes from neonatal mice or rats and hiPSC-CMs. Meanwhile, immature cardiomyocytes have been demonstrated to predominantly rely on glycolysis, transitioning to oxidative phosphorylation through maturation, which enhances electrical stability, contractility, and structural organization. LCFAs play a key role in the cardiomyocyte maturation process by serving as key metabolic factors that generate ATP through mitochondrial β-oxidation, thereby improving metabolic efficiency. Additionally, LCFAs are involved in activating cytoskeletal components and signaling pathways integral to cardiomyocyte contractility. Importantly, studies suggest that when multiple biochemical and biophysical stimuli are simultaneously applied, various aspects of cardiomyocyte maturation are synergistically accelerated. Therefore, future studies focusing on the coordinated application of these regulatory factors are expected to enhance the maturation process, ultimately contributing to the generation of mature cardiomyocytes suitable for regenerative medicine and other advanced applications.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 114-127"},"PeriodicalIF":4.2,"publicationDate":"2025-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144184526","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development and validation of the Azu-Eos as a fluorescence-free staining method for enhanced bright-field microscopy in the comet assay","authors":"Penyo Ivanov,&nbsp;Dessislava Staneva,&nbsp;Milena Georgieva,&nbsp;George Miloshev","doi":"10.1016/j.ymeth.2025.05.011","DOIUrl":"10.1016/j.ymeth.2025.05.011","url":null,"abstract":"<div><div>The comet assay is a valuable technique for assessing cellular DNA damage due to intrinsic and environmental factors, particularly in studies of genotoxicity and mutagenesis. However, its broader application is limited by the need for fluorescent staining, the requirement for expensive fluorescent microscopes, and the inability to preserve and re-examine slides over time. We developed the Azure B-Eosin Y (Azu-Eos) staining method to visualise the comets under a standard bright-field microscope to overcome these limitations. The newly developed DNA-staining technique eliminates the need for fluorescence and allows unlimited slide storage and re-examination without quality deterioration. We optimised various factors to ensure the reliability and quality of bright-field stained comets, achieving results comparable to and even better than those obtained with traditional fluorescent dyes. This cost-effective, sensitive, and fluorescence-free method has broad potential applications in medicine, ecology, and environmental monitoring, enabling fast and on-site genotoxicity testing.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 103-113"},"PeriodicalIF":4.2,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144179830","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}