ArticleBMC bioinformatics2020
FocAn: automated 3D analysis of DNA repair foci in image stacks acquired by confocal fluorescence microscopy.
Article in BMC bioinformatics, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed, 40 citations in OpenAlex.
- Automated machine learning profiling with MAP-HR for quantifying homologous recombination foci in patient samples.NAR cancer · 2025Article
- Chronic lymphocytic leukemia (CLL) screening and abnormality detection based on multi-layer fluorescence imaging signal enhancement and compensation.Journal of cancer research and clinical oncology · 2025Article
- Article
- Evaluation of the antibacterial activity ofFrontiers in microbiology · 2023Article
- HARLEY mitigates user bias and facilitates efficient quantification and co-localization analyses of foci in yeast fluorescence images.Scientific reports · 2022Article
- A computational approach to quantifying miscounting of radiation-induced double-strand break immunofluorescent foci.Communications biology · 2022Article
- A deep learning model (FociRad) for automated detection of γ-H2AX foci and radiation dose estimation.Scientific reports · 2022Article
- DNA damage and repair in peripheral blood mononuclear cells after internal ex vivo irradiation of patient blood withEuropean journal of nuclear medicine and molecular imaging · 2022Article
- Article
- DeepFoci: Deep learning-based algorithm for fast automatic analysis of DNA double-strand break ionizing radiation-induced foci.Computational and structural biotechnology journal · 2021Article
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Authors and funding
7 authors at 3 institutions in 2 countries.
Funding
Abstract
backgroundPhosphorylated histone H2AX, also known as γH2AX, forms μm-sized nuclear foci at the sites of DNA double-strand breaks (DSBs) induced by ionizing radiation and other agents. Due to their specificity and sensitivity, γH2AX immunoassays have become the gold standard for studying DSB induction and repair. One of these assays relies on the immunofluorescent staining of γH2AX followed by microscopic imaging and foci counting. During the last years, semi- and fully automated image analysis, capable of fast detection and quantification of γH2AX foci in large datasets of fluorescence images, are gradually replacing the traditional method of manual foci counting. A major drawback of the non-commercial software for foci counting (available so far) is that they are restricted to 2D-image data. In practice, these algorithms are useful for counting the foci located close to the midsection plane of the nucleus, while the out-of-plane foci are neglected.
resultsTo overcome the limitations of 2D foci counting, we present a freely available ImageJ-based plugin (FocAn) for automated 3D analysis of γH2AX foci in z-image stacks acquired by confocal fluorescence microscopy. The image-stack processing algorithm implemented in FocAn is capable of automatic 3D recognition of individual cell nuclei and γH2AX foci, as well as evaluation of the total foci number per cell nucleus. The FocAn algorithm consists of two parts: nucleus identification and foci detection, each employing specific sequences of auto local thresholding in combination with watershed segmentation techniques. We validated the FocAn algorithm using fluorescence-labeled γH2AX in two glioblastoma cell lines, irradiated with 2 Gy and given up to 24 h post-irradiation for repair. We found that the data obtained with FocAn agreed well with those obtained with an already available software (FoCo) and manual counting. Moreover, FocAn was capable of identifying overlapping foci in 3D space, which ensured accurate foci counting even at high DSB density of up to ~ 200 DSB/nucleus.
conclusionsFocAn is freely available an open-source 3D foci analyzer. The user-friendly algorithm FocAn requires little supervision and can automatically count the amount of DNA-DSBs, i.e. fluorescence-labeled γH2AX foci, in 3D image stacks acquired by laser-scanning microscopes without additional nuclei staining.
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