shape and size), as well as chromatin organization. We previously developed a workflow called NucleusJ2.0, designed to compute nuclear morphometric parameters (e.g. Three-dimensional (3D) imaging methods are widely used to investigate nuclear morphology. Changes in the location of heterochromatic domains (also known as chromocenters in Arabidopsis thaliana ) can have a drastic effect on gene expression and on the maintenance of heterochromatin itself. The spatial arrangement of chromatin within the nucleus has fundamental consequences for the accessibility and activity of regions of the genome. The nucleus is a dynamic and complex structure that changes morphology and organization of its DNA content during development. The images used in this study are publicly available at and. NODeJ can be downloaded from with source code, documentation and further information avaliable at. NODeJ is written in Java and provided as an ImageJ plugin with a command line option to perform more high-throughput analyses. NODeJ allows for efficient automated analysis of subnuclear structures by avoiding the semi-automated steps, resulting in reduced processing time and analytical bias. NODeJ is also able to detect signals in nuclei from DNA FISH experiments, allowing for the analysis of specific targets of interest. We reanalyzed public datasets and determined that NODeJ is able to accurately identify heterochromatin domains from a diverse set of Arabidopsis thaliana nuclei stained with DAPI or Hoechst. NODeJ performs a Laplacian convolution on the mask of a nucleus to enhance the contrast of intra-nuclear objects and allow their detection. This program segments and analyzes high intensity domains in nuclei from 3D images. We present an automated method, Nuclear Object DetectionJ (NODeJ), developed as an imageJ plugin. Three-dimensional imaging technology allows to follow these dynamic changes, but only a few semi-automated processing methods currently exist for quantitative analysis of the 3D chromatin organization. Chromatin organization is a dynamic process that can be affected by biotic and abiotic stresses. The three-dimensional nuclear arrangement of chromatin impacts many cellular processes operating at the DNA level in animal and plant systems.
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