<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>The three-dimensional nuclear arrangement of chromatin impacts many cellular processes operating at the DNA level in animal and plant systems. Chromatin organization is a dynamic process that can be affected by biotic and abiotic stresses. 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.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>We present an automated method, Nuclear Object DetectionJ (NODeJ), developed as an imageJ plugin. This program segments and analyzes high intensity domains in nuclei from 3D images. NODeJ performs a Laplacian convolution on the mask of a nucleus to enhance the contrast of intra-nuclear objects and allow their detection. We reanalyzed public datasets and determined that NODeJ is able to accurately identify heterochromatin domains from a diverse set of <jats:italic>Arabidopsis thaliana</jats:italic> nuclei stained with DAPI or Hoechst. NODeJ is also able to detect signals in nuclei from DNA FISH experiments, allowing for the analysis of specific targets of interest.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusion and availability</jats:title> <jats:p>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 written in Java and provided as an ImageJ plugin with a command line option to perform more high-throughput analyses. NODeJ can be downloaded from <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://gitlab.com/axpoulet/image2danalysis/-/releases">https://gitlab.com/axpoulet/image2danalysis/-/releases</jats:ext-link> with source code, documentation and further information avaliable at <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://gitlab.com/axpoulet/image2danalysis">https://gitlab.com/axpoulet/image2danalysis</jats:ext-link>. The images used in this study are publicly available at <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://www.brookes.ac.uk/indepth/images/">https://www.brookes.ac.uk/indepth/images/</jats:ext-link> and <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://doi.org/10.15454/1HSOIE">https://doi.org/10.15454/1HSOIE</jats:ext-link>.</jats:p> </jats:sec>