Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images.Abbreviations: 3D FISH: three-dimensional fluorescence in situ hybridization; 3D: three-dimensional; ASY1: ASYNAPTIC 1; CC: chromocenters; CO: Crossover; DAPI: 4',6-diamidino-2-phenylindole; DMC1: DNA MEIOTIC RECOMBINASE 1; DSB: Double-Strand Break; FISH: fluorescence in situ hybridization; GFP: GREEN FLUORESCENT PROTEIN; HEI10: HUMAN ENHANCER OF INVASION 10; NCO: Non-Crossover; NE: Nuclear Envelope; Oligo-FISH: oligonucleotide fluorescence in situ hybridization; RNPII: RNA Polymerase II; SC: Synaptonemal Complex; SIM: structured illumination microscopy; ZMM (ZIP: MSH4: MSH5 and MER3 proteins); ZYP1: ZIPPER-LIKE PROTEIN 1.

Central European Institute of Technology and Department of Experimental Biology Masaryk University Brno Czech Republic

Central European Institute of Technology and National Centre for Biomolecular Research Masaryk University Brno Czech Republic

Centre of the Region Haná for Biotechnological and Agricultural Research Olomouc Czech Republic

Department of Molecular Mechanisms of Disease DMMD University of Zürich Zürich Switzerland

Department of Plant and Microbial Biology Zürich Basel Plant Science Center University of Zürich Zürich Switzerland

Institut Génétique Reproduction et Développement Université Clermont Auvergne CNRS INSERM 63001 Clermont Ferrand France

Institute of Biology Freie Universität Berlin Germany

Leibniz Institute of Plant Genetics and Crop Plant Research Gatersleben D 06466 Seeland Germany

Service and Support for Science IT Universität Zürich Zürich Switzerland

The James Hutton Institute Errol Road Invergowrie DD2 5DA Scotland UK

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Insight into chromatin compaction and spatial organization in rice interphase nuclei