Image analysis workflows to reveal the spatial organization of cell nuclei and chromosomes

Nucleus. 2022 Dec;13(1):277-299. doi: 10.1080/19491034.2022.2144013.


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.

Keywords: 3D organization; Nucleus; RNA Pol II; SIM; STED imaging; chromatin; chromosome; crossovers; image analysis; meiosis; metaphase; mitosis; nuclear bodies; nuclear speckles; oligo FISH; pachytene; quantification; segmentation; spatial distribution; transcription factories.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Nucleus*
  • Chromatin*
  • Green Fluorescent Proteins
  • Humans
  • In Situ Hybridization, Fluorescence
  • Microscopy, Fluorescence
  • Workflow


  • Chromatin
  • Green Fluorescent Proteins

Grants and funding

This work was funded by grants from the Swiss National Science Foundation [310030_185186 and IZCOZ0_198171 to CB, 310030_201268 to RS]; the Velux Stiftung (project 1107 to CB); the Ricola foundation (to CB); the ERC Grant Shuffle (Project ID: 669182) and Biotechnology and Biological Sciences Research Council (BB/T008636/1) to IC; the Deutsche Forschungsgemeinschaft to VS (Schu 762/11-1); the UZH-FUB Global Strategy and Partnerships Funding Scheme (grant to K); the Deutsche Akademische Austauschdienst Dienst (grant to K); the International Max Planck Research School for Biology and Computation (project IMPRS-BAC to CJ); the Czech Science Foundation (project 18-20134Sto MAL, 21-02929S to AP); the Operational Programme Research, Development and Education – “Project Internal Grant Agency of Masaryk University” (No.CZ.02.2.69/0.0/0.0/19_073/0016943 to MAL and MK) and ‘Plants as a tool for sustainable global development’ (No. CZ.02.1.01/0.0/0.0/16_019/0000827 to KK and AP); the German Federal Ministry of Education and Research (BMBF) in frame of the grant HERBY (FKZ-031B0188 to SH); the Purkyně fellowship to AP; the European Research Council (H2020 European Research Council ERC-AdG-787074-NucleolusChromatin to R.S.); CT, AS, SM and SD are supported by CNRS, INSERM, Université Clermont Auvergne (UCA), Agence Nationale de la Recherche (project grants 16-IDEX-0001 CAP 20-25 CIR1 and CIR3).