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

ABSTRACT

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.