Fig. 1

Workflow for quantitative mapping of specific FISH signals on 3D chromatin compaction maps. a Representative part of a section from an original 3D-SIM image stack of a whole nucleus acquisition shown by the example of a BJ1 nucleus. Chromatin counterstained with DAPI (gray), two adjacent DNA targets visualized by green and red fluorescent signals (arrow). The arrowhead points to an additional small green fluorescent (background) signal. Scale bar 2 µm. b Segmented fluorescent voxels of FISH signals within DAPI mask after defined parameter settings. The small isolated green signal seen in (arrowhead in a) is discarded due to a distance >0.5 µm from the nearest red signal centroid, set as limit between two differently labeled targets. c Same section after classification of DAPI signals into seven intensity classes as proxy for chromatin compaction visualized as color heat map. d Inset magnification from framed area in b and c with outlined green and red segmented signals. e Relative distribution of green and red fluorescent voxels in this nucleus mapped on DAPI intensity-defined chromatin compaction classes (gray). f Assignment of the active and inactive nuclear compartment (ANC/INC) linked to chromatin compaction classes, for explanation see text