Interphase-fluorescence in situ hybridization (iFISH) is a robust and powerful method to detect chromosomal aberrations and rearrangements at single-cell resolution. Automated image analysis methods, based on iFISH spot detection at the single cell level, could help to better describe tumor heterogeneity. However, methods to segment spot- and nuclear images and to analyze spatially resolved single-cell features in tissue sections are still missing.