Accurate quantification of spatial distances between fluorescent signals in multi-channel 3D microscopy is essential for understanding genomic organization and gene regulation. However, chromatic aberration introduces systematic spatial offsets between channels that significantly bias distance measurements, particularly at short genomic distances. We present FISH-Dist, an automated computational pipeline for precise distance measurements in 3D fluorescence in situ hybridization (FISH) experiments acquired on standard confocal microscopes. Our method combines deep learning-based spot segmentation, 3D Gaussian fitting for sub-pixel localization, and two complementary chromatic aberration correction approaches, affine (ACC) and linear (LCC). We validated measurement accuracy using DNA origami nanorulers and systematically evaluated FISH probe design parameters, including probe spacing, density, and target sequence length. The pipeline achieves sub-pixel accuracy in signal detection and substantially reduces inter-channel distance measurement errors. This enables robust quantification of spatial relationships in 3D FISH datasets. Unlike existing tools optimized for long-range chromosomal interactions or requiring super-resolution microscopy, FISH-Dist specifically addresses the technical challenges of standard confocal imaging at short genomic distances, where chromatic aberration has a proportionally greater impact on measurement accuracy.

Accurate quantification of spatial distances between fluorescent signals in multi-channel 3D microscopy is essential for understanding genomic organization and gene regulation. However, chromatic aberration introduces systematic spatial offsets between channels that significantly bias distance measurements, particularly at short genomic distances. We present FISH-Dist, an automated computational pipeline for quantitative distance measurements in 3D fluorescence in situ hybridization (FISH) experiments acquired on standard confocal microscopes. Our method combines deep learning-based spot segmentation, 3D Gaussian fitting for sub-pixel localization, and two complementary chromatic aberration correction approaches: affine (ACC) and linear (LCC). We validated the pipeline by measuring the lengths of DNA origami nanorulers and systematically evaluated FISH probe design parameters, including probe spacing, density, and target sequence length. FISH-Dist achieves sub-pixel accuracy in signal detection and substantially reduces inter-channel distance measurement errors. This enables a reproducible quantification of spatial relationships in 3D FISH datasets. Unlike existing tools optimized for long-range chromosomal interactions or requiring super-resolution microscopy, FISH-Dist specifically addresses the technical challenges of standard confocal imaging at short genomic distances, where chromatic aberration has a proportionally greater impact on measurement accuracy.