# FISH-Dist: An Automated Pipeline for 3D Genomic Spatial Distance Quantification in FISH Imaging

## TL;DR

FISH-Dist is a new tool that improves the accuracy of measuring distances between DNA signals in 3D microscope images.

## Contribution

FISH-Dist introduces a pipeline for accurate 3D genomic distance quantification in FISH imaging using deep learning and chromatic aberration correction.

## Key findings

- FISH-Dist achieves sub-pixel accuracy in detecting fluorescent signals.
- The pipeline reduces inter-channel distance measurement errors caused by chromatic aberration.
- Validation on DNA origami nanorulers confirms the pipeline's accuracy and reproducibility.

## Abstract

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.