Image-based 3D genomics through chromatin tracing

Correct organization of higher-order genome folding is essential for the regulation of gene expression, DNA replication and other genomic functions. Technological advances in high-throughput sequencing-based methods have allowed for systematic profiling of the fundamental architectural features of chromatin organization at the genome level. However, how chromatin is folded in 3D space at single-cell and single-chromosome-copy resolution in intact cells and tissues has been a long-standing question owing to a lack of appropriate methodology. Recent advances in chromatin labelling, imaging and automated fluidics technologies have led to the development of chromatin tracing, enabling direct mapping of the 3D chromatin folding trajectory in situ at the single-cell and single-molecule level. Within nearly a decade of its development, chromatin tracing has been applied at different genomic scales and to a spectrum of cell types and model organisms, improving our understanding of the structures, mechanisms and functions of chromatin organization in various biological and medical areas. In this Primer, we introduce the experimental principles, data analysis procedures and current applications of chromatin tracing. We describe how chromatin tracing can be combined with multimodal imaging and genetic screening technologies and provide a perspective on the limitations of current chromatin tracing approaches and the direction of technological developments for filling major gaps in discoveries.