Accurate and controlled DNA replication during S phase is pivotal for cellular genomic integrity and proliferation.
Initiation and progression of S phase is tightly regulated and spatiotemporally organized in a non-random manner1,2. Gene-rich regions (euchromatin) replicate first (early-S phase), whereas facultative and constitutive heterochromatin replicate in mid and late-S phase, respectively.
Although S phase progression and regulation have been extensively studied and knowledge on the mechanisms is established at the macromolecular level, still information on the organization and structure of replicons (replicated chromatin) is lacking.
3D-Structured-Illumination-Microscopy (3D-SIM) enabled the visualization of chromatin structural changes/conformation previously not assessable due to the diffraction limit of conventional fluorescence microscopy3.
Combined with optimized bioinformatics solutions for analyzing multidimensional data sets we established an evaluation pipeline for quantifying spatial properties of replication foci (RF).
We were able to analyze volume differences of newly synthesized DNA compared to compacted already replicated chromatin. In addition, with the use of a precisely tailored voxel segmentation of DNA labeling intensities reflecting compaction4,5, we mapped RF onto chromatin classes at different S phase stages (early, mid, late).
This work showed for the first time super-resolved active molecular processes occurring at spatially-defined, less compacted chromatin regions, regardless of the original compaction state of these regions (euchromatin, heterochromatin).
This approach could potentially be applied for the study of a wide-range of active processes, such as transcription or DNA repair.