Nanoscale Imaging of Chromatin Structure

ORCA builds on recent innovations in RNA and DNA FISH, taking advantage of array-derived oligonucleotide (oligo) probes (Oligopaints)^{9 ,13 (link),18 (link),20 ,21} . ORCA reconstructs the trajectory of a genomic region of interest (100–700 kb), by tiling the region in short sections (2–10 kb) with primary probes that have unique barcodes²⁰ (Fig. 1a, Extended Data Fig. 1a-c, Supplementary Data Tables 1-5). These barcodes are labelled with a fluorophore and imaged. The signal is then removed via strand displacement (Supplementary Data Table 6). The process repeats for each barcode. This is conceptually similar to recent^{18 (link)} and concurrent work^{17 (link),19 (link)}, though with improved genomic resolution (Fig. 1b). With high-precision fiducial registration (Extended Data Fig. 1a-c), sequential imaging allows barcoded sections within a diffraction-limited volume to be resolved, as in STORM, while adding sequence resolution across the domain (Fig. 1b). We represent the measured 3D positions of the barcodes as spheres, pseudo-coloured per barcode and linked with a smooth polymer (Fig. 1c), and as distance maps (Fig. 1d).
We applied ORCA to visualize the nanoscale DNA path of the BX-C at 10-kb and 2-kb resolution in 6-µm cryosections of Drosophila embryos 10–12 hours post-fertilization (hpf). The 10-kb step size allowed a 700-kb region, including flanking domains of the BX-C, to be traced with 70 barcodes (Fig 1d, e). The 2-kb step size enhanced the resolution over a 130-kb regulatory region spanning abd-A to Abd-B (Fig.1a, g, h). Missed detection events (Fig. 1d, g, grey lines) were largely stochastic – showing no significant variation between embryos, cell types, or probesets, and limited variation between barcodes (Extended Data Fig. 2). Replicate experiments yielded reproducible measurements (Pearson’s r > 0.95) (Extended Data Fig. 1d). Comparisons to published Hi-C^{22 (link)} across a range of contact thresholds revealed qualitatively similar features and quantitatively similar contact distributions (Fig. 1e, f, h, i, Extended Data Fig.1 e, f). In Drosophila chromosomes, which are predominantly paired in interphase, our ORCA images reveal that paired homologs follow a common trajectory to within ~50 nm. (Extended Data Fig. 3). Additional ORCA experiments in mouse embryonic stem cells tracing the region containing Sox2 at 5-kb resolution (Extended Data Fig. 4) exhibited strong correspondence with published Hi-C data^{23 (link)} (Pearson’s r = 0.96), illustrating versatility across cell types.