All other steps were performed as described in and ~60 nm in over the course of the experiment, with displacements measured less than 1 h apart being essentially indistinguishable from those measured at the end of a 96-h experiment (Extended Data Fig. cell types during development requires precise interactions between genes and Neurod1 distal Pyraclonil regulatory sequences. Our understanding of how these interactions look in three dimensions, vary across cell types in complex tissue, and relate to transcription remains limited. Here we describe optical reconstruction of chromatin architecture (ORCA), a method to trace the DNA path in single cells with nanoscale accuracy and genomic resolution reaching 2 kilobases. We applied ORCA to a Hox gene cluster in cryosectioned embryos Pyraclonil and labelled ~30 RNA species in parallel. We identified cell-type-specific physical borders between active and Polycomb-repressed DNA, and unexpected Polycomb-independent borders. Deletion of Polycomb-independent borders led to ectopic enhancer-promoter contacts, aberrant gene expression, and developmental defects. Together, these results illustrate an approach for high-resolution, single-cell DNA domain analysis and revealed partitioning of the genome into topological associated domains (TADs), where intra-domain contacts are enriched over inter-domain contacts. TADs frequently span developmental genes and their and resolve large numbers of individual. Accordingly, multicolour fluorescent hybridization (FISH)6-12, oligo-stochastic optical reconstruction microscopy (oligo-STORM)9,13-17, and sequential FISH17-19 have revealed cell-type-specific chromatin packaging, compartmentalization, and long-range mapping of TADs and resolution of enhancer-promoter interactions achieved by recent Hi-C with the single-cell resolution and tissue organization provided by Seafood. Like Hi-C, the strategy can detect parts of the genome which preferentially connect to promoters Like microscopy strategies, the approach can detect sub-populations of cells with common properties without needing cell sorting or dissection. To tell apart cell types within a cells and relate enhancer-promoter contacts to gene expression, the method should be compatible with simultaneous measurement of mRNAs and nascent transcription in each cell. Finally, to provide a sufficiently sampled view of the tissue, the method should process thousands of individual cells per run. Here, we describe optical reconstruction of chromatin architecture (ORCA), an approach that simultaneously achieves these goals, and apply it to test several predictions about chromatin structure and embryos. Principle of the method ORCA builds on recent innovations in RNA and DNA FISH, taking advantage of array-derived oligonucleotide (oligo) probes (Oligopaints)9,13,18,20,21. ORCA reconstructs the trajectory of a genomic region of interest (100C700 kb), by tiling the region in short sections (2C10 kb) with primary probes that have unique barcodes20 (Fig. 1a, Extended Data Fig. 1a-?-c,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 recent18 and concurrent work17,19, though with improved genomic resolution (Fig. 1b). With high-precision fiducial registration (Extended Data Fig. 1a-?-c),c), sequential imaging Pyraclonil 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). Open in a separate window Figure 1 O Optical reconstruction of chromatin architecture (ORCA).a, A region of interest is labelled with primary probes, partitioning the region into 70 segments with unique barcodes. Each barcode is imaged by sequentially introducing a complementary readout oligo carrying a fluorophore, which is removed after imaging, and the process repeats. b, Example data from imaged barcodes. Centers from 3D-Gaussian installing of the real stage pass on function are represented while + on places. c, Diffraction-limited picture of the complete site, overlaid with colored places indicating the positions of every barcode. Zoomed-in look at displays the same places connected to be able of genomic placement (ORCA picture). Similar pictures were collected for many 19,103 cells analysed in (e). d, Maps from two specific cells from wild-type embryos 10-12 hpf, displaying pairwise ranges between all barcodes that tracked a 700-kb area (chr3R:12.20-12.90 Mb (dm3)) at 10-kb quality (range maps). e, Rate of recurrence across all cells in the.