[Combinatorial barcoding for high-throughput spatial genomics. This figure demonstrates the concept by showing oligonucleotide probes hybridized with target DNA. The oligo probes have single-strand overhangs. Those overhangs serve as the target for a second oligonucleotide probe, this one conjugated with a fluorescent marker. Successive rounds of targeting fluorescent tagged markers to the single-strand overhanges then allows the same position to be targeted multiple times with different colors over time. The combination of colors forms a unique barcode and can be used to identify which genetic loci is being observed in 3D space. ]FIGURE 1: Combinatorial barcoding. The illustration above demonstrates how combinatorial barcoding works. Individual loci can be distinguished through successive rounds of imaging, with each round presenting an opportunity to change fluorescent tags through various methods. Most often, probes targeting the template strand are designed with overhanging stretches of single-stranded DNA. These overhangs can then be targeted by fluorescently labeled probes, imaged, and then dissociated to make room for successive rounds, each with a new set of fluorescently labeled probes. Note that the absence of color (indicated by white circles) can be used as part of the barcode. High-quality, large-scale oligo synthesis makes it possible to apply this technique genome wide for high-throughput spatial genomics studies. Created with BioRender.com [https://app.biorender.com/].