Imaging the Genome


Figure 4: Probe design and workflow of OligoSTORM. (A) Oligopaint probe design. Probes contain a region of genomic homology and two flanking sequences, referred to as “Mainstreet” and “Backstreet,” which carry barcodes used for OligoSTORM and OligoDNA-PAINT. The design of the probes allows for each genomic region to be imaged twice if necessary. (B) Workflow of sequential rounds of hybridization. The sample begins with all oligopaint probes containing unique barcodes bound to the sample. Oligonucleotides complementary to barcode 1 and imaging strands are introduced to the sample, and barcode 1 is imaged. Oligonucleotides complementary to barcode 2, imaging strands, and toehold oligos for removing barcode 1 oligos are applied to the sample, and barcode 2 is imaged. This process is repeated, changing the toehold oligos to remove the previous round’s barcode, until all barcodes have been imaged. (C) The overall workflow of an OligoSTORM experiment. It begins with the design and generation of a probe library. All probes are then hybridized to the sample in one round of hybridization. Mainstreet and Backstreet barcodes are then used to image each barcode sequentially using single-molecule localization microscopy. Data are drift corrected, and clusters are extracted using DBSCAN cluster analysis, and the final image is assembled. Figure adapted with permission from its original version [14].


22 www.microscopy-today.com • 2020 November


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