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. 2023 Dec 27;21(2):331–341. doi: 10.1038/s41592-023-02115-3

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 5 — a, Orthogonality matrix of the barcode domains for 15 designed thermal probes. b, Orthogonality matrix of the quencher regions for the 15 designed thermal probes. The designed sequence is orthogonal to each other and none of them has strong interactions predicted to affect the imaging. The sequence interactions were evaluated with NUPACK software to calculate the yield at equilibrium. c, The validation of 15 RNA targets with smFISH in Hela cells. Each RNA was bound individually with ISH probes and then the imager was applied to visualize the RNA in cells. Scale bars, 10 µm. At least three independent repeats were performed to validate the results.