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. 2022 Oct 17;5:1100. doi: 10.1038/s42003-022-04040-1

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© The Author(s) 2022

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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Fig. 1 — a Scheme of the splitSMLM imaging system and the overall workflow. b Super-resolution images of a single-labeled sample, reconstructed (from left to right) from localizations within the λ_S channel, from localizations within the λ_L channel or after demixing in SplitViSu, demonstrating removal of spurious localizations and resolution improvement. c Image of the two spectrally different channels, λ_S and λ_L (left and right, respectively), with localizations originating either from AF647 (green), CF680 (orange) or from background noise (blue). d Bivariate histogram of photon counts I_S and I_L originating from the λ_S and λ_L channels with three sectors corresponding to three fluorophore species. e Univariate histogram of ratios r on a semi-log plot with three peaks corresponding to three fluorophore species. f Fluorescence spectra of AF647, CF660C and CF680 calculated within the λ_S and λ_L channels (left), without image splitter (top right), or equivalent spectra after demixing in SplitViSu (bottom right). Vertical lines represent average wavelengths for the corresponding fluorophores in the corresponding channels. Average wavelengths after demixing in SplitViSu are calculated as the mean values of the average wavelengths of the fluorophores in the λ_S and λ_L channels. Arrows indicate maximum spectral difference within the triplet of fluorophores and reflect the amount of chromatic aberrations in the corresponding setting. Scale bars, 100 nm (b) and 500 nm (c).