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. 2022 Jan 10;13:169. doi: 10.1038/s41467-021-27798-0

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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. 3 — a Transcripts of three different target genes are tagged using two probes with different spectra. Targets 1 and 3 are tagged each with one probe, Target 2 is tagged with both simultaneously. b, c The fluorescence is collected in the two expected spectral channels for the known emission of the two probes (representative small regions of a whole 3D field of view). d The maximum projection of the two channels is shown and pseudo-colored depending on the presence in the respective channels (as an inset within the whole field of view. e The actual counts of each target within the whole field of view. f As a parallel example, transcripts of three different target genes are tagged using two probes with different lifetime. Targets 1 and 3 are tagged each with one probe, Target 2 is tagged with both simultaneously. g The phasor plot presents three populations, corresponding to the pixels with the three combinations; the two components by themselves plus the linear combination falling in the middle. h) Machine learning clustering technique is used to identify the groups (Gaussian mixture model). i) The multicomponent method is used to extract the fraction of one of the components in each detected puncta. j The same inset is shown with the pseudocoloring now depending on the lifetime clustering. k The counts for each lifetime cluster in the whole field of view. l The combination of the information in both the spectral and the lifetime dimension yields a final 6-plex. m The overall counts for the 6-plex detection of transcripts including POLR2A (Alexa647 & ATTO565), MTOR (ATTO647 & ATTO565), KI67 (Alexa647 & ATTO647), BRCA1 (Alexa647), NCOA2 (ATTO647), NCOA3 (ATTO565) with the appropriate expressed genes that correspond to each combination. Experiments were conducted with cultures of mNeon green cells. Scale bar 10 µm in large image and 2 µm in insets. Source data are provided as a Source Data file.