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. 2018 Mar 15;8:4590. doi: 10.1038/s41598-018-22892-8

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© The Author(s) 2018

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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Immunofluorescence imaging of five subcellular structures. Immunofluorescence imaging using the five selected BAA fluorophores was completed on separate samples to demonstrate the structural and spectral separation of the flurophores and cellular targets. (A) The nuclear membrane was labeled using indirect immunofluorescence with BAA-37a. (B) The outer mitochondrial membrane was labeled using indirect immunofluorescence with BAA-22a. (C) Tubulin was labeled using indirect immunofluorescence with BAA-5a. (D) The extracellular membrane was labeled using BAA-2a conjugated wheat germ agglutinin. (E) Vimentin was labeled using indirect immunofluorescence with BAA-39a. (F) The in situ normalized spectral emission of each BAA fluorophore was collected using the confocal laser scanning microscopy and used for spectral unmixing in (G) simultaneously stained samples created using the same five selected BAA fluorophores conjugated to the validated subcellular tagging reagents. The unmixed, individual spectral channels show the (H) nuclear membrane, (I) mitochondrial membrane, (J) tubulin, (K) extracellular membrane, and (L) vimentin. Scale bar = 10 µm.