{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T00:30:00Z","timestamp":1786408200838,"version":"build-2736575974"},"reference-count":32,"publisher":"Wiley","issue":"2","license":[{"start":{"date-parts":[[2026,7,19]],"date-time":"2026-07-19T00:00:00Z","timestamp":1784419200000},"content-version":"vor","delay-in-days":18,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T00:00:00Z","timestamp":1782864000000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["The Plant Journal"],"published-print":{"date-parts":[[2026,7]]},"abstract":"<jats:title>SUMMARY<\/jats:title>\n                  <jats:p>\n                    Genetically encoded biosensors are one of the essential tools in biological research. They enable visualization of molecules of interest from the subcellular level to entire organism level\n                    <jats:italic>in vivo<\/jats:italic>\n                    and can be used to monitor the presence of small molecules, gene expression, protein activity, and protein degradation. However, multiplexing fluorescent biosensors in plants is notoriously difficult due to signal bleed\u2010through and strong autofluorescence from chlorophyll. In this study, we investigated the potential of multiplexing biosensors based on the selection of reporter fluorescent proteins. We characterized the emission spectra, fluorescence lifetimes, and relative brightness of diverse fluorescent proteins in plant leaves. We show that selected proteins exhibit comparable brightness, supporting their use in co\u2010expression experiments and reliable quantification of individual signals. To separate three overlapping signals, we applied two different linear unmixing approaches and compared them to results obtained without unmixing. We identified the channel separation unmixing approach as the most suitable for biosensors. Additionally, we show how unmixing with the selected approach can be applied to separate autofluorescence and five fluorescent proteins. We further validated this approach in virus\u2010infected cells by following organelle dynamics\n                    <jats:italic>in vivo<\/jats:italic>\n                    . Finally, we demonstrate the feasibility of high\u2010throughput segmentation and quantification with a custom MATLAB workflow for nuclei, chloroplasts, and cytoplasm signal analysis. Overall, our work demonstrates that biosensors can be multiplexed, even when their emission spectra overlap.\n                  <\/jats:p>","DOI":"10.1111\/tpj.71034","type":"journal-article","created":{"date-parts":[[2026,7,19]],"date-time":"2026-07-19T20:21:46Z","timestamp":1784492506000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Robust quantification of multiplexed fluorescent protein\u2010based biosensors in plant tissues"],"prefix":"10.1111","volume":"127","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5975-4032","authenticated-orcid":false,"given":"Valentina","family":"Levak","sequence":"first","affiliation":[{"name":"Department of Biotechnology and Systems Biology National Institute of Biology  Ljubljana Slovenia"},{"name":"Jo\u017eef Stefan International Postgraduate School  Ljubljana Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3303-9086","authenticated-orcid":false,"given":"An\u017ee","family":"\u017dupani\u010d","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Systems Biology National Institute of Biology  Ljubljana Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5190-7217","authenticated-orcid":false,"given":"Karmen","family":"Poga\u010dar","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Systems Biology National Institute of Biology  Ljubljana Slovenia"},{"name":"Jo\u017eef Stefan International Postgraduate School  Ljubljana Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nastja","family":"Marondini","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Systems Biology National Institute of Biology  Ljubljana 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