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All-optical strategies to minimize photobleaching in reversibly switchable fluorescent proteins
KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0003-1769-972x
KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0003-3368-0017
KTH Royal Inst Technol, SciLifeLab, Solna, Sweden.
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2025 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 16, no 1, article id 10843Article in journal (Refereed) Published
Abstract [en]

Photobleaching is a general hurdle of fluorescence-based techniques especially in high-resolution microscopy that relies on prolonged and complex illumination. Strategies to reduce photobleaching require chemical modifications of the cell medium, which often compromise physiological cellular conditions. Here, we outline an all-optical strategy to minimize photobleaching in reversibly switching fluorescent proteins (RSFPs), a class of probes used in super-resolution and protein-multiplexing imaging techniques. By identifying the photobleaching pathways, we develop imaging schemes to increase the number of on-off photoswitching cycles, either modulating the on-switching light or co-irradiating the RSFPs with light at longer wavelengths with respect to fluorescence excitation. We apply the optimized imaging scheme to achieve imaging multiplexing at high-spatiotemporal resolutions and to record longer time-lapse imaging of sub-cellular structures with both confocal microscopy and parallelized RESOLFT nanoscopy.

Place, publisher, year, edition, pages
Springer Nature , 2025. Vol. 16, no 1, article id 10843
National Category
Biophysics
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URN: urn:nbn:se:kth:diva-376328DOI: 10.1038/s41467-025-67009-8ISI: 001629548500001PubMedID: 41326380Scopus ID: 2-s2.0-105023572508OAI: oai:DiVA.org:kth-376328DiVA, id: diva2:2038106
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QC 20260212

Available from: 2026-02-12 Created: 2026-02-12 Last updated: 2026-02-12Bibliographically approved

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Marin-Aguilera, GuillemPennacchietti, FrancescaVolpato, AndreaKulkarni, AbhilashBagheri, NiushaMinet, GuillaumeWidengren, JerkerTesta, Ilaria

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Marin-Aguilera, GuillemPennacchietti, FrancescaVolpato, AndreaKulkarni, AbhilashBagheri, NiushaMinet, GuillaumeWidengren, JerkerTesta, Ilaria
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Science for Life Laboratory, SciLifeLabBiophysicsBio-Opto-Nano Physics
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