All-Optical Strategies to Minimize Photo-Bleaching in Reversibly Switchable Fluorescent Proteins

Marin-Aguilera, G.; Pennacchietti, F.; Volpato, A.; Papalini, A.; Kulkarni, A.; Bagheri, N.; Minet, G.; Widengren, J.; Testa, I.

doi:10.1101/2025.01.20.633912

bioRxiv · 10.1101/2025.01.20.633912

All-Optical Strategies to Minimize Photo-Bleaching in Reversibly Switchable Fluorescent Proteins

Marin-Aguilera, G.·Pennacchietti, F.·Volpato, A.·Papalini, A.·Kulkarni, A.·Bagheri, N.·Minet, G.·Widengren, J.·Testa, I.

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Abstract

Photo-bleaching is a general hurdle of fluorescence-based techniques that becomes even more severe in high- resolution microscopy that relies on prolonged, focused and complex illumination sequences. Strategies to reduce photo-bleaching require chemical modifications of the cell media, which often stave off physiological cellular conditions. Here, we outline an all-optical strategy to minimize photo-bleaching in reversibly switching fluorescent proteins (RSFPs), a class of probes used in several super-resolution and protein-multiplexing imaging techniques. By identifying the photobleaching pathways, we developed novel imaging schemes to increase the number of ON- OFF photo-switching cycles based on a designed modulation of the on-switching light or a co-irradiation with red- shifted light. By rationalizing the photo-cycle, we expand multiplexing strategies with RSFPs to high- spatiotemporal resolutions while maintaining the accuracy and recording longer time-lapse imaging of sub-cellular structures with both confocal microscopy and parallelized RESOLFT nanoscopy.

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Marin-Aguilera, G., Pennacchietti, F., Volpato, A., Papalini, A., Kulkarni, A., Bagheri, N., Minet, G., Widengren, J., Testa, I.. 2025-01-22. All-Optical Strategies to Minimize Photo-Bleaching in Reversibly Switchable Fluorescent Proteins. https://doi.org/10.1101/2025.01.20.633912

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