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Published on: March 26, 2019
All-optical strategies to minimize photobleaching in reversibly switchable fluorescent proteins
Guillem Marín-Aguilera1, Francesca Pennacchietti2, Andrea Volpato1
1SciLifeLab, KTH Royal Institute of Technology, Solna, Sweden.
Abstract:
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.
Insights
This study presents an all-optical method to reduce photobleaching in reversibly switching fluorescent proteins (RSFPs). This technique enhances super-resolution microscopy by increasing photoswitching cycles without altering cellular conditions.
Area of Science:
- Biophysics
- Microscopy
- Cell Biology
Background:
- Photobleaching limits fluorescence microscopy, especially super-resolution techniques requiring extensive illumination.
- Current methods to mitigate photobleaching involve chemical changes to cell media, potentially disrupting cell physiology.
Purpose of the Study:
- To develop an all-optical strategy to minimize photobleaching in reversibly switching fluorescent proteins (RSFPs).
- To enable advanced imaging techniques like super-resolution and protein multiplexing without compromising cellular health.
Main Methods:
- Investigated photobleaching pathways in RSFPs.
- Developed imaging schemes involving modulation of on-switching light or co-irradiation with longer wavelengths.
- Applied optimized schemes to confocal microscopy and parallelized RESOLFT nanoscopy.
Main Results:
- Successfully minimized photobleaching in RSFPs through optical manipulation.
- Achieved high-spatiotemporal resolution imaging multiplexing.
- Enabled extended time-lapse imaging of sub-cellular structures.
Conclusions:
- An all-optical strategy effectively reduces photobleaching in RSFPs, overcoming a major limitation in fluorescence microscopy.
- This method supports advanced imaging applications, including super-resolution and long-term live-cell imaging, while maintaining physiological conditions.
