Photoactivatable Xanthone (PaX) Dyes Enable Quantitative, Dual Color, and Live-Cell MINFLUX Nanoscopy.

Area of Science:

• Super-resolution microscopy
• Biophysics
• Chemical biology

Background:

• MINFLUX nanoscopy requires specific fluorophore properties for high-resolution imaging.
• Photoswitchable dyes are crucial for MINFLUX, enabling temporal control of fluorescence.
• Existing fluorophores for 561 nm excitation in MINFLUX have limitations.

Purpose of the Study:

• To evaluate novel photoactivable xanthone (PaX) dyes for MINFLUX nanoscopy.
• To compare the performance of PaX dyes against existing fluorophores like mMaple.
• To introduce new PaX dyes for advanced MINFLUX applications, including dual-color and live-cell imaging.

Main Methods:

• Quantitative comparison of MINFLUX localization success rates for PaX dyes and mMaple.
• Analysis of effective labeling efficiency in endogenously tagged nuclear pore complexes.
• Development and application of PaX₅₉₅ for dual-color imaging and assessment of PaX photoactivation characteristics.

Main Results:

• PaX dyes demonstrate superior localization efficiency compared to mMaple in MINFLUX.
• PaX dyes perform comparably to established reversible molecular switches in single-molecule localization microscopy.
• PaX dyes enable robust dual-color 561 nm MINFLUX imaging and fast live-cell imaging.

Conclusions:

• PaX dyes are highly effective fluorophores for 561 nm MINFLUX nanoscopy.
• These dyes meet the demands for precise label positioning and quantitative analysis.
• PaX dyes significantly advance the capabilities of MINFLUX imaging, particularly for live-cell applications.