Simple and robust 3D MINFLUX excitation with a variable phase plate.

Area of Science:

• Optical microscopy
• Nanotechnology
• Biophysics

Background:

• MINFLUX (MInimal reMoval oF foLd structure) microscopy offers exceptional resolution for superresolution imaging and single fluorophore tracking.
• Existing MINFLUX implementations are complex, costly, and limited in speed and robustness.

Purpose of the Study:

• To develop a high-performance, cost-effective, and robust excitation module for MINFLUX microscopy.
• To enhance the speed and versatility of MINFLUX localization through rapid phase scanning.

Main Methods:

• Utilized an electro-optical modulator combined with a spatial light modulator to create a variable phase plate.
• Generated MHz timescale phase scanning for rapid probing of single fluorophores.
• Developed bisected and top-hat phase patterns for compact excitation point-spread functions in 3D.

Main Results:

• Achieved microsecond timescale scanning of excitation patterns across fluorophores.
• Demonstrated switching times within 60 microseconds and alternation between excitation wavelengths.
• Presented a robust 3D and multi-color MINFLUX excitation module design.

Conclusions:

• The developed module significantly improves MINFLUX speed, robustness, and cost-effectiveness.
• This innovation paves the way for wider adoption of high-performance open-source MINFLUX systems.
• The module is envisioned as a key component for future advanced microscopy platforms.