Accelerated MINFLUX Nanoscopy, through Spontaneously Fast-Blinking Fluorophores

Area of Science:

• Super-resolution microscopy
• Biophysics
• Chemical biology

Background:

• MINFLUX nanoscopy offers high precision (1 nm) but is limited by slow localization times (milliseconds).
• Existing fluorescent probes are optimized for high photon yield and long on-times, unsuitable for rapid MINFLUX imaging.
• Current MINFLUX applications prioritize precision over speed, limiting dynamic biological studies.

Purpose of the Study:

• To develop and implement accelerated MINFLUX nanoscopy with significantly reduced localization times.
• To design novel, spontaneously blinking fluorescent markers with fast on-times (1-3 ms) compatible with MINFLUX.
• To optimize imaging protocols for enhanced speed in live-cell super-resolution microscopy.

Main Methods:

• Design and synthesis of novel silicon rhodamine-based fluorophores with fused (benzo)thiophene spirolactam fragments.
• Characterization of single-molecule blinking behavior and photophysical properties of new fluorophores.
• Implementation of customized localization algorithms for fast-blinking fluorophores on a commercial MINFLUX microscope.

Main Results:

• Achieved up to a 30-fold increase in MINFLUX nanoscopy localization speed.
• Developed fluorescent markers exhibiting fast on-times (1-3 ms) and live-cell permeability.
• Demonstrated successful accelerated MINFLUX imaging with optimized routines and new probes.

Conclusions:

• Novel fluorescent markers and optimized routines enable significantly faster MINFLUX nanoscopy.
• Accelerated MINFLUX opens new possibilities for studying fast dynamic processes in live cells.
• This advancement broadens the applicability of super-resolution microscopy for biological discovery.