MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope.

Area of Science:

• Super-resolution microscopy
• Biophysics
• Cell biology

Background:

• The minimal photon fluxes (MINFLUX) concept advanced fluorescence microscopy resolution to molecular scales.
• Early MINFLUX implementations required specialized, custom-built microscopes, limiting broad applicability.

Purpose of the Study:

• To demonstrate that MINFLUX can achieve molecular-scale resolution using a standard microscope stand.
• To assess the general availability and applicability of MINFLUX for biological imaging.

Main Methods:

• Implementation of MINFLUX on a standard microscope stand with synchronized electro-optical and galvanometric beam steering.
• Development of a sub-nanometer precision sample stabilization system relative to the microscope stand.
• Real-time, nanometer-precise localization of single fluorophores.

Main Results:

• Achieved 1-3 nm 3D resolution with MINFLUX on a standard microscope.
• Demonstrated localization precision of 2.2 nm with ~800 photons and <1 nm with ~2500 photons.
• Attained 3D imaging precision of ~2.4 nm in-plane and ~1.9 nm axially.
• Established spatio-temporal resolution for tracking (<20 nm within ~100 µs).

Conclusions:

• MINFLUX microscopy is now widely applicable for achieving molecule-scale resolution using standard equipment.
• The developed methods enable precise imaging and tracking of biological structures and dynamics at the nanoscale.
• Successful application to imaging cell/neurobiological samples and tracking lipid diffusion in model membranes validates the technique.