- MINFLUX (Minimal Photon Fluxes) is an advanced single-molecule localization microscopy technique that achieves nanometer-scale resolution with unprecedented efficiency in photon usage.
- Developed as a further step beyond established super-resolution methods such as STED (Stimulated Emission Depletion) and PALM/STORM (Photoactivated Localization Microscopy/Stochastic Optical Reconstruction Microscopy), MINFLUX uses a targeted excitation strategy that allows the precise localization of fluorescent molecules with far fewer detected photons. This leap in efficiency means MINFLUX can capture dynamic molecular processes at sub-millisecond timescales and at resolutions of 1–3 nanometers, which approaches the scale of individual biomolecules.
- The core principle of MINFLUX relies on positioning a spatially modulated excitation pattern—specifically, a donut-shaped laser beam with a central intensity minimum—directly over the molecule of interest. Because fluorescence emission is weakest when the molecule is exactly at the beam’s dark center, detecting minimal photon counts during beam shifts allows the algorithm to triangulate the molecule’s position with extreme precision. Instead of relying on broad Gaussian point spread functions and collecting thousands of photons for statistical accuracy, MINFLUX iteratively positions the excitation minimum close to the molecule, reducing the number of required photons by an order of magnitude compared to traditional localization microscopy. This results in faster acquisition times and less photobleaching, making it ideal for studying fast and delicate biological processes.
- In practice, MINFLUX combines this targeted illumination approach with photo-switchable or photo-activatable fluorescent labels, enabling controlled activation of individual molecules in crowded fields. Once a molecule is activated, the excitation pattern is rapidly repositioned in a few predefined locations around it, and photon counts are recorded. Using mathematical fitting, the molecule’s coordinates are calculated with nanometer precision. Because the beam’s central zero acts as a reference point, the accuracy of localization is largely independent of the diffraction limit, which typically constrains optical microscopy to ~200 nm resolution.
- MINFLUX has transformative applications in cell biology, neuroscience, and structural biology. It enables direct visualization of the nanoscale organization of proteins in live cells, tracking of molecular motors along cytoskeletal filaments, and even real-time monitoring of conformational changes in macromolecular complexes.
- Its ability to combine ultra-high spatial resolution with high temporal resolution makes it uniquely suited for studying the molecular mechanisms that underlie life at the most fundamental level. However, the method requires highly stable instrumentation, advanced beam-shaping optics, and precise sample preparation, meaning its adoption is still largely concentrated in specialized laboratories.
