Superresolution microscopy methods such as STED and PALM/STORM have revolutionized far-field optical fluorescence microscopy by manipulating state transitions of the emitters, offering potentially unlimited resolution. In practice, however, the resolution of an image is ultimately limited by the finite photon budget of fluorescent probes. Single-emitter tracking applications also suffer from spatial-temporal resolution limitations imposed by the finite photon emission rate.
In this colloquium I will present MINFLUX (Maximally INFormative Luminescence eXcitation), a technique that tackles the localization problem by rendering each fluorescent photon more informative. For a given photon budget, an improved localization precision is obtained by repeatedly probing an emitter location with a zero of intensity. Conversely, it is possible to attain a given localization precision by using fewer photon than conventional centroid-localization techniques.
I will present results of superresolution imaging and single protein tracking. Images of DNA origami labeled with Atto647N achieve ~1nm precision, resolving molecules only 6 nm apart. Tracking of 30S ribosomal subunits protein in living E. coli fused with the photoconvertible protein mEOS2 demonstrate a 22-fold reduction of the required photon detections and increase the temporal resolution and the number of localizations per track by 100-fold, which naturally improves the estimation of the diffusion coefficient.