A common framework for single-molecule localization using sequential structured illumination.

Localization of single fluorescent molecules is key for physicochemical and biophysical measurements, such as single-molecule tracking and super-resolution imaging by single-molecule localization microscopy. Over the last two decades, several methods have been developed in which the position of a single emitter is interrogated with a sequence of spatially modulated patterns of light. Among them, the recent MINFLUX technique outstands for achieving a ∼10-fold improvement compared with wide-field camera-based single-molecule localization, reaching ∼1-2 nm localization precision at moderate photon counts. Here, we present a common framework for this type of measurement. Using the Cramér-Rao bound as a limit for the achievable localization precision, we benchmark reported methods, including recent developments, such as MINFLUX and MINSTED, and long-established methods, such as orbital tracking. In addition, we characterize two new proposed schemes, orbital tracking and raster scanning, with a minimum of intensity. Overall, we found that approaches using an intensity minimum have a similar performance in the central region of the excitation pattern, independent of the geometry of the excitation pattern, and that they outperform methods featuring an intensity maximum.