Camera-based localization microscopy optimized with calibrated structured illumination

Until very recently, super-resolution localization and tracking of fluorescent particles used camera-based wide-field imaging with uniform illumination. Then it was demonstrated that structured illuminations encode additional localization information in images. The first demonstration of this uses scanning and hence suffers from limited throughput. This limitation was mitigated by fusing camera-based localization with wide-field structured illumination. Current implementations, however, use effectively only half the localization information that they encode in images. Here we demonstrate how all of this information may be exploited by careful calibration of the structured illumination. Our approach achieves maximal resolution for given structured illumination, has a simple data analysis, and applies to any structured illumination in principle. We demonstrate this with an only slightly modified wide-field microscope. Our protocol should boost the emerging field of high-precision localization with structured illumination. The structure and processes of life’s molecules at the nanoscale are probed with optical super resolution techniques. The authors present a method that combines conventional localization information and information from structured illumination, which outperforms other methods by localizing single molecules with a theoretically optimal doubling of precision.