A multi‐emitter fitting algorithm for potential live cell super‐resolution imaging over a wide range of molecular densities

Multi‐emitter fitting algorithms have been developed to improve the temporal resolution of single‐molecule switching nanoscopy, but the molecular density range they can analyse is narrow and the computation required is intensive, significantly limiting their practical application. Here, we propose a computationally fast method, wedged template matching (WTM), an algorithm that uses a template matching technique to localise molecules at any overlapping molecular density from sparse to ultrahigh density with subdiffraction resolution. WTM achieves the localization of overlapping molecules at densities up to 600 molecules μm^–2 with a high detection sensitivity and fast computational speed. WTM also shows localization precision comparable with that of DAOSTORM (an algorithm for high‐density super‐resolution microscopy), at densities up to 20 molecules μm^–2, and better than DAOSTORM at higher molecular densities. The application of WTM to a high‐density biological sample image demonstrated that it resolved protein dynamics from live cell images with subdiffraction resolution and a temporal resolution of several hundred milliseconds or less through a significant reduction in the number of camera images required for a high‐density reconstruction. WTM algorithm is a computationally fast, multi‐emitter fitting algorithm that can analyse over a wide range of molecular densities. The algorithm is available through the website. https://doi.org/10.17632/bf3z6xpn5j.1