Global fitting for high-accuracy multi-channel single-molecule localization

Multi-channel SMLM imaging is powerful. Here the authors report globLoc, a GPU-based global fitting algorithm, to extract maximum information from multichannel single molecule data; this gives improved localisation precision for biplane and 4Pi-SMLM and colour assignment in multi-colour astigmatic SMLM. Multi-channel detection in single-molecule localization microscopy greatly increases information content for various biological applications. Here, we present globLoc, a graphics processing unit based global fitting algorithm with flexible PSF modeling and parameter sharing, to extract maximum information from multi-channel single molecule data. As signals in multi-channel data are highly correlated, globLoc links parameters such as 3D coordinates or photon counts across channels, improving localization precision and robustness. We show, both in simulations and experiments, that global fitting can substantially improve the 3D localization precision for biplane and 4Pi single-molecule localization microscopy and color assignment for ratiometric multicolor imaging.