BackgroundStochastic optical reconstruction microscopy (STORM), a super-resolution microscopy technique based on single-molecule localizations, has become popular to characterize sub-diffraction limit targets. However, due to lengthy image acquisition, STORM recordings are prone to sample drift. Existing cross-correlation or fiducial marker-based algorithms allow correcting the drift within each channel, but misalignment between channels remains due to interchannel drift accumulating during sequential channel acquisition. This is a major drawback in multi-color STORM, a technique of utmost importance for the characterization of various biological interactions.ResultsWe developed RegiSTORM, a software for reducing channel misalignment by accurately registering STORM channels utilizing fiducial markers in the sample. RegiSTORM identifies fiducials from the STORM localization data based on their non-blinking nature and uses them as landmarks for channel registration. We first demonstrated accurate registration on recordings of fiducials only, as evidenced by significantly reduced target registration error with all the tested channel combinations. Next, we validated the performance in a more practically relevant setup on cells multi-stained for tubulin. Finally, we showed that RegiSTORM successfully registers two-color STORM recordings of cargo-loaded lipid nanoparticles without fiducials, demonstrating the broader applicability of this software.ConclusionsThe developed RegiSTORM software was demonstrated to be able to accurately register multiple STORM channels and is freely available as open-source (MIT license) athttps://github.com/oystein676/RegiSTORM.gitand 10.5281/zenodo.5509861 (archived), and runs as a standalone executable (Windows) or via Python (Mac OS, Linux).Supplementary InformationThe online version contains supplementary material available at 10.1186/s12859-023-05320-1.