splitSMLM, a spectral demixing method for high-precision multi-color localization microscopy applied to nuclear pore complexes.

Single molecule localization microscopy (SMLM) with a dichroic image splitter can provide invaluable multi-color information regarding colocalization of individual molecules, but it often suffers from technical limitations. Classical demixing algorithms tend to give suboptimal results in terms of localization precision and correction of chromatic errors. Here we present an image splitter based multi-color SMLM method (splitSMLM) that offers much improved localization precision and drift correction, compensation of chromatic distortions, and optimized performance of fluorophores in a specific buffer to equalize their reactivation rates for simultaneous imaging. A novel spectral demixing algorithm, SplitViSu, fully preserves localization precision with essentially no data loss and corrects chromatic errors at the nanometer scale. Multi-color performance is further improved by using optimized fluorophore and filter combinations. Applied to three-color imaging of the nuclear pore complex (NPC), this method provides a refined positioning of the individual NPC proteins and reveals that Pom121 clusters act as NPC deposition loci, hence illustrating strength and general applicability of the method.