Fig. 1: NP-Cloud extracts nanoscale spatial shifts between two sets of single-molecule localizations.

a Left: Zoom-in view of a small region of simulated single-molecule localizations in two channels (red and blue) with initially identical single-molecule positions but added Gaussian scattering of standard deviations σ = 10 nm to each localization. Right: Corresponding nearest-paired (NP) analysis result: The 1600 localizations in the red channel across the entire field were each searched for their nearest paired localization in the blue channel with a search radius of 50 nm, and the calculated vectorial distances (displacements) were plotted as Δx and Δy in 2D. b Left: Simulated data as (a), but with all the single-molecule positions in the red channel uniformly shifted by (+20 nm, +10 nm). Right: Corresponding NP-analysis result. c Left: Simulated data as (a), but with 9-fold additional random single-molecule localizations added to each channel. Right: Corresponding NP-analysis result. d Left: Simulated data as (c), but with all the single-molecule positions in the red channel uniformly shifted by (+20 nm, +10 nm). Right: Corresponding NP-analysis result. e NP-analysis result of Iteration 2, after shifting the localizations in the red channel by the averaged displacements of the NP-analysis results in d. f NP-analysis result after 5 more iterations of shifting and NP analysis. g Stepwise (open symbols) and total (filled symbols) shifts in xy through the above iteration process.