A simple method to estimate the average localization precision of a single-molecule localization microscopy experiment

The localization precision is a crucial and important parameter for single-molecule localization microscopy (SMLM) and directly influences the achievable spatial resolution. It primarily depends on experimental imaging conditions and the registration potency of the algorithm used. We propose a new and simple routine to estimate the average experimental localization precision in SMLM, based on the nearest neighbor analysis. By exploring different experimental and simulated targets, we show that this approach can be generally used for any 2D or 3D SMLM data and that reliable values for the localization precision σ _SMLM are obtained. Knowing σ _SMLM is a prerequisite for consistent visualization or any quantitative structural analysis, e.g., cluster analysis or colocalization studies.