Single‐molecule localization microscopy goes quantitative

In the last few years, single-molecule localization (SMLM) techniques have been used to address biological questions in different research fields. More recently, super-resolution has also been proposed as a quantitative tool for quantifying protein copy numbers at the nanoscale level. In this scenario, quantitative approaches, mainly based on stepwise photobleaching and quantitative SMLM assisted by calibration standards, offer an exquisite tool for investigating protein complexes. This primer focuses on the basic concepts behind quantitative super-resolution microscopy, also providing strategies to overcome the technical hurdles that could limit their application.