Nanometer resolved single-molecule colocalization of nuclear factors by two-color super resolution microscopy imaging.

In order to study the detailed assembly and regulation mechanisms of complex structures and machineries in the cell, simultaneous in situ observation of all the individual interacting components should be achieved. Multi-color Single-Molecule Localization Microscopy (SMLM) is ideally suited for these quantifications. Here, we build on previous developments and thoroughly discuss a protocol for two-color SMLM combining PALM and STORM, including sample preparation details, image acquisition and data postprocessing analysis. We implement and evaluate a recently proposed colocalization analysis method (aCBC) that allows single-molecule colocalization quantification with the potential of revealing fine, nanometer-scaled, structural details of multicomponent complexes. Finally, using a doubly-labeled nuclear factor (Beaf-32) in Drosophila S2 cells we experimentally validate the colocalization quantification algorithm, highlight its advantages and discuss how using high molecular weight fluorescently labeled tags compromises colocalization precision in two-color SMLM experiments.