Analysis methods for interrogating spatial organisation of single molecule localisation microscopy data

Single-molecule localisation microscopy (SMLM) gives access to biological information below the diffraction limit, allowing nanoscale cellular structures to be probed. The data output is unlike that of conventional microscopy images, instead consisting of an array of molecular coordinates. These represent a spatial point pattern that attempts to approximate, as closely as possible, the underlying positions of the molecules of interest. Here, we review the analysis methods that can be used to extract biological insight from SMLM data, in particular for the application of quantifying nanoscale molecular clustering. We review how some of the common artefacts inherent in SMLM can corrupt the acquired data, and therefore, how the output of SMLM cluster analysis should be interpreted.