Membrane Topography and the Overestimation of Protein Clustering in Single Molecule Localisation Microscopy – Identification and Correction

According to single-molecule localisation microscopy (SMLM) almost all plasma membrane proteins are clustered. We demonstrate that clusters can arise from variations in membrane topography where the local density of a randomly distributed membrane molecule to a degree matches the variations in the local amount of membrane. Further, we demonstrate that this false clustering can be differentiated from genuine clustering by using a membrane marker to report on local variations in the amount of membrane. In dual colour live cell SMLM using the membrane probe DiI alongside either the transferrin receptor (TfR) or the GPI-anchored protein CD59, we found that pair correlation (PC) analysis reported both proteins and DiI as being clustered, as did its derivative PC-PALM and nearest neighbour analyses. After converting the localisations into images and using the DiI image to factor out topography variations, no CD59 clusters were visible, suggesting that the clustering reported by the other methods is an artefact. However, the TfR clusters persisted after topography variations were factored out. We demonstrate that membrane topography variations must be considered before concluding that membrane molecules cluster and present a method to this end.