Statistical analysis of 3D localization microscopy data for quantification of protein distribution upon clot formation

Extension of localization microscopy super-resolution methods to the third dimension as well as its multicolor application became state-of-the-art in the last decade. Most of recent studies show the co-localization of three-dimensionally arranged proteins within cells. However, only a few publications focused on analysis of local protein density and there mostly in 2D. Additionally, there are still limits regarding the quantitative and comparative analysis methods. Here we present a platform called “SM2SA” (Single Molecule Localization for 2 Samples Analysis) that addresses the quantitative analysis of protein distributions at nano- and microscopic level. It allows comparative multi-parameter cluster analysis of proteins in three dimensions. Software tools are provided for filtering of outliers and for analysis of two independent samples as well as drift correction. Two sets of the three-dimensional positions of single molecules are resampled and combined in clusters via hierarchical positional clustering algorithm ranging from nanometers up to micrometers. Features like average cluster density, curvature and Ripley K/H function values are extracted and statistically compared. The methodology allows a quantitative comparison of protein cluster parameters. Exemplarily, we used this platform to analyze the distribution and clustering of membrane proteins (CD41 /CD62p) inside human platelet clots via two-color immunostaining.