Analysis of Nanoscale Protein Clustering with Quantitative Localization Microscopy

Abstract

Dynamic clustering of proteins on the nanoscale is a vital step in many signaling processes and other cellular functions. Localization microscopy techniques such as PALM and dSTORM provide methods to localize complexes to nanoscale resolution. Quantification of the number of underlying protein subunits is more difficult, however, due to the complex photophysics of the fluorophore labels. Re-activation, stochastic blinking, and incomplete detection all contribute to over- and under-counting artifacts.In order to generate an accurate quantification of protein subunit numbers we have developed a method based on fluorophore blinking kinetics captured in PALM microscopy. This approach takes advantage of both spatial and temporal information to form adaptive discrimination criteria and avoids both over- and under-counting quantification errors. The technique presented can correctly identify and quantify molecular clusters in simulated data with 98% accuracy. We will present additional data on the application of this technique in quantifying the nature of clusters of proteins involved in the immune synapse and immune signaling.