PALM imaging and cluster analysis of protein heterogeneity at the cell surface

Abstract

The authors employed photoactivatable localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM) imaging and image analysis based on Ripley's K ‐function to quantify the distribution and heterogeneity of proteins at the cell plasma membrane. The membrane targeting sequence of the N‐terminal region of the T cell receptor‐pathway kinase Lck fused to the photo‐convertible fluorescent protein tdEos (Lck_N10‐tdEos), clusters into sub‐100 nm regions which cover ∼7% of the cell surface. 2‐channel PALM imaging of Lck_N10‐tdEos and the N‐terminus of the kinase Src (Src_N15‐PS‐CFP2) are demonstrated. Finally, T cell microclusters at the immune synapse are imaged at super‐resolution using dSTORM, showing that conventional TIRF images contain unresolved, small clusters. These methods are generally applicable to other cell and fluorophore systems to quantify 2‐D molecular clustering at nanometer scales.