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

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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 (LckN10-tdEos), clusters into sub-100 nm regions which cover ∼7% of the cell surface. 2-channel PALM imaging of LckN10-tdEos and the N-terminus of the kinase Src (SrcN15-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. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

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