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PALM imaging and cluster analysis of protein heterogeneity at the cell surface
Author(s) -
Owen Dylan M.,
Rentero Carles,
Rossy Jérémie,
Magenau Astrid,
Williamson David,
Rodriguez Macarena,
Gaus Katharina
Publication year - 2010
Publication title -
journal of biophotonics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.877
H-Index - 66
eISSN - 1864-0648
pISSN - 1864-063X
DOI - 10.1002/jbio.200900089
Subject(s) - fluorophore , biophysics , chemistry , cell , microbiology and biotechnology , immunological synapse , cell membrane , biology , biochemistry , t cell , immune system , t cell receptor , fluorescence , optics , physics , immunology
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. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)