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J Biophotonics. 2010 Jul;3(7):446-54. doi: 10.1002/jbio.200900089.

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

Author information

1
Centre for Vascular Research, University of New South Wales, Sydney, New South Wales, Australia. dylan.owen@unsw.edu.au

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 approximately 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.

PMID:
20148419
DOI:
10.1002/jbio.200900089
[Indexed for MEDLINE]
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