PALM Imaging and Cluster Analysis of Protein Heterogeneity at the Cell Surface

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

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Adaptor Proteins, Signal Transducing / analysis*
  • Animals
  • Anthozoa
  • Cell Membrane / chemistry*
  • Cluster Analysis
  • Computer Simulation
  • HeLa Cells
  • Humans
  • Image Processing, Computer-Assisted*
  • Jurkat Cells
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / analysis*
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / genetics
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / metabolism
  • Membrane Proteins / analysis*
  • Microscopy, Fluorescence / methods*
  • Optics and Photonics / methods
  • Stochastic Processes
  • T-Lymphocytes / chemistry*
  • T-Lymphocytes / cytology
  • T-Lymphocytes / metabolism
  • Transfection
  • src-Family Kinases / chemistry*
  • src-Family Kinases / genetics
  • src-Family Kinases / metabolism

Substances

  • Adaptor Proteins, Signal Transducing
  • LAT protein, human
  • Luminescent Proteins
  • Membrane Proteins
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
  • src-Family Kinases