Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

J Vis Exp. 2017 Jun 24:(124):55997. doi: 10.3791/55997.

Abstract

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

Publication types

  • Video-Audio Media

MeSH terms

  • Fluorescent Antibody Technique / methods*
  • Fluorescent Dyes / chemistry
  • Humans
  • Jurkat Cells
  • Lymphocyte Activation
  • Microscopy, Fluorescence / methods*
  • Receptors, Antigen, T-Cell / metabolism
  • Single Molecule Imaging / methods*
  • Synaptosomal-Associated Protein 25 / metabolism
  • T-Lymphocytes / cytology*
  • T-Lymphocytes / metabolism

Substances

  • Fluorescent Dyes
  • Receptors, Antigen, T-Cell
  • SNAP25 protein, human
  • Synaptosomal-Associated Protein 25