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  3. Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species

Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species

  • Cell Chem Biol. 2016 Nov 17;23(11):1417-1427. doi: 10.1016/j.chembiol.2016.10.006.
Stephen R Adams 1,  Mason R Mackey 2,  Ranjan Ramachandra 2,  Sakina F Palida Lemieux 3,  Paul Steinbach 4,  Eric A Bushong 2,  Margaret T Butko 3,  Ben N G Giepmans 2,  Mark H Ellisman 5,  Roger Y Tsien 6
Affiliations

Affiliations

  • 1 Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address: [email protected].
  • 2 National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA 92093, USA.
  • 3 Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA.
  • 4 Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093, USA.
  • 5 National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA 92093, USA; Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093, USA.
  • 6 Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA; Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093, USA; Department of Chemistry & Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA.
PMID: 27818300 DOI: 10.1016/j.chembiol.2016.10.006
Abstract

Electron microscopy (EM) remains the primary method for imaging cellular and tissue ultrastructure, although simultaneous localization of multiple specific molecules continues to be a challenge for EM. We present a method for obtaining multicolor EM views of multiple subcellular components. The method uses sequential, localized deposition of different lanthanides by photosensitizers, small-molecule probes, or peroxidases. Detailed view of biological structures is created by overlaying conventional electron micrographs with pseudocolor lanthanide elemental maps derived from distinctive electron energy-loss spectra of each lanthanide deposit via energy-filtered transmission electron microscopy. This results in multicolor EM images analogous to multicolor fluorescence but with the benefit of the full spatial resolution of EM. We illustrate the power of this methodology by visualizing hippocampal astrocytes to show that processes from two astrocytes can share a single synapse. We also show that polyarginine-based cell-penetrating Peptides enter the cell via endocytosis, and that newly synthesized PKMζ in cultured neurons preferentially localize to the postsynaptic membrane.

Keywords

diaminobenzidine photooxidation; electron energy-loss spectroscopy; electron microscopy; lanthanide chelate; multicolor; peroxidase; photosensitizer.

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