Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species.
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.
Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address: sadams@ucsd.edu.
Stephen R
SR
Adams
National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA 92093, USA.
Mason R
MR
Mackey
National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA 92093, USA.
Ranjan
R
Ramachandra
Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA.
Sakina F
SF
Palida Lemieux
Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093, USA.
Paul
P
Steinbach
National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA 92093, USA.
Eric A
EA
Bushong
Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA.
Margaret T
MT
Butko
National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA 92093, USA.
Ben N G
BNG
Giepmans
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.
Mark H
MH
Ellisman
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.
Roger Y
RY
Tsien
D000818:Animals; D001253:Astrocytes / Q000648:ultrastructure; D057846:Cell-Penetrating Peptides / Q000032:analysis; D002478:Cells, Cultured; D004285:Dogs; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D028581:Lanthanoid Series Elements / Q000032:analysis*; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D008807:Mice, Inbred BALB C; D046032:Microscopy, Energy-Filtering Transmission Electron / Q000379:methods*
D057846:Cell-Penetrating Peptides; D028581:Lanthanoid Series Elements
diaminobenzidine photooxidation; electron energy-loss spectroscopy; electron microscopy; lanthanide chelate; multicolor; peroxidase; photosensitizer