1. Click-EM for imaging metabolically tagged nonprotein biomolecules
- Author
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Ngo, John T, Adams, Stephen R, Deerinck, Thomas J, Boassa, Daniela, Rodriguez-Rivera, Frances, Palida, Sakina F, Bertozzi, Carolyn R, Ellisman, Mark H, and Tsien, Roger Y
- Subjects
Analytical Chemistry ,Chemical Sciences ,Aminobutyrates ,Click Chemistry ,DNA ,Fluorescent Dyes ,HEK293 Cells ,HeLa Cells ,Humans ,Lipids ,Listeria monocytogenes ,Microscopy ,Electron ,Molecular Structure ,Neurons ,Peptidoglycan ,RNA ,Singlet Oxygen ,Hela Cells ,Medicinal and Biomolecular Chemistry ,Biochemistry and Cell Biology ,Biochemistry & Molecular Biology ,Biochemistry and cell biology ,Medicinal and biomolecular chemistry - Abstract
EM has long been the main technique for imaging cell structures with nanometer resolution but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce click-EM, a labeling technique for correlative light microscopy and EM imaging of nonprotein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal 'click chemistry' ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of click-EM in imaging metabolically tagged DNA, RNA and lipids in cultured cells and neurons and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes.
- Published
- 2016