Click-electron microscopy for imaging metabolically tagged non-protein biomolecules

Autor: Ngo, John T., Adams, Stephen R., Deerinck, Thomas J., Boassa, Daniela, Rodriguez-Rivera, Frances, Palida, Sakina F., Bertozzi, Carolyn R., Ellisman, Mark H., Tsien, Roger Y.
Jazyk: angličtina
Rok vydání: 2016
Předmět:
Zdroj: Nature chemical biology
ISSN: 1552-4469
1552-4450
Popis: Electron microscopy (EM) has long been the main technique to image 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 non-protein 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.
Databáze: OpenAIRE
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