Click-EM for imaging metabolically tagged nonprotein biomolecules.
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.
Department of Pharmacology, University of California, San Diego, La Jolla, California, USA.
John T
JT
Ngo
Department of Pharmacology, University of California, San Diego, La Jolla, California, USA.
Stephen R
SR
Adams
National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, California, USA.
Thomas J
TJ
Deerinck
National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, California, USA.
Daniela
D
Boassa
Department of Chemistry, University of California, Berkeley, California, USA.
Frances
F
Rodriguez-Rivera
Department of Pharmacology, University of California, San Diego, La Jolla, California, USA.
Sakina F
SF
Palida
Department of Chemistry, Stanford University, Stanford, California, USA.|Howard Hughes Medical Institute, Stanford University, Stanford, California, USA.
Carolyn R
CR
Bertozzi
National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, California, USA.|Department of Neurosciences, University of California, San Diego, La Jolla, California, USA.
Mark H
MH
Ellisman
Department of Pharmacology, University of California, San Diego, La Jolla, California, USA.|National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, California, USA.|Department of Chemistry &Biochemistry, University of California, San Diego, La Jolla, California, USA.|Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California, USA.
Roger Y
RY
Tsien
D000613:Aminobutyrates / Q000737:chemistry; D057930:Click Chemistry; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000032:analysis* / Q000737:chemistry; D008089:Listeria monocytogenes / Q000378:metabolism; D008854:Microscopy, Electron / Q000379:methods*; D015394:Molecular Structure; D009474:Neurons / Q000737:chemistry / Q000378:metabolism; D010457:Peptidoglycan / Q000032:analysis* / Q000096:biosynthesis; D012313:RNA / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D026082:Singlet Oxygen / Q000737:chemistry
D000613:Aminobutyrates; D005456:Fluorescent Dyes; D008055:Lipids; D010457:Peptidoglycan; D026082:Singlet Oxygen; C005959:2,4-diaminobutyric acid; D012313:RNA; D004247:DNA