Genetic code expansion enables live-cell and super-resolution imaging of site-specifically labeled cellular proteins

Chayasith Uttamapinant, Jonathan D Howe, Kathrin Lang, Václav Beránek, Lloyd Davis, Mohan Mahesh, Nicholas P Barry, Jason W Chin

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (β-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells.

Original languageEnglish
Pages (from-to)4602-5
Number of pages4
JournalJournal of the American Chemical Society
Issue number14
Publication statusPublished - 15 Apr 2015

Keywords / Materials (for Non-textual outputs)

  • Actins
  • Animals
  • Binding Sites
  • COS Cells
  • Cell Survival
  • Cercopithecus aethiops
  • Genetic Code
  • HEK293 Cells
  • Humans
  • Lysine
  • Optical Imaging
  • Protein Engineering
  • Vimentin


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