Saal, Kim-A. Richter, Frank Rehling, Peter Rizzoli, Silvio O. Combined Use of Unnatural Amino Acids Enables Dual-Color Super-Resolution Imaging of Proteins <i>via</i> Click Chemistry Recent advances in optical nanoscopy have brought the imaging resolution to the size of the individual macromolecules, thereby setting stringent requirements for the fluorescent labels. Such requirements are optimally fulfilled by the incorporation of unnatural amino acids (UAAs) in the proteins of interest (POIs), followed by fluorophore conjugation <i>via</i> click chemistry. However, this approach has been limited to single POIs in mammalian cells. Here we solve this problem by incorporating different UAAs in different POIs, which are expressed in independent cell sets. The cells are then fused, thereby combining the different proteins and organelles, and are easily imaged by dual-color super-resolution microscopy. This procedure, which we termed Fuse2Click, is simple, requires only the well-established Amber codon, and allows the use of all previously optimized UAAs and tRNA/RS pairs. This should render it a tool of choice for multicolor click-based imaging. fluorophore conjugation;Click Chemistry;cell sets;protein;dual-color super-resolution microscopy;optimized UAAs;POI;click chemistry;Unnatural Amino Acids Enables Dual-Color Super-Resolution Imaging;click-based imaging;Such requirements;Amber codon;Fuse 2Click;imaging resolution 2018-12-10
    https://acs.figshare.com/articles/journal_contribution/Combined_Use_of_Unnatural_Amino_Acids_Enables_Dual-Color_Super-Resolution_Imaging_of_Proteins_i_via_i_Click_Chemistry/7464572
10.1021/acsnano.8b06047.s002