10.1021/acsnano.8b06047.s002 Kim-A. Saal Kim-A. Saal Frank Richter Frank Richter Peter Rehling Peter Rehling Silvio O. Rizzoli Silvio O. Rizzoli Combined Use of Unnatural Amino Acids Enables Dual-Color Super-Resolution Imaging of Proteins <i>via</i> Click Chemistry American Chemical Society 2018 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 00:00:00 Journal contribution 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 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.