Fluorescent ncAA are desired by many for in vitro and in vivo work. The down sides to using fluorescent ncAA is that the size of the amino acid side chain is currently limited and this results in structures with fluorescent abilities that are not ideal for most applications. Another drawback to fluorescent ncAA for in vivo work is that the ncAA need to be added to the growing cells at high concentrations at the stage of ncAA-protein expression, which means the residual free amino acid need to be washed out prior to imaging. Since these amino acids have limited availability we often recommend that those desiring site-specifically fluorescently labeled protein approach this with bioorthogonal ligations.

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