LSSA12 was more stable in guanidinium HCl (GdnHCl) than mT-Sapphire, mAmetrine, and eGFP (Extended Data Fig. 6e). Site-directed mutagenesis confirmed the functional importance of the E222D and G65S mutations in LSSA12 (Extended Data Fig. 6f). LSSmGFP and LSSA12 enjoy similar advantages as hfYFP, including the absence of cysteine residues, low pKa, tolerance of fixatives, high chemical and thermal stability, and a single excitation band.