Benefiting from the hfYFP crystal structure, we eliminated hfYFP’s 514-nm excitability and produced two exclusively 405-nm excitable GFPs with [a large Stokes shift], LSSA12 and LSSmGFP. These LSS FPs overcome the cross-excitation problem of mT-Sapphire (Fig. 5c–e) without sacrificing molecular brightness (Table 1). LSSmGFP has high chemical and thermodynamic stability and the same molecular brightness as mAmetrine, while lasting twice as long under laser-scanning confocal illumination before photobleaching (Extended Data Fig. 7g).