Fig. 1. Rational design of photostabilizer unnatural amino acids and synthetase screening. (A) Structural alignment of the β-barrels and highly conserved chromophore frameworks of representative FPs (yellow: Hyperfolder YFP (PDB: 7UGR); green: mNeonGreen (PDB: 5LTR); blue: ECFP (PDB: 2WSN); red: mCherry (PDB: 2H5Q)). (B) Schematic illustration of the “intramolecular photostabilization” concept migration from self-healing dyes to FPs. (C) Photophysical processes of the chromophore and the photostabilization mechanism of the NBA moiety. Upon photon absorption, the S₀ chromophore transitions to the S₁ state and primarily returns to S₀via fluorescence emission. However, a fraction of the excited chromophores undergoes ISC to form a reactive, long-lived T₁. In the presence of ambient oxygen, the T₁ state can transfer energy to generate reactive singlet oxygen, which subsequently attacks the chromophore and causes irreversible photobleaching. The incorporated NBA moiety acts as an efficient quencher, rapidly depleting the T₁ state via an intramolecular redox cycle (ox/red) to provide a safe energy relaxation pathway back to S₀, thereby effectively suppressing photobleaching. (D) Chemical structures of the designed photostabilizer unnatural amino acids, 1-NBAK and 2-NBAK. (E) Screening of a MbPylRS mutant capable of recognizing both 1-NBAK and 2-NBAK. (F) Fluorescence characterization confirming the site-specific incorporation of 1-NBAK and 2-NBAK into the GFPuv-N149TAG reporter. Schematic diagrams were created with BioRender.com. FPs, fluorescent proteins; NBA, nitrobenzyl; S₀, ground state; S₁, first excited singlet state; T₁, triplet state; ISC, intersystem crossing; MbPylRS, Methanosarcina barkeri pyrrolysyl-tRNA synthetase.