Fig. 3. Photostability screening of mNG-NBAK variants in live E. coli. (A) High-throughput photostability screening workflow for the bright variants via LED irradiation in an ice bath, with samples placed in the central position of wells to ensure uniform illumination. (B) Schematic of the in vivo photostability assay evaluating fluorescence retention, where higher retention under identical irradiation time indicates greater photostability. (C) and (D) In vivo photobleaching kinetics of the 105 mNG-1-NBAK (C) and 87 mNG-2-NBAK (D) bright variants expressed in E. coli. In both panels, curves for the WT and photostability-reduced variants are colored green and yellow, respectively. In (D), curves for the 67 favorable variants are colored light pink, with the top 12 variants highlighted in dark pink. (see Fig. S6 for detailed positions of the 67 favorable variants) (E) fluorescence retention rates at 30 minutes for the top 12 mNG-2-NBAK variants (dark pink) and the WT (green). (F) Spatial distribution of the top 12 photostable 2-NBAK modification sites mapped onto the mNG β-barrel (PDB: 5LTR). Schematic diagrams were created with BioRender.com. mNG, mNeonGreen; WT, wild type.