Fig. 4. In vitro photostability validation and characterization of purified top 12 mNG-2-NBAK variants. (A) Schematic of the in vitro photostability assay for the purified protein variants, where higher retention under identical irradiation time indicates greater photostability. (B) Fluorescence spectra of the top 12 mNG-2-NBAK variants (yellow lines) and the WT (green lines), demonstrating preserved spectral properties without significant shifts. (C) In vitro photobleaching kinetics of five representative variants (2 µM) under continuous irradiation. The full dataset for the top 12 variants is shown in Fig. S7. The legend matches the top-to-bottom order of the curves. The control represents non-irradiated samples at the identical concentration. (D) Relative brightness and photostability of the top 12 variants. Relative brightness represents the initial fluorescence (t = 0) of purified proteins at 2 µM normalized to the WT (100%). The photobleaching half-life (t_1/2) indicates the time required for this fluorescence to decay by 50%. Schematic diagrams were created with BioRender.com. mNG, mNeonGreen; WT, wild type.