After three rounds of directed evolution (Methods), we isolated mSandy2 (Table 1 and Fig. 2), a variant whose emission wavelength is not significantly altered but that displays an absolute quantum yield increase of 0.09 relative to mSandy1, making it approximately 33% brighter than its parent. mSandy2 is also 40% brighter than mCherry, an RFP that emits at a similar wavelength. However, chromophore maturation in mSandy2 is less efficient than in either mSandy1 or mCherry, as evidenced by the peaks at approximately 390 and 510 nm in its absorption spectrum (Fig. 2d) that correspond to neutral and anionic green chromophores (ESI Fig. 5†), respectively.