A palette of bright and photostable monomeric fluorescent proteins for bacterial time-lapse imaging

Fluorescent proteins (FPs) are pivotal for examining protein production, localization, and dynamics in live bacterial cells. However, the use of FPs in time-lapse imaging is frequently constrained by issues such as oligomerization or limited photostability. Here, we report the engineering of novel cyan, green, yellow, and red FPs that exhibit improved photostability and aggregation properties while retaining high in vivo brightness. We first derived superfolder green fluorescent protein into mChartreuse, a brighter, more photostable, and monomeric fluorophore. mChartreuse was further derived into cyan and yellow variants with enhanced photostability and dispersibility. We also report a mutation that eliminates residual oligomerization in red FPs derived from <i>Discosoma</i> sp., such as mCherry or mApple. Incorporation of this mutation in mApple among other substitutions yielded mLychee, a bright and photostable monomeric red FP. These novel FPs advance fluorescence time-lapse analysis in bacteria, and their spectral properties match current imaging standards, ensuring seamless integration into existing research workflows.