Abstract
Fluorescent proteins (FPs) are essential tools for live imaging, with brightness and photostability being key parameters when selecting an FP for experiments. Recently developed variants of monomeric StayGold (mSG) and mGold2 promise improvements in these properties, yet their performance can vary across model organisms and tissue contexts. To guide the selection of FPs for zebrafish imaging, we expressed membrane-targeted FP fusions in embryos and systematically compared their in vivo brightness and photostability against the widely used mNeonGreen (mNG). Among the mSG variants, mSG(A) displayed brightness comparable to mNG but with markedly increased photostability. In contrast, mSG(BJ) and mSG(E138D) showed reduced brightness, while retaining improved photostability. The mGold2 variants exhibited no improvement in photostability over mNG, but their increased brightness at 514 nm excitation allowed effective imaging at lower laser intensities, thereby extending usable imaging times. There were no significant differences detected between the two mGold2 variants. Overall, mSG(A) emerges as an optimal choice for long-term imaging, while mGold2 variants are advantageous when maximal brightness is required. These results provide practical benchmarks for FP selection in zebrafish embryonic imaging.