Cyan variants of green fluorescent protein are widely used as donors in Förster resonance energy transfer experiments. The popular, but modestly bright, Enhanced Cyan Fluorescent Protein (ECFP) was sequentially improved into the brighter variants Super Cyan Fluorescent Protein 3A (SCFP3A) and mTurquoise, the latter exhibiting a high-fluorescence quantum yield and a long mono-exponential fluorescence lifetime. Here we combine X-ray crystallography and excited-state calculations to rationalize these stepwise improvements. The enhancement originates from stabilization of the seventh β-strand and the strengthening of the sole chromophore-stabilizing hydrogen bond. The structural analysis highlighted one suboptimal internal residue, which was subjected to saturation mutagenesis combined with fluorescence lifetime-based screening. This resulted in mTurquoise2, a brighter variant with faster maturation, high photostability, longer mono-exponential lifetime and the highest quantum yield measured for a monomeric fluorescent protein. Together, these properties make mTurquoise2 the preferable cyan variant of green fluorescent protein for long-term imaging and as donor for Förster resonance energy transfer to a yellow fluorescent protein.

Infrared fluorescent proteins (IFPs) are ideal for in vivo imaging and monomeric versions of these proteins can be advantageous as protein tags or for sensor development. In contrast to GFP, which requires only molecular oxygen for chromophore maturation, phytochrome-derived IFPs incorporate biliverdin (BV) as the chromophore. However, BV varies in concentration in different cells and organisms. Here we engineered cells to express the heme oxygenase responsible for BV biosynthesys and a brighter monomeric IFP mutant (IFP2.0). Together, these tools improve the imaging capabilities of IFP2.0 compared to monomeric IFP1.4 and dimeric iRFP. By targeting IFP2.0 to the plasma membrane, we demonstrate robust labeling of neuronal processes in Drosophila larvae. We also show that this strategy improves the sensitivity when imaging brain tumors in whole mice. Our work shows promise in the application of IFPs for protein labeling and in vivo imaging.