Visible light excited ratiometric-GECIs for long-term in-cellulo monitoring of calcium signals.

Genetically Encoded Calcium Indicators (GECIs) are powerful molecular tools for monitoring calcium (Ca2+) signaling in the cytosol and organellar compartments. However, currently available ratiometric indicators that allow measurements of resting Ca2+ levels have limitations in long-term Ca2+ imaging. They either are ultraviolet (UV)-excited ones with strong photo-toxicity, or have poor performance. To overcome this hurdle, we developed a set of visible light excited ratiometric-GECIs (VR-GECIs) based on existing mono-colored GECIs. With performance comparable to their corresponding mono-color prototypes, this set of VR-GECIs enables long-term measurements of intra-cellular or intra-organellar Ca2+ signals. Using these VR-GECIs together with a newly developed off-line analysis tool, we achieved long-term measurements of Ca2+ homeostasis of moving or dividing cells. Our tools may find broad applications in decoding Ca2+-modulated physiological or pathological processes.