Exploration of mScarlet for development of a red lifetime sensor for calcium imaging

The past decades, researchers have worked on the development of genetically encoded biosensors, including over 60 genetically encoded calcium indicators (GECIs) containing a single fluorescent protein (FP). Red fluorescent GECIs provide advantages in terms of imaging depths and reduced cell toxicity. Most of GECIs respond with a fluorescence intensity change, and researchers have strived to improve the sensors in terms of brightness and fold-change. Unfortunately, fluorescence intensity is influenced by many factors other than the desired sensor response. GECIs with a fluorescence lifetime contrast overcome this drawback, but so far, no bright red GECI has been developed that shows a fluorescence lifetime contrast. We tried to tackle this challenge by using the brightest red fluorescent proteins from the mScarlet family to develop a new sensor. We did succeed in creating remarkable bright probes, but the fluorescence lifetime contrast we observed in bacterial lysates was lost in mammalian cells. Based on our results, and the success of others to develop a pH and a voltage sensor of mScarlet, we are confident that a GECI with mScarlet is feasible. To this end, we propose to continue development using a mammalian cell-based screening, instead of screening in bacterial lysates.