PinkyCaMP: an mScarlet-based calcium sensor with enhanced brightness, photostability and multiplexing capabilities

Abstract Genetically encoded calcium (Ca 2+ ) indicators (GECIs) are essential tools for monitoring neuronal activity, but the performance of red fluorescent GECIs has remained limited. In particular, many red indicators are relatively dim, produce low signal-to-noise ratios and can undergo unwanted photoswitching when exposed to blue light, restricting their use in all-optical experiments that combine imaging with optogenetics or multicolor imaging. Here we show the development of PinkyCaMP, a Ca 2+ sensor based on the bright red fluorescent protein mScarlet. PinkyCaMP exhibits markedly improved brightness, photostability and signal-to-noise ratio compared to existing red GECIs, while remaining fully compatible with blue-light-based optogenetic and dual-color imaging approaches. PinkyCaMP is well-tolerated by neurons, showing no detectable toxicity or aggregation, both in vitro and in vivo. PinkyCaMP enables a broad spectrum of imaging modalities, including single-photon methods, such as fiber photometry, widefield imaging and miniature microscopy imaging, as well as two-photon imaging in awake mice.