Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca2+) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca2+ indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca2+ imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca 2+ was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca2+ and adenosine 5′-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca2+ imaging.
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