Quantitative imaging of dynamic concentrations of cellular ions and metabolites is fundamental to understanding signaling networks. However, applications of intensity-based biosensors are fundamentally confounded by their dependence on probe concentration and excitation intensity. To address this challenge, we report a class of time-resolved genetically encoded calcium indicators (tr-GECIs)tr-CCaMP, tr-GCaMP, and tr-RCaMP. These sensors convert calcium levels into changes in fluorescence lifetime, a photophysical parameter intrinsically insensitive to variations in probe concentration or excitation light intensity. Our design maintains high brightness in both Ca2+-bound and Ca2+-free states, enabling robust detection via fluorescence lifetime imaging microscopy (FLIM). Engineering key residues near the chromophore and optimizing linkers yielded broad lifetime dynamic ranges: 2.14 ns for tr-CCaMP, 1.32 ns for tr-GCaMP, and 1.57 ns for tr-RCaMP. The sensors cover complementary Ca2+ affinity ranges with Kd values from 23.8 nM to 416 nM at 37 °C, allowing concentration determination from tens of nanomolars to micromolars. We applied tr-GECIs to map resting Ca2+ in proximity to multiple organelles and uncovered localized microdomains near organelle membranes, suggesting sites of active Ca2+ exchange. Unlike ratiometric or FRET sensors, tr-GECIs require only a single optical channel, simplifying multiplexed imaging. This work establishes a generalizable platform for developing lifetime-based biosensors, facilitating quantitative analysis of cellular signaling events with high spatial precision.
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