Time-Resolved Genetically Encoded Indicators toward Quantitative Imaging of Calcium Dynamics in Living Cells.

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.