Rapid fluorescence lifetime sensor development of LifeCamp enables transient and baseline absolute calcium measurements

Abstract

Genetically encoded calcium sensors (GECIs) have been instrumental for studying neuronal activity and intracellular signaling. GECIs are typically fluorescence-intensity sensors that change brightness upon calcium binding. Iterative improvements in GECIs have yielded indicators that report action potential-evoked calcium entry with high sensitivity and temporal resolution, enabling measurement of network activity across thousands of neurons. However, fluorescence intensity-based measurements generally cannot report baseline or absolute calcium levels and may confound neuromodulatory regulation of calcium handling with changes in action potential firing. Fluorescence lifetime sensors are insensitive to many artifacts that plague intensity-based measures and report absolute substrate levels, including those at rest. However, relatively few lifetime sensors for neuronal signals exist, and developing new sensors is typically difficult and low-yield. Here, we introduce a new rapid lifetime sensor development (RALISED) platform, which we use to build a new GCaMP8m-based high-speed lifetime calcium sensor, termed LifeCamp. We show that LifeCamp enables comparison of baseline calcium signals in cell culture, brain slices, and mice. In addition, we show that LifeCamp enables the detection of fast action potential-evoked calcium transients in single neurons from brain slices and in behaving mice. Using LifeCamp, we discovered calcium baseline changes associated with neuronal activity in brain slices and behaving mice, as well as slow average calcium changes in neuronal populations of freely moving mice. Altogether, this study highlights the utility of the RALISED method to rapidly develop new lifetime sensors and the application of the LifeCamp calcium lifetime sensor to study fast and slow calcium signaling. Significance statementWe developed a new high-speed, sensitive calcium lifetime sensor (LifeCamp) using a novel rapid lifetime sensor development (RALISED) platform. LifeCamp has high sensitivity and a large substrate-dependent lifetime change (<1ns), allowing for the capture of baseline calcium levels, transient calcium changes, and neuronal firing in vitro and behaving animals. LifeCamp lifetime measurement is insensitive to artifacts that plague conventional intensity imaging and enables absolute comparison of baseline and transient calcium changes across cells, brain regions, and experimental conditions. Hence, LifeCamp is a powerful tool that enables a more accurate and in-depth understanding of neuronal activity and calcium signaling.