Abstract
The most successful genetically encoded calcium indicators (GECIs) employ an intensity or ratiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biological range. Here, we report on a sensing strategy in which a conformational change directly modifies the fluorescence quantum yield and fluorescence lifetime of a circular permutated turquoise fluorescent protein. The fluorescence lifetime is an absolute parameter that enables straightforward quantification, eliminating intensity-related artifacts. An engineering strategy that optimizes lifetime contrast led to a biosensor that shows a 3-fold change in the calcium-dependent quantum yield and a fluorescence lifetime change of 1.3 ns. We dub the biosensor Turquoise Calcium Fluorescence LIfeTime Sensor (Tq-Ca-FLITS). The response of the calcium sensor is insensitive to pH between 6.2–9. As a result, Tq-Ca-FLITS enables robust measurements of intracellular calcium concentrations by fluorescence lifetime imaging. We demonstrate quantitative imaging of calcium concentrations with the turquoise GECI in single endothelial cells and human-derived organoids.
Highlights
The most successful genetically encoded calcium indicators (GECIs) employ an intensity or ratiometric readout
Intensity of a single fluorescent protein (FP) is inherently unsuitable for quantitative measurements, as intensity is influenced by many factors including movement, concentration, bleaching, and sample thickness
We looked into ratiometric sensors, either based on Förster resonance energy transfer (FRET) imaging or on the ratio between a cpFP calcium sensor and a second FP
Summary
The most successful genetically encoded calcium indicators (GECIs) employ an intensity or ratiometric readout. The calciumdependent interaction between CaM and the peptide results in a conformation change that is converted into a change in fluorescence intensity of the cpGFP These probes are known as GCaMP or GECO6. The pKa of GECIs is near the physiological pH and a change in intracellular pH changes the intensity Some of these effects can be corrected by ratio imaging using a second fluorescent protein (FP), usually an orange FP (OFP) or red FP (RFP)[12,13]. The ratio depends on the intensity of the second FP, which in turn is determined by its maturation[12] These sensors present the same pH sensitivity as their parents[12,13]. GECI probes based on lifetime contrast would enable true quantitative calcium imaging, independent of equipment
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