Novel Genetically Encoded Ratiometric Calcium Indicators

Abstract

Genetically encoded calcium indicators (GECIs) have recently undergone dramatic improvements in brightness, dynamic range, kinetics, and calcium sensitivity and now are capable of reporting single neuronal action potentials - comparing favorably to synthetic calcium dyes. Nevertheless, GECIs still lag behind synthetic calcium dyes in applications requiring quantitation of absolute intracellular calcium concentration. Here, we describe a novel approach to achieve dual-color ratiometric calcium measurements.When expressed individually, GCaMP and mCherry, respectively, exhibit calcium-dependent and -independent fluorescence intensities; and when present at the same level, their fluorescence intensity ratio should provide an estimate of absolute calcium concentration. Unfortunately, the two proteins do not always express equally in all locations in a cell. A simple tandem construct ensures equal levels everywhere of the two proteins, but non-radiative energy transfer from GCaMP to mCherry reduces the dynamic range of GCaMP signals and leads to apparent calcium-dependent fluorescence changes of mCherry. We introduced a rigid alpha helix, the ER/K helix, between the fluorophores, in order to prevent significant Forster resonance energy transfer (FRET). In parallel experiments, we demonstrated in vitro and in cellulo that the tandem GCaMP-ER/K-mCherry construct provides quantitative estimates of calcium concentration, without suffering reduction in dynamic range due to FRET activity.