A blue-shifted genetically encoded Ca²⁺ indicator with enhanced two-photon absorption

Genetically encoded calcium ion (Ca²⁺) indicators (GECIs) are powerful tools for monitoring intracellular Ca²⁺ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca²⁺ concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy.

We describe the development and applications of T-GECO1 - a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1.

We used protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using one-photon excitation fiber photometry in mice, and ex vivo using two-photon Ca²⁺ imaging in hippocampal slices.

The Ca²⁺-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49,300 M^-1cm^-1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca²⁺-dependent fluorescence increase is 15-fold and the apparent K_d for Ca²⁺ is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant.

T-GECO1 is a high performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.