A blue-shifted genetically encoded Ca indicator with enhanced two-photon absorption.

Significance: Genetically encoded calcium ion (Ca) indicators (GECIs) are powerful tools for monitoring intracellular Ca concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca 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 and 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.

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

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

Results: The Ca-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 Mcm, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca-dependent fluorescence increase is 15-fold and the apparent for Ca 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.

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