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

Area of Science:

• Neuroscience
• Molecular Biology
• Biochemistry

Background:

• Genetically encoded calcium ion (GECI) indicators are crucial for monitoring intracellular calcium dynamics in living systems.
• Existing GECIs for neuronal activity imaging are limited, necessitating new variants with distinct photophysical properties.
• Blue-shifted GECIs are particularly promising for enhanced two-photon brightness, facilitating multiphoton microscopy.

Purpose of the Study:

• To develop and characterize T-GECO1, a high-performance blue-shifted GECI.
• To expand the palette of GECIs for advanced fluorescence imaging of neuronal activity.
• To assess T-GECO1's utility in various experimental settings, including in vitro, in vivo, and ex vivo applications.

Main Methods:

• Protein engineering and directed evolution were employed to develop T-GECO1.
• Purified T-GECO1 protein was characterized for its photophysical properties.
• Performance was assessed in cultured rat hippocampal neurons, mouse brains, and ex vivo hippocampal slices using one- and two-photon microscopy.

Main Results:

• T-GECO1 exhibits an excitation peak at 468 nm and an emission peak at 500 nm, with a high quantum yield (0.83).
• It possesses approximately double the two-photon brightness of EGFP and a 15-fold dynamic range for calcium detection.
• T-GECO1 demonstrated superior signal-to-noise ratio for action potential detection compared to a late-generation GCaMP variant under two-photon excitation.

Conclusions:

• T-GECO1 is a high-performance blue-shifted GECI.
• It offers significant advantages over existing GCaMP variants for neuronal activity imaging, particularly under two-photon excitation.
• T-GECO1 expands the toolkit for advanced in vitro and in vivo neuroscience research.