A modular chemigenetic calcium indicator for multiplexed in vivo functional imaging

Area of Science:

• Neuroscience
• Biochemistry
• Molecular Biology

Background:

• Genetically encoded fluorescent calcium indicators are crucial for cellular physiology research.
• Existing indicators lack brightness and multiplexing capabilities for simultaneous in vivo imaging of multiple signals.

Purpose of the Study:

• To develop a modular chemigenetic calcium indicator (WHaloCaMP) for bright, genetically targetable, and multiplexed in vivo imaging.
• To enable simultaneous recording of multiple physiological signals with enhanced sensitivity.

Main Methods:

• WHaloCaMP was engineered using bright dye-ligands and protein sensor domains with a tryptophan-based quenching mechanism.
• Compatibility with rhodamine dye-ligands spanning green to near-infrared wavelengths was established.
• Calcium-induced fluorescence intensity and lifetime changes were characterized.

Main Results:

• WHaloCaMP demonstrated a 7x fluorescence intensity and 2.1-ns fluorescence lifetime increase upon calcium binding with near-infrared dyes.
• Successful in vivo Ca2+ imaging was achieved in flies and mice.
• Three-color multiplexed functional imaging of neurons and astrocytes in zebrafish larvae was performed.
• Quantification of Ca2+ concentration using fluorescence lifetime imaging microscopy (FLIM) was demonstrated.

Conclusions:

• WHaloCaMP offers a versatile platform for advanced in vivo calcium imaging.
• Its modularity and compatibility with various dyes facilitate multiplexed recordings and quantitative measurements.
• This indicator significantly enhances the capability for simultaneous physiological signal monitoring in complex biological systems.