Large-scale deep tissue voltage imaging with targeted-illumination confocal microscopy.

Area of Science:

• Neuroscience
• Biomedical Engineering
• Optical Imaging

Background:

• Genetically encoded voltage indicators enable cellular specificity in voltage imaging.
• High kilohertz imaging rates result in weak signals, limiting resolution.
• Out-of-focus fluorescence and tissue scattering reduce signal-to-noise ratio and cause crosstalk in dense tissues.

Purpose of the Study:

• To develop a microscope overcoming signal weakness and background noise in voltage imaging.
• To improve signal-to-noise ratio and reduce crosstalk for in vivo imaging.
• To enable high-fidelity voltage imaging at large scales and penetration depths.

Main Methods:

• Developed a novel microscope combining targeted illumination and confocal gating.
• Maximized signal detection efficiency.
• Quantified improvements in signal-to-noise ratio and crosstalk reduction experimentally and theoretically.

Main Results:

• Significantly enhanced signal-to-noise ratio compared to conventional methods.
• Substantially reduced crosstalk between cells in densely labeled tissue.
• Demonstrated high-fidelity in vivo voltage imaging across diverse conditions and indicator types.

Conclusions:

• The developed microscope offers a versatile solution for challenging in vivo voltage imaging.
• Enables high-fidelity imaging at greater depths and scales.
• Facilitates advanced neuroscience research requiring precise cellular voltage monitoring.