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Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

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

Sheng Xiao¹, William J Cunningham², Krishnakanth Kondabolu²

¹Department of Biomedical Engineering, Boston University, Boston, MA, USA. shengx@bu.edu.

|June 5, 2024

Summary

New microscopy overcomes weak signals and background noise in cellular voltage imaging. This advance enables high-fidelity in vivo imaging for neuroscience research.

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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.