Optical Estimation of Absolute Membrane Potential Using One- and Two-Photon Fluorescence Lifetime Imaging Microscopy

Area of Science:

• Neuroscience
• Biophysics
• Optical Imaging

Background:

• Membrane potential (Vm) is critical for physiological functions across various scales.
• Accurate measurement of absolute Vm is essential for understanding cellular processes.
• Existing methods often provide relative, not absolute, Vm measurements.

Purpose of the Study:

• To develop and validate a method for accurately measuring absolute membrane potential.
• To assess the robustness and portability of fluorescence lifetime imaging microscopy (FLIM) with VF2.1.Cl dye.
• To improve the resolution of lifetime-based Vm measurements.

Main Methods:

• Utilized a voltage-sensitive dye, VF2.1.Cl, for fluorescence lifetime imaging.
• Performed measurements on single-photon counting instruments to test consistency.
• Evaluated VF2.1.Cl performance under one-photon and two-photon (2P) illumination.

Main Results:

• VF2.1.Cl lifetime measurements showed high consistency across different instruments (<0.5 ps/mV slope difference).
• VF-FLIM achieved better than 20 mV Vm resolution under 2P illumination, a significant improvement.
• Demonstrated VF-FLIM as a robust and portable Vm metric across platforms and illumination types.

Conclusions:

• VF-FLIM provides a reliable method for quantifying absolute membrane potential.
• This technique offers a significant advancement for optical recording of Vm.
• VF-FLIM serves as a foundational tool for studying Vm in complex biological tissues.