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


ABSTRACT: Background: Membrane potential (V mem) exerts physiological influence across a wide range of time and space scales. To study V mem in these diverse contexts, it is essential to accurately record absolute values of V mem, rather than solely relative measurements. Materials and Methods: We use fluorescence lifetime imaging of a small molecule voltage sensitive dye (VF2.1.Cl) to estimate mV values of absolute membrane potential. Results: We test the consistency of VF2.1.Cl lifetime measurements performed on different single-photon counting instruments and find that they are in striking agreement (differences of <0.5 ps/mV in the slope and <50 ps in the y-intercept). We also demonstrate that VF2.1.Cl lifetime reports absolute V mem under two-photon (2P) illumination with better than 20 mV of V mem resolution, a nearly 10-fold improvement over other lifetime-based methods. Conclusions: We demonstrate that VF-FLIM is a robust and portable metric for V mem across imaging platforms and under both one-photon and 2P illumination. This work is a critical foundation for application of VF-FLIM to record absolute membrane potential signals in thick tissue.

SUBMITTER: Lazzari-Dean JR 

PROVIDER: S-EPMC8558063 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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

Lazzari-Dean Julia R JR   Miller Evan W EW  

Bioelectricity 20210909 3


<b><i>Background:</i></b> Membrane potential (<i>V</i> <sub>mem</sub>) exerts physiological influence across a wide range of time and space scales. To study <i>V</i> <sub>mem</sub> in these diverse contexts, it is essential to accurately record absolute values of <i>V</i> <sub>mem</sub>, rather than solely relative measurements. <b><i>Materials and Methods:</i></b> We use fluorescence lifetime imaging of a small molecule voltage sensitive dye (VF2.1.Cl) to estimate mV values of absolute membrane  ...[more]

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