Optical estimation of absolute membrane potential using fluorescence lifetime imaging

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Abstract

All cells maintain ionic gradients across their plasma membranes, producing transmembrane potentials (V_mem). Mounting evidence suggests a relationship between resting V_mem and the physiology of non-excitable cells with implications in diverse areas, including cancer, cellular differentiation, and body patterning. A lack of non-invasive methods to record absolute V_mem limits our understanding of this fundamental signal. To address this need, we developed a fluorescence lifetime-based approach (VF-FLIM) to visualize and optically quantify V_mem with single-cell resolution in mammalian cell culture. Using VF-FLIM, we report V_mem distributions over thousands of cells, a 100-fold improvement relative to electrophysiological approaches. In human carcinoma cells, we visualize the voltage response to growth factor stimulation, stably recording a 10-15 mV hyperpolarization over minutes. Using pharmacological inhibitors, we identify the source of the hyperpolarization as the Ca²⁺-activated K⁺ channel K_Ca3.1. The ability to optically quantify absolute V_mem with cellular resolution will allow a re-examination of its signaling roles.

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All data presented in the manuscript is available in the supporting / supplementary information.

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Julia Rose Lazzari-Dean

Department of Chemistry, University of California, Berkeley, Berkeley, United States

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0003-2971-5379
Anneliese M M Gest

Department of Chemistry, University of California, Berkeley, Berkeley, United States

Competing interests
No competing interests declared.
Evan W Miller

Department of Chemistry, University of California, Berkeley, Berkeley, United States

For correspondence
evanwmiller@berkeley.edu

Competing interests
Evan W Miller, is listed as an inventor on a patent describing voltage-sensitive fluorophores. This patent (US20170315059) is owned by the Regents of the University of California.

"This ORCID iD identifies the author of this article:" 0000-0002-6556-7679

Funding

National Science Foundation (GRFP)

Julia Rose Lazzari-Dean

National Institutes of Health (R35GM119855)

Evan W Miller

Alfred P. Sloan Foundation (FG-2016-6359)

Evan W Miller

March of Dimes Foundation (5-FY-16-65)

Evan W Miller

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.