Optical estimation of absolute membrane potential using fluorescence lifetime imaging

Abstract

All cells maintain ionic gradients across their plasma membranes, producing transmembrane potentials (Vmem). Mounting evidence suggests a relationship between resting Vmem 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 Vmem 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 Vmem with single-cell resolution in mammalian cell culture. Using VF-FLIM, we report Vmem 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 KCa3.1. The ability to optically quantify absolute Vmem with cellular resolution will allow a re-examination of its signaling roles.

Modulation of the Cell Membrane Potential and Intracellular Protein Transport by High Magnetic Fields

“…A pilot experiment is required to study the predicted effect of HMFs on the membrane potential. For example, fast changes in the membrane voltage under a pulsed HMF could be detected experimentally using fluorescence lifetime imaging [Lazzari-Dean et al, 2019]. A possibility to control the cell membrane potential remotely with magnetic fields could represent a new biomedical tool for magnetogenetics, whose goal is to genetically engineer (manipulate proteins, DNA, etc.)…”

Aktivitätsbasierte Sensorik: ein synthetisch‐methodischer Ansatz für die selektive molekulare Bildgebung und darüber hinaus.

“…Chemie rung, [204] Esterasespaltung [205] oder Lichtaktivierung [206] angesprochen und die Spannung durch Fluoreszenzlebensdauermikroskopie (FLIM, fluorescence lifetime imaging microscopy) gemessen. [207] In der Arbeitsgruppe von New wurden Flavine als reversible Redoxschalter biologischen Ursprungs eingesetzt, welche die dynamischen Flüsse von Oxidationsund Reduktionsereignissen in Zellen überwachen kçnnen. [208] Innovative Ansätze mit redoxempfindlichen niedermolekularen Substanzen, gekoppelt an Sensorplattformen, ergaben Nicotinimidium-gekoppelte MRT-Kontrastmittel, [209] ebenso wie Naphthalimid-, [210] subzellulärl okalisierbare [211] und ratiometrische, [212] an fluoreszierende Gerüste gekoppelte Flavinredoxsensoren.…”

“…Von diesem Ausgangspunkt aus wurde eine vielgestaltige Familie von Spannungssensoren mit unterschiedlichen Anregungs‐/Emissionsprofilen entwickelt . Die molekularen Drähte wurden dabei mit Styryl‐ und Fluorenresten abgestimmt, Zellen durch bioorthogonale Markierung, Esterasespaltung oder Lichtaktivierung angesprochen und die Spannung durch Fluoreszenzlebensdauermikroskopie (FLIM, fluorescence lifetime imaging microscopy) gemessen . In der Arbeitsgruppe von New wurden Flavine als reversible Redoxschalter biologischen Ursprungs eingesetzt, welche die dynamischen Flüsse von Oxidations‐ und Reduktionsereignissen in Zellen überwachen können .…”

Activity‐Based Sensing: A Synthetic Methods Approach for Selective Molecular Imaging and Beyond

“…[196][197][198][199][200][201][202] This approach has enabled the tuning of molecular wires from styryl to fluorene, [203] targeting of cells by bio-orthogonal labeling, [204] esterase cleavage, [205] or light activation, [206] as well as quantitative voltage measurements by fluorescence lifetime imaging microscopy (FLIM). [207] The New group has exploited flavins as bioderived reversible redox switches,w hich can monitor dynamic fluxes of oxida- tion and reduction events in cells. [208] Innovative work using redox-sensitive small molecules coupled to sensor platforms led to nicotinimidium-coupled MRI contrast agents, [209] as well as naphthalimide, [210] subcellular-targetable, [211] and ratiometric [212] fluorescent scaffold-coupled flavin redox sensors.…”