Fluorescence Imaging of Cell Membrane Potential: From Relative Changes to Absolute Values

Abstract

Membrane potential is a fundamental property of biological cells. Changes in membrane potential characterize a vast number of vital biological processes, such as the activity of neurons and cardiomyocytes, tumorogenesis, cell-cycle progression, etc. A common strategy to record membrane potential changes that occur in the process of interest is to utilize organic dyes or genetically-encoded voltage indicators with voltage-dependent fluorescence. Sensors are introduced into target cells, and alterations of fluorescence intensity are recorded with optical methods. Techniques that allow recording relative changes of membrane potential and do not take into account fluorescence alterations due to factors other than membrane voltage are already widely used in modern biological and biomedical studies. Such techniques have been reviewed previously in many works. However, in order to investigate a number of processes, especially long-term processes, the measured signal must be corrected to exclude the contribution from voltage-independent factors or even absolute values of cell membrane potential have to be evaluated. Techniques that enable such measurements are the subject of this review.

Keywords: cell membrane potential; genetically-encoded voltage indicators; membrane potential imaging; potentiometric sensors; voltage-sensitive dyes.

Figure 1

Commonly-used classes of molecular and biomolecular potentiometric sensors. (a) Electrochromic organic dyes. Upper. Electrochromic organic dyes are characterized by different electron distributions in the ground and excited states. Interaction with an external electric field E alters the energies of the ground and excited states to a different extent, resulting in the shift of absorption, excitation and emission bands. Lower. Spectral shift of electrochromic dye upon membrane voltage change can be utilized for membrane potential imaging. The depicted band can represent absorption, excitation, or emission spectral band. (b) FRET-based organic dyes. Upper. Fluorescence resonance energy transfer (FRET) can occur between two molecules (donor and acceptor) with overlapping excitation and emission bands. After photoexcitation, the donor can either re-emit a photon or transfer energy to the acceptor via FRET. If the acceptor is fluorescent, it can emit a photon with a longer wavelength. Lower. FRET pair includes an immobile donor fluorophore (green oval) attached to the outer surface of the cell membrane and a mobile lipophilic ion (acceptor) located inside the membrane (red circle). The fluorescence of the donor is quenched via FRET. Upon depolarization the acceptor moves further from the donor, resulting in the decrease of FRET efficiency and an increase in donor fluorescence. (c) Molecular wire-based voltage-sensitive dyes. A fluorophore (green oval) is attached to the outer surface of the cell membrane. Its fluorescence is quenched by electron transfer from an electron donor (red circle) through a molecular wire. Depolarization decreases the rate of electron transfer, resulting in the enhancement of fluorescence. (d) Redistribution voltage-sensitive dyes. At equilibrium the ratio of extracellular and intracellular concentrations of charged membrane-permeable dyes is determined by membrane potential in accordance with the Nernst equation. Upon depolarization the intracellular concentration of anionic dyes (green circles) increases leading to the enhancement of fluorescence detected from the cell interior. (e) Voltage-sensitive domain-based genetically-encoded voltage indicators. Upper. Voltage-induced structural reorganization of a voltage-sensitive domain (VSD, blue cylinders) is passed to the voltage-independent fluorescent protein (FP, green cylinder) through a peptide linker. The fluorescence of the construct is voltage-dependent. Lower Two FPs with overlapping excitation and emission bands are attached to the VSD. Voltage-induced structural reorganization of VSD changes the relative position of FPs, resulting in the change of FRET efficiency and, therefore, fluorescence intensities of both donor and acceptor. (f) FRET-opsin genetically-encoded voltage indicators. Fluorescent protein (FP) is attached to microbial rhodopsin (Rh). At positive voltages the chromophore of Rh is protonated and its absorption efficiently quenches FP emission via FRET. Upon depolarization, the concentration of Rh with protonated chromophore decreases, leading to the lowering of the red-shifted absorption band, decrease of FRET efficiency, and the enhancement of detected FP fluorescence. (g) Rhodopsin-based genetically-encoded voltage indicators. Several microbial rhodopsins, such as archaerhodopsin-3 (Arch), demonstrate intrinsic linear voltage dependence of fluorescence intensity.

Figure 2

Elimination of fluorescence changes caused by factors other than voltage. (a) Ratio of two signals recorded from a single emission band. The voltage-induced shift of the emission band of electrochromic dyes results in the opposite changes of emission intensities corresponding to wavelengths at the left and right wings of the band. The ratio of fluorescence intensities at two wavelengths from the left and right wings of the emission band does not depend on sensors concentration and was shown to be linearly dependent on voltage [31,107]. (b) Ratio of two signals recorded from two fluorophores of the same sensor. Sensor is constructed as a fusion of a voltage-sensitive domain (VSD) and two fluorescent proteins—green (GFP) and red (RFP). Voltage-dependent structural reorganization of VSD is passed to FPs, resulting in linear fluorescence voltage-dependence of GFP. The fluorescence intensity of RFP remains voltage-independent. The ratio of two signals can be used to cancel out concentration dependence [28,106]. (c) Ratio of two signals from different voltage-dependent distributions of sensors. Microbial rhodopsin Arch D95H demonstrates linear fluorescence response to membrane potential changes attributed to the voltage-dependent equilibrium between the fluorescent (F) and non-fluorescent states of the protein [114]. In the proposed protocol the whole population of sensors was converted into state F with blue light illumination and the corresponding fluorescence intensity $F_{all}$ was measured. Afterward, orange light was used to establish an equilibrium with voltage-dependent concentration of state F, and the fluorescence intensity $F_{eq}$ was measured. The relative decrease of fluorescence intensity was shown to be a robust concentration-corrected metrics. (d) Excited state lifetime can be used for membrane potential imaging. This property was shown to be independent of fluorophore bleaching and illumination conditions, largely independent of fluorophore concentration. Voltage may affect the excited state lifetime by altering the rate constant of non-radiative decay ($k_{nr}$) [41]. (e) Estimation of voltage-independent component of fluorescence intensity for redistribution dyes. The fluorescence intensity detected from the cell interior is determined by the intracellular dye concentration, which should be directly related to membrane potential value in accordance with the Nernst equation. However, a part of dye molecules are bound to intracellular interaction sites and provide equal contribution to detected fluorescence at any membrane potential value. To determine the binding constant $K_b$ and derive the corrected equation for estimating membrane potential the difference in fluorescence intensity detected from cell interior $F_{in}$ and extracellular medium $F_{out}$ at 0 mV was measured [115]. (f) Eliminating cell size dependence of fluorescence signal for redistribution dyes. At high concentrations cationic dyes demonstrate two emission bands—a green band attributed to dye monomers and a red-shifted band attributed to dye aggregates. At saturating concentration all intracellular interaction sites are occupied at any voltage by dye monomers and green fluorescence is not affected by voltage changes. Upon depolarization the concentration of intracellular dye aggregates decreases, resulting in the decrease of red signal. Both green and red signals are proportional to cell size and their ratio can be used to cancel out cell size dependence [116].

Figure 3

Methods used to calibrate sensors for membrane voltage imaging to measure optical signals for a series of established membrane voltage values. (a) Patch-clamp technique. The specific value of cell membrane potential can be established using the patch-clamp device. Actual cell membrane potential is measured as the voltage difference between the recording electrode patched to the cell membrane and the reference electrode placed into an electrolyte or extracellular medium. The value is passed to the feedback module, which determines the difference between the actual and desired values set by a signal generator. The difference is compensated by the injection of an electric current into the cell through the current-passing electrode. (b) Generating electric field with microelectrodes. A cell is placed between two microelectrodes that generate a uniform electric field E. The corresponding changes in cell membrane potential are proportional to the magnitude of the electric field and the cosine of the angle between the electric field vector and membrane surface. (c) Ionophore-based calibration techniques. The addition of valinomycin ionophore makes membrane voltage determined solely by the extracellular concentration of potassium ions. For calibration, fluorescence intensities are measured for a series of extracellular potassium concentrations. (d) Calibration of charged organic dyes using completely depolarized cells. For charged dyes, membrane voltage can be derived from the ratio of intracellular and extracellular concentrations in accordance with the Nernst equation. The relationship between detected fluorescence intensity and dye concentration can be derived by the measurement of fluorescence intensity for a set of extracellular dye concentrations using cells depolarized to 0 mV, when intracellular and extracellular concentrations are equal. Afterward, ${\left[Dye\right]}_{in}$ and ${\left[Dye\right]}_{out}$ can be obtained from the calibration plot.