Measuring Absolute Membrane Potential Across Space and Time

Abstract

Membrane potential (V_mem) is a fundamental biophysical signal present in all cells. V_mem signals range in time from milliseconds to days, and they span lengths from microns to centimeters. V_mem affects many cellular processes, ranging from neurotransmitter release to cell cycle control to tissue patterning. However, existing tools are not suitable for V_mem quantification in many of these areas. In this review, we outline the diverse biology of V_mem, drafting a wish list of features for a V_mem sensing platform. We then use these guidelines to discuss electrode-based and optical platforms for interrogating V_mem. On the one hand, electrode-based strategies exhibit excellent quantification but are most effective in short-term, cellular recordings. On the other hand, optical strategies provide easier access to diverse samples but generally only detect relative changes in V_mem. By combining the respective strengths of these technologies, recent advances in optical quantification of absolute V_mem enable new inquiries into V_mem biology.

Keywords: electrophysiology; fluorescence; fluorescence lifetime; membrane potential; microscopy; quantitative imaging.

Figure 1

Membrane potential (V_mem) signals and measurement techniques across time and space. (a) A schematic of cellular V_mem, with examples of seven biological processes where V_mem signaling plays a role. The resistance to ionic flow between compartments sets the length scale of V_mem signals; V_mem can scale across tissues or be compartmentalized within organelles or dendritic spines. The properties of ion channels and pumps that determine V_mem, as well as ion diffusion between compartments, dictate the time duration of a V_mem response. (b) Biological space accessible to the three most common strategies for absolute V_mem measurement (shaded regions), overlaid with example biological process of interest (outlined boxes, numbered as in panel a). While many techniques can report cellular V_mem on the scale of milliseconds or seconds, longer recordings or recordings across large areas are difficult to access. Shaded areas indicate regions where single trial recordings retain voltage resolution on the scale of biological V_mem changes (≤20 mV) and minimally alter the biological sample. Recordings with fluorescence electrochromic dyes require recalibration with an electrode on each cell, whereas fluorescence lifetime and patch-clamp electrophysiology can be applied as stand-alone measurements that are comparable between cells. Shaded areas are restricted to demonstrated application space for each technique and do not indicate the full extent of its possible use.

Figure 2

Electrophysiological configurations for V_mem recording. (a) General schematic for electrode-based V_mem recordings, in which a cell comes into direct contact with an electrode. Voltage is measured as the difference between the recording electrode and a reference electrode in the bath solution. (b–d) Close-up of the interface between the membrane and the electrode in different electrophysiology configurations. (b) In whole-cell patch-clamp electrophysiology, the plasma membrane is ruptured, and the cytosol mixes with the recording electrode solution. (c) In the cell-attached configuration, the plasma membrane is left intact. (d) In perforated patch, the membrane is not ruptured, but ionophores introduced into the recording solution allow ionic exchange across the membrane.

Figure 3

Optical strategies for reporting absolute V_mem. (a) Single-color fluorescence intensity recordings detect changes in V_mem by changes in a sensor’s fluorescence quantum yield (shown), extinction coefficient, or cellular concentration. This technique generally cannot report absolute V_mem optically, although estimates can be made if calibrations are performed for every cell of interest. (b) Single-color fluorescence lifetime can report absolute V_mem, particularly if the V_mem sensor operates via photoinduced electron transfer.(c) FRET-based sensors show differences in the FRET ratios of two fluorophores, often resulting from V_mem-dependent changes in the distance between the two. The diagram shows a FRET-oxonol system. (d) The excitation and/or emission spectra of electrochromic dyes depend on the electric field in the plasma membrane. (e) Certain GEVIs show V_mem-related differences in the temporal dynamics of the absorption of their excited state. (f) SRS imaging reports changes in V_mem-dependent vibrational frequencies. Abbreviations:λ, wavelength; depol, depolarization; FRET, Förster resonance energy transfer; GEVI, genetically encoded voltage indicator; hyperpol, hyperpolarization; SRS, stimulated Raman scattering; V_mem, membrane potential.