Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Abstract

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera. Video LinkThe video component of this article can be found at

Toward Better Genetically Encoded Sensors of Membrane Potential

“…with two similarly bright FPs) have the advantage that the ratio removes common mode noise due to movement, e.g., respiration and blood flow. However, the fluorescent intensities of the donor and acceptor FPs are often different, which means the shot noise of the ratio will be higher than that of either individual signal [35, 36]. FRET quenching microbial rhodopsin signals are not ratiometric because there is only a single optical signal (from the fluorescent donor).…”

Biophysical Parameters of GEVIs: Considerations for Imaging Voltage

“…The rhodopsin-based GEVIs exhibit a near-linear voltage response ( 9 , 15 , 20 , 21 , 22 ). Other GEVIs exhibit a sigmoidal fluorescence change as a function of voltage ( 4 , 6 , 23 , 24 , 25 , 26 ). A linear slope will spread the dynamic fluorescence change over a broad voltage range.…”

Optogenetic Monitoring of Synaptic Activity with Genetically Encoded Voltage Indicators

“…An example of this is shown in Figure 2 . The HEK cell in Figure 2 is expressing a GEVI from which the ΔF and the ΔF/F traces from three different light levels are shown (Lee et al, 2016 ). As can be seen from this comparison, a high ΔF/F value can be achieved by a large change in fluorescence or a small change in fluorescence when the probe is dim.…”

“… Varying light levels affect ΔF and ΔF/F values. (A) An HEK 293 cell expressing a single fluorescent protein (FP) based GEVI, Bongwoori, is shown in resting light intensity (RLI), (B) the fluorescence traces of averaged ΔF (F x -F 0 ) values from three different regions with varying intensity, (C) the fluorescence traces showing averaged ΔF/F values from the same regions in ( B ; modified with permission from Lee et al, 2016 , Figure 6). …”