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. Author manuscript; available in PMC: 2022 May 6.
Published in final edited form as: Annu Rev Biophys. 2021 Mar 2;50:447–468. doi: 10.1146/annurev-biophys-062920-063555

Table 2.

Comparison of optical Vmem recording strategies

Performance attribute Single-color fluorescence intensitya Single-color fluorescence lifetimeb FRET-basedc Electrochromic (ratio-based)d GEVI temporal dynamicse Stimulated Raman scatteringf
Spatial resolutiong +++
(<1 μm)		 ++
(~1 μm)h 		+++
(<1 μm)		 +++
(<1 μm)		 ++
(~1 μm)h 		+
(>1 μm)h
Temporal resolution ++
(1 ms)		 +
(5 ms)h 		+
(1 ms or >10 ms)i 		++
(1 ms)		 −
(s)h 		++
(1 ms)h
Voltage resolutionj −
(≫00 mV)		 ++
(VF: 20 mV, GEVI: >100 mV)		 ND +
(~100 mV)		 ++
(10 mV)		 ND
Stability +
(minutes)		 ++
(hours)		 ++
(hours)		 +
(minutes)		 ND ND
Noninvasiveness +++ +++ +++ +++ +++ +++
In situ capabilities +++ +++ +++ +++ +++ ++
Access to subcellular structuresk ++ ++ ++ ++ ++ ++
Throughput and multiplexing +++ ++ +++ +++ + ++
Ease of use +++ + ++ ++ − −
a

Many single-color fluorescence intensity Vmem sensors have been designed, although they are seldom used for absolute Vmem quantification. Data are compiled from References 57, 77, 115, and 116.

b

Single-color fluorescence lifetime recordings have been shown for genetically encoded voltage indicators (16) and for small-molecule VFs (59).

c

FRET-based systems may use conformational changes of the indicator (4), changes in the absorption spectrum of a rhodopsin derivative (3, 125), or translocation of charged groups in the membrane (15, 35, 72) to report Vmem. Many properties are similar across these architectures; metrics in this case represent all types of FRET-based strategies unless otherwise noted.

d

Performance metrics are aggregated from References 17, 33, 34, 68, 79, and 122.

e

Demonstrated in Reference 41.

f

Demonstrated in References 61 and 62.

g

Spatial resolution is determined by the diffraction limit, as well as optics used in the microscope. Techniques rated ++ rather than +++ generally require moderate spatial binning to obtain adequate signal.

h

For fluorescence lifetime, GEVI temporal dynamics, and stimulated Raman scattering measurements, there is a trade-off between spatial and temporal resolution, and one may be sacrificed to improve the other. We list the best values of either spatial or temporal resolution shown in the literature or that we have demonstrated in our laboratory.

i

FRET sensors based on protein conformational changes or dye translocation exhibit temporal resolution of >10 ms, whereas electrochromic FRET-based sensors can resolve millisecond-time Vmem events.

j

Voltage resolution listed is for applying a calibration determined on one cell onto another individual cell; more precise Vmem measurements can be obtained if measurements from multiple cells are averaged. Most techniques have substantially better resolution for quantifying the magnitude of a Vmem change on an individual cell (e.g., VF-FLIM has 5-mV resolution for quantifying Vmem changes on a single cell versus 20-mV resolution for quantifying absolute Vmem alone).

k

Limited by delivery of sensor to intracellular structures and/or the ability to isolate signal from only the subcellular membrane structure of interest when all membrane structures are stained.

Technique performance in each category is ranked as follows: −, poor; +, fair; ++, good; +++, excellent. Abbreviations: FLIM, fluorescence lifetime imaging; FRET, Förster resonance energy transfer; GEVI, genetically encoded voltage indicator; ND, not determined; VF, VoltageFluor; Vmem, membrane potential.