Optical voltage imaging in neurons: moving from technology development to practical tool


A central goal in neuroscience is to determine how the brain’s neuronal circuits generate perception, cognition and emotions and how these lead to appropriate behavioural actions. A methodological platform based on genetically encoded voltage indicators (GEVIs) that enables the monitoring of large-scale circuit dynamics has brought us closer to this ambitious goal. This Review provides an update on the current state of the art and the prospects of emerging optical GEVI imaging technologies.

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Fig. 1: Structural features and optical reporting mechanism of selected GEVIs and hybrid GEVIs.
Fig. 2: Isolation of individual neurons for single-cell-level voltage imaging.
Fig. 3: Spatial scales and level of analysis.
Fig. 4: Experimental setup configurations for different levels of GEVI imaging, from behavioural to cellular-level analysis.


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The authors thank S. Antic for suggestions and a set of figures for an earlier version of this article. Work in our laboratory is supported by grants from the BRAIN initiative (US National Institutes of Health grants U01MH109091 and U01NS099573).

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Nature Reviews Neuroscience thanks M. Hoppa and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Correspondence to Thomas Knöpfel.

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Submitted to a process that (reversibly) deactivates fluorescence emission.

2p cross-section

A measure describing how well a fluorescent dye is excited by light of a given intensity; similar to the one-photon absorption extinction coefficient, but two-photon absorption increases with the square of the light intensity.

Signal-to-noise ratio

(SNR). A measure that compares the level of a desired signal (for example, voltage-dependent change in fluorescence) to the level of background noise (in this case, random fluctuations of measured fluorescence). The SNR is defined as the ratio of signal power to noise power.


A term standing for ‘picture element’; the light detected by one pixel of the detector may come from anywhere within the corresponding area in the object plane.

Bessel beam

A laser beam with a profile shaped in the form of a Bessel function that can be used to generate an axially elongated excitation volume.

Light sheet illumination

A method in which a thin slice (usually from a few hundred nanometres to a few micrometres) of a sample is illuminated. Compared with conventional epifluorescence microscopy, light sheet illumination produces reduced out-of-focus background fluorescence.

Light field deconvolution

A technique for high-speed volumetric imaging. Using an array of lenses, the object is imaged at different angles, providing 3D information about a sample. The 3D structure is reconstructed by mathematical operations termed deconvolution. This technique allows for imaging in three dimensions simultaneously with a 2D detector.

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Knöpfel, T., Song, C. Optical voltage imaging in neurons: moving from technology development to practical tool. Nat Rev Neurosci 20, 719–727 (2019). https://doi.org/10.1038/s41583-019-0231-4

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