Box 1 Standards for testing genetic voltage indicators

Direct comparison between different types or families of voltage sensors is difficult in many cases because of a lack of accepted standardized guidelines for the measurement of parameters (see [49] for an attempt). To help developers and users of these tools, we propose a short guideline for standardized testing of genetic voltage indicators, to enable comparing a previously designed voltage sensor with a new one. Although meant for neurons, it could be adapted to other excitable cells. Standard parameters to be reported could include:

(1) Basic measurement parameters, using a light source (halogen lamps, LEDs, one or two photon lasers), power measurements (irradiance, in W/cm²) and signal to noise ratio (SNR). Fluorescence changes upon physiological voltage steps, and rise and decay times are also necessary, since they reflect speed and sensitivity of detection. Additionally, optimal absorption/emission spectra for fluorophores and range of light intensity used for testing should be reported.

(2) Photostability is a general benchmark for fluorophores and could determine the temporal range of an experimental design. Measurement of the fluorescence half-life should be included in the same range of light intensities tested.

(3) ∆F/F responses to spontaneous and triggered action potentials (up to 100 Hz) should be reported to predict voltage sensor behavior for measurement of neuronal activity.

(4) As the vast majority of proteins do not have a unique localization in the cellular membrane, providing a detailed description of subcellular localization could help researchers to choose properly and consider any later image analysis for discarding signals from intracellular compartments. For neurons, targeting voltage sensors to somatic, dendritic, or axonal domains is also highly desired and should be highlighted