This primer reviews the practical considerations for designing voltage imaging experiments using genetically encoded voltage indicators (GEVIs), covering indicator selection, excitation wavelengths, microscopy configurations, construct expression strategies, and data analysis.
The guide outlines key trade-offs in voltage imaging: one-photon vs. two-photon excitation, signal-to-noise ratio determinants, and the use of structured illumination to improve tissue contrast — enabling resolution of both subthreshold dynamics and action potentials from single ion channels to large-scale population activity in behaving animals.
As a primer/review, this paper presents no original experimental data, so claims are not directly validated by a new study. Rapidly evolving instrumentation and indicator development may limit the longevity of specific recommendations.
Labs setting up voltage imaging should use this guide to systematically match their GEVI choice, microscopy configuration, and expression strategy to their biological question. Pay particular attention to the SNR trade-offs between one-photon and two-photon excitation when imaging in intact tissue.
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