Imaging Neuronal Activity with Fast and Sensitive Red-Shifted Electrochromic FRET Indicators

Genetically encoded voltage indicators (GEVIs) allow optical recording of neuronal activities with high spatial resolution. While most existing GEVIs emit in the green range, red-shifted GEVIs are highly sought after because they would enable simultaneous stimulation and recording of neuronal activities when paired with optogenetic actuators, or two-color imaging of signaling and neuronal activities when used along with GFP-based indicators. In this study, we present several improved red-shifted GEVIs based on the electrochromic Förster resonance energy transfer (eFRET) between orange/red fluorescent proteins/dyes and rhodopsin mutants. Through structure-guided mutagenesis and cell-based sensitivity screening, we identified a mutant rhodopsin with a single mutation that exhibited more than 2-fold improvement in voltage sensitivity. Notably, this mutation has been independently discovered by Pieribone et al. ( Pieribone, V. A. et al. Nat Methods 2018 , 15 ( 12 ), 1108 - 1116 ). In cultured rat hippocampal neurons, our sensors faithfully reported action potential waveforms and subthreshold activities. We also demonstrated that this mutation could enhance the sensitivity of hybrid indicators, thus providing insights for future development.