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Genetically encoded voltage indicators (GEVIs) could potentially be used for mapping neural circuits at the plane of synaptic potentials and plateau potentials-two blind spots of GCaMP-based imaging. In the last year alone, several laboratories reported significant breakthroughs in the quality of GEVIs and the efficacy of the voltage imaging equipment. One major obstacle of using well performing GEVIs in the pursuit of interesting biological data is the process of transferring GEVIs between laboratories, as their reported qualities (e.g., membrane targeting, brightness, sensitivity, optical signal quality) are often difficult to reproduce outside of the laboratory of the GEVI origin. We have tested eight available GEVIs (Archon1, ArcLightD, ASAP1, ASAP2s, ASAP3b, Bongwoori-Pos6, FlicR1, and chi-VSFP-Butterfly) and two voltage-sensitive dyes (BeRST1 and di-4-ANEPPS). We used the same microscope, lens, and optical detector, while the light sources were interchanged. GEVI voltage imaging was attempted in the following three preparations: (1) cultured neurons, (2) HEK293 cells, and (3) mouse brain slices. Systematic measurements were successful only in HEK293 cells and brain slices. Despite the significant differences in brightness and dynamic response (ON rate), all tested indicators produced reasonable optical signals in brain slices and solid in vitro quality properties, in the range initially reported by the creator laboratories. Side-by-side comparisons between GEVIs and organic dyes obtained in HEK293 cells and brain slices by a "third party" (current data) will be useful for determining the right voltage indicator for a given research application.

In VitroTesting of Voltage Indicators: Archon1, ArcLightD, ASAP1, ASAP2s, ASAP3b, Bongwoori-Pos6, BeRST1, FlicR1, and Chi-VSFP-Butterfly