Multimodal optical imaging combining voltage-sensitive dye ElectroFluor630 with genetically encoded calcium, glutamate, or voltage indicators.

Significance: Combining genetically encoded neuronal activity indicators (GENIs), restricted to only one neuron type, with a voltage-sensitive dye (VSD) that reports pan-neuronal activity, could be beneficial for understanding neural circuits. Recently introduced far-red EF-630 may be compatible with green GENIs and could serve as an internal reference of neuronal activity for multiple GENIs. Aim: Here, we assess the EF-630 compatibility with several green-fluorescent-protein-based GENIs, including the calcium indicator GCaMP6f, the glutamate indicator iGluSnFR, and two voltage indicators ASAP2s, and ASAP5-Kv, for recording neuronal aggregate responses. Approach: Mouse brain slices expressing each GENI were stained with EF-630, and then extracellular stimulation and population optical imaging were sequentially performed at two wavelengths. In addition, cre-dependent ASAP5-Kv transgenic mice were generated and characterized. Results: Dual recordings provided population signals in both channels for all combinations. For each indicator pair, we quantified Conclusions: These kinetic differences underscore how both the choice of indicator pair and the targeted cell type influence the interpretation of neural population activity. Overall, our work provides the first systematic characterization of paired VSD-GENI measurements, establishing practical considerations and performance benchmarks for dual optical imaging of neuronal populations.