ElectroFluor Voltage‐Sensitive Dyes: Comprehensive Analysis of Wavelength‐Dependent Sensitivity and Cross‐Channel Bleed‐Through

New voltage-sensitive ElectroFluor (EF) dyes that emit across the visible and near-infrared spectrum (e.g., 730 nm) were recently developed. We evaluated EF-530, EF-630, and EF-730p-dyes spectrally orthogonal to green fluorescent protein (GFP)-at excitation wavelengths outside the conventional 470 nm range used for GFP-based indicators. Although previously applied in cardiac voltage imaging, their performance in neuronal tissue remains untested. We performed side-by-side comparisons using population voltage imaging in mouse cerebral cortex slices at optimal excitation wavelengths (530, 630, and 730 nm) and assessed cross-channel signal bleed-through across four excitation wavelengths (475, 530, 630, and 730 nm). All dyes produced robust optical signals at their optimal wavelengths, though non-preferred channels exhibited bleed-through with distinct amplitudes, polarities, and photobleaching patterns. These results provide detailed quantifications of EF dye performance for neuronal population imaging.