Figure 2. Optical mapping system featuring detection of red-shifted PGH1 voltage-sensitive dye signals, concurrent with orthogonal excitation of either ChR2 or eNpHR3.0 optogenetic constructs.

(a) Optical layout with dual-pathway excitation modules placed above NRVM monolayer. Fluorescence emission is captured from below, through a 253-channel fiber optic bundle. (b) Top, Exemplar NRVM (without optogenetic expression) action potential averaged from multiple channels, using customary di-4-ANEPPS voltage-sensitive dye. Bottom, corresponding activation map of uniform propagation. The color bar represents activation time in ms defined as the time of the stimulus to 50% of peak. (c) Top, Action-potential morphology was largely maintained with use of the red-shifted PGH1 dye. APDs were modestly prolonged compared to di-4-ANEPPS. Bottom, Propagation with PGH1 was also uniform throughout the monolayer, though PGH1-stained monolayers exhibited somewhat slower conduction. (d, e) Blue- or green-light pulses had no collateral effects on NRVMs lacking optogenetic expression, as demonstrated by exemplar PGH1 traces from a single recording channel. Blue or green illumination caused baseline shifts of the fluorescence signal, requiring crosstalk compensation, which is described in more detail in Method “Gap compensation for blue- and green-light bleed-through.” All blue illumination at 7.2 mW/mm². All green illumination at 1.4 mW/mm².