Fig. 3: Finding the optimal excitation power density and pulse width for red-light optogenetic pacing of ReaChR-expressing Drosophila.

a M-mode images acquired from a late pupa with a resting heart rate (RHR) ~3 Hz, showing the HR following a pacing rate (PR) of 5 Hz with pulse intensities tuned from 1.86 to 7.26 mW mm⁻², and the HR deviation from the PR with pulse densities lower than 0.91 mW mm⁻². b The effect of excitation power densities on pacing probabilities for larva (n = 14), early pupae (n = 17), late pupae (n = 11), and adult (n = 12) stages, showing that decreased excitation power densities lower than 3.63 mW mm⁻² led to lower pacing probabilities. c 2D M-mode images acquired from a late pupa with an RHR ~2.5 Hz, showing the heartbeat following the pacing rate (PR) of 5.5 Hz as the pulse widths are tuned from 5 ms to 40 ms, and the heart rate (HR) deviation from the PR with a pulse width of 2 ms. d The influence of stimulation pulse width on pacing probabilities for larva (n = 14), early pupa (n = 17), late pupa (n = 11), and adult (n = 12) stages. Pulse widths of 10 ms or less resulted in lower pacing probabilities for the first two stages. Width of 5 ms lowered the pacing probabilities for the late two stages. e–h Plots of the average RHR (black solid line), the average HRs from tuning the pacing frequencies (red curve), and the fitting line of the average HRs (blue dashed line) characterizing the HR adjustable ranges for larva (n = 14), early pupa (n = 16), late pupa (n = 11), and adult (n = 10). The fly heart reliably followed the pacing frequency up to ~167%, ~218%, ~219% and ~143% of the RHR at the four developmental stages, respectively. Results are presented as mean ± SD. Red traces on the M-mode images illustrate the intensity change of excitation light. Red and black fonts in M-mode images represent PRs and HRs respectively, as in Fig. 2. Scale bars throughout the figure, 30 μm.