Figure 3.

Single-camera dual V_m measurements. The V_m dye was excited with alternating excitation bands, using a 660/10 excitation BP filter in even frames. Odd frames were acquired using off-the-shelf excitation BP filters selected to closely match the semasbestic points (λ_Sem) corresponding to the λ_LPF of the four LPFs. λ_Exc wavelengths are effective excitation wavelengths of the BP filters. Their nominal center wavelengths are listed in Supplementary Figure 3. ${\lambda }_{\mathrm{\text{Exc}}}^{nom}$ = 525 nm is the nominal center wavelength of 20 nm wide (OD6) BP filter (Semrock). Shown optical action potential signals are obtained using the stacking procedure from a single pixel, averaging at least 400 periods without any filtering. The stacking resolves small V_m signal changes buried under the noise level, which could be interpreted as no change in V_m signal in the odd frames otherwise. The cross-talk, the presence of V_m signal due to the differences between the filters effective excitation wavelength and corresponding semasbestic points is less than 0.25% for most LPF. The sensitivity to the difference is the lowest for λ_LPF = 740.0 nm, resulting in ΔF/F amplitude of less than 0.25% using excitation wavelength of more than 5 nm different from the ideal semasbestic point.