Figure 1.

Mechanisms of measurement with an electrochromic V_m dye. (A) The Gaussian absorption (blue) and emission curves (red) are good fits for a given electrochromic V_m dye for illustration purposes, with solid lines for polarized membrane and leftward shifted dotted lines for maximally depolarized membrane by a propagating AP. For excitation at the isosbestic point and for a given LPF on the camera side passing only part of the emission spectra, as illustrated with the solid (black) vertical lines, ΔF/F is always negative. ΔF/F = 0 can only be achieved if the entire emission spectrum is obtained (solid blue vertical line), thereby not dependent on the optical setup, the λ_LPF value. Excitation left of the isosbestic point results in an amplitude increase of the emission spectra due to absorption coefficient increase with the shift of the absorption spectra. Depending on the filter λ_LPF, overall ΔF/F sign can be negative or positive, and in between, there is a particular λ_LPF such that positive change cancels the negative change resulting in ΔF/F = 0, for specific λ_Exc, termed semasbestic wavelength. (B) Theoretically calculated map of ΔF/F magnitude values as a function of λ_Exc and λ_LPF, showing the transition from positive to negative ΔF/F, and continuous line of semasbestic wavelengths, ΔF/F = 0 isochrone line, for each λ_Exc - λ_LPF pair. (C) Illustration of the experimental setup. (D) APs from optical mapping measurements on isolated rabbit heart near a semasbestic wavelength. A 10 nm wide BP excitation filter was used of 540 nm nominal center wavelength along different LPFs on the camera's side. Due to low ΔF/F values SNR is low. However, ensemble averaging (stacking) increases SNR without filtering in post-processing. (E) Quadratic fit curves from ΔF/F simulated values (B), for four different LPFs of the same λ_LPF values as LPF used in ΔF/F measurements on isolated hearts. (F) Quadratic fit curves from ΔF/F magnitude values for four different LPFs. Optical mapping recordings were performed on isolated rabbit heart for across a wide range of excitation wavelengths, from 500 to 660 nm. Zero crossings correspond to the semasbestic wavelengths. All λ_LPF values of LPFs are experimentally measured.