Methodology for Cross-Talk Elimination in Simultaneous Voltage and Calcium Optical Mapping Measurements With Semasbestic Wavelengths

Figures

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  Figure 1. Mechanisms of measurement with an electrochromic Vm dye. (A) The Gaussian absorption (blue) and emission curves (red) are good fits for a given electrochromic Vm 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 λLPF 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 λLPF, overall ΔF/F sign can be negative or positive, and in between, there is a particular λLPF such that positive change cancels the negative change resulting in ΔF/F = 0, for specific λExc, termed semasbestic wavelength. (B) Theoretically calculated map of ΔF/F magnitude values as a function of λExc and…

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  Figure 2. The semasbestic wavelengths across all experiments for JPW-6003 Vm dye. (A) Box plots are a statistical representation of semasbestic wavelength averaged for each species. The ends of each box are the upper and lower quartiles, with the median marked as a horizontal line inside the box. The whiskers lines represent the upper and lower extremity. The P-values represent results of one-way ANOVA analysis with and without the isosbestic points from isolated pig hearts experiments. (B) The mean values and uncertainties of experimentally obtained semasbestic points across all species for different LFPs used on the camera. (C) A linear curve fit relating the range of semasbestic wavelengths corresponding to different λLPF. (D) Sensitivity analysis near the isosbestic wavelength for different LPFs. Experimentally obtained ΔF/F magnitude values were averaged across all species for the same LPF-BP filter pairs. Excitation wavelengths for up to 10 nm off the semasbestic wavelength result in less than 0.5% in the fractional change of the Vm signal.

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  Figure 3. Single-camera dual Vm measurements. The Vm 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 (λSem) corresponding to the λLPF of the four LPFs. λExc wavelengths are effective excitation wavelengths of the BP filters. Their nominal center wavelengths are listed in Supplementary Figure 3. λExcnom = 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 Vm signal changes buried under the noise level, which could be interpreted as no change in Vm signal in the odd frames otherwise. The cross-talk, the presence of Vm 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 λLPF = 740.0 nm, resulting in ΔF/F amplitude of less than 0.25% using excitation wavelength of…

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  Figure 4. Single-camera dual Vm−Ca measurements in isolated hearts of different species. (A) Arrhythmic effects under dynamic pacing, shown as spatial dispersion of APD and CaD across tissue for different species for even and odd beats. The spatial dispersion indicates an increased susceptibility to arrhythmia. APD values are obtained from 50% signal rise in amplitude till 50% AP repolarization. Numbers to the right indicate 3rd and 97th percentile APD values expressed in milliseconds. CaD values are obtained as the integral from 50% rise in amplitude till 50% decrease. Blue-red patterns show variations in APD and CaD (ΔAPD, ΔCaD) between even and odd beats (discordant alternans), showing regions alternating out of phase and separated with the nodal lines (white lines). Spatially discordant alternans are the counterpart of T-wave ECG alternans, a well-known marker for arrhythmia susceptibility. Although all species do develop arrhythmia under dynamic pacing, the electrophysiology across species is very different. Spatially discordant alternans are the most pronounced in rabbits, while pig hearts show very little APD and CaD dispersion, with insignificant differences between even and odd…

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  Figure 5. Simultaneous single-camera-based measurements of Vm and Ca signals in isolated cell culture monolayers of neonatal rats. (A) Time-snapshots of Vm - Ca dynamic at different time points illustrating the presence of anchored spiral wave and spatial loss of correlation between propagating Vm wavefront from Ca dynamics. (B) Normalized Vm and Ca signals obtained from processed raw recordings showing Ca signal lagging in time. (C) Representative unfiltered Vm and Ca signal traces with no filtering expressed as a relative change. In cell culture monolayers, Ca signals have significantly higher SNR than Vm signals originating only from the dye bound to the cell membrane. The amount of cross-talk and the presence of Vm signal in Ca signal with excitation at near the semasbestic point (Exc550) results is a small yet negligible cross-talk compared to the amplitudes of the signals.