A novel approach to dual excitation ratiometric optical mapping of cardiac action potentials with di-4-ANEPPS using pulsed LED excitation

Abstract

We developed a new method for ratiometric optical mapping of transmembrane potential (V(m)) in cardiac preparations stained with di-4-ANEPPS. V(m)-dependent shifts of excitation and emission spectra establish two excitation bands (<481 and >481 nm) that produce fluorescence changes of opposite polarity within a single emission band (575-620 nm). The ratio of these positive and negative fluorescence signals (excitation ratiometry) increases V(m) sensitivity and removes artifacts common to both signals. We pulsed blue (450 ± 10 nm) and cyan (505 ± 15 nm) light emitting diodes (LEDs) at 375 Hz in alternating phase synchronized to a camera (750 frames-per-second). Fluorescence was bandpass filtered (585 ± 20 nm). This produced signals with upright (blue) and inverted (cyan) action potentials (APs) interleaved in sequential frames. In four whole swine hearts with motion chemically arrested, fractional fluorescence for blue, cyan, and ratio signals was 1.2 ± 0.3%, 1.2 ± 0.3%, and 2.4 ± 0.6%, respectively. Signal-to-noise ratios were 4.3 ± 1.4, 4.0 ± 1.2, and 5.8 ± 1.9, respectively. After washing out the electromechanical uncoupling agent, we characterized motion artifact by cross-correlating blue, cyan, and ratio signals with a signal with normal AP morphology. Ratiometry improved cross-correlation coefficients from 0.50 ± 0.48 to 0.81 ± 0.25, but did not cancel all motion artifacts. These findings demonstrate the feasibility of pulsed LED excitation ratiometry in myocardium.

Figure 1

Model of di-4-ANEPPS voltage sensitivity (A) Vm-dependent spectral shift of excitation spectrum from Vm = rest (solid) to ΔVm = +100 mV (dashed). The black circle shows the isosbestic wavelength at which excitation is the same during depolarization and rest. (B) Difference spectrum taken by subtracting the resting excitation spectrum from the shifted excitation spectrum (top). Normalized power density spectrum for blue and cyan LEDs (bottom). (C) Emission spectrum peak as a function of excitation wavelength. Published peaks (squares) as well as peaks estimated for 450 nm and 505 nm excitation (circles) are shown. (D) Blue and cyan excitation result in two resting emission spectra (solid gray and black lines, respectively). The emission spectra undergo a Vm-dependent shift and amplitude modulation resulting from the Vm-dependent excitation shift (dashed lines). The black circles show the emission isosbestic points. (E) Difference spectra taken by subtracting the resting emission spectra from their respective shifted and amplitude modulated emission spectra. The gray box is the emission passband used in this study. (F) When emitted fluorescence is filtered with the passband shown in (E), upright OAPs result from blue excitation and inverted OAPs result from cyan excitation.

Figure 2

Example signals. Top: Blue-elicited fluorescence. Middle: Cyan-elicited fluorescence. Bottom: Ratio signal. The vertical scale bar indicates ΔF/F.

Figure 3

Successful (left column) and unsuccessful (right column) correction of motion artifact. The model signal, e(t) is gray. XC is the cross-correlation coefficient between the respective gray and black signals. The vertical scale bars are 2% of background fluorescence.

Figure 4

Histogram of cross-correlation coefficients of model signal e(t) with blue-elicited b(t) (dark), cyan-elicited c(t) (light), and ratio r(t) (hatched) signals from beating hearts.