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

摘要

We developed a new method for ratiometric optical mapping of transmembrane potential (Vm) in cardiac preparations stained with di-4-ANEPPS (DI-4). The method uses pulsed light emitting diode (LED) excitation. Vm-dependent shifts of excitation and emission spectra establish two excitation bands (481 nm) that produce positive and negative changes in fluorescence within a single emission band (570-615 nm). The ratio of these positive and negative fluorescence signals (excitation ratiometry) increases Vm sensitivity and removes artifacts common to both signals. We pulsed blue (460±20 nm) and cyan (505±20 nm) LEDs at 500 Hz in alternating phase synchronized to a photodetector (1 kHz sampling) equipped with a 585±20 nm band pass emission filter. This produced signals with upright (blue) and inverted (cyan) optical action potentials (OAPs) interleaved in sequential frames. In whole rabbit heart with chemically arrested motion, we measured mean±SD of fractional fluorescence (DF/F) for blue, cyan, and ratio (blue/cyan) signals of 1.4±.21%, -1.1±0.18%, and 2.8±0.54%, respectively. Signal to noise ratios were 6.4±1.6, 4.4±1.2, and 7.5±1.8, respectively. Blue, cyan, and ratio signals were correlated to an averaged representative signal to determine the extent of motion correction by ratio. Correlation coefficients were 0.33±0.57 before ratio and 0.72±0.32 after ratio with motion present. These findings establish the feasibility of using pulsed LEDs for excitation ratiometric optical mapping of Vm in cardiac muscle.