Aims: Transmembrane voltage (Vm) sensitive dyes convert Vm signal changes to fluorescence changes. This study aims to quantify the effect of nonlinearities in optical mapping measurement comparing it to standard microelectrode measurements of the action potential (AP). Nonlinearity impacts are studied on AP duration variations and AP upstroke shape. Further, we aim to show existence of a single excitation wavelength, termed isosbestic point, at which nonlinear factors in optical mapping measurements are minimized.
Methods: Optical mappings were performed on isolated rat and rabbit hearts with Di-4-ANBDQPQ Vm dye with excitation wavelengths ranging from 500 to 660 nm. Fluorescence was collected though long pass (LP) filters ranged from 600 to 775 nm avoiding overlapping with excitation. This enabled a complete assessment of Vm fluorescent signal changes due to two independent variables, emission fluorescence spectral shift and amplitude change. Microelectrode measurements of AP were obtained by standard microelectrode techniques.
Results: Nonlinearities in optical mapping measurement were quantified by second order polynomial fit with microelectrode measurements. Obtained results indicated that nonlinearities in optical mapping could lead to misshaped AP upstroke with either longer initial rise time or prolonged time reaching maximum of the AP and results indicated a single excitation point (isosbestic) with maximal linear correlation with microelectrode measurements. These results were consistent for both rat and rabbit hearts. Nonlinearity in optical mapping did not affect AP duration measurements in rabbit hearts, while in rat hearts different excitation wavelength and LP filters introduced up to 15% variability, which is minimized at isosbestic point.
Conclusions: Optical mapping methods are invaluable tool and variances in measurements of major physiological parameters as conduction velocity and AP duration should not be biased by the method. Deviations can be attributed to optical mapping measurement nonlinearities and can be elevated with Vm dye excitation at isosbestic point.