Isosbestic Point in Optical Mapping; Theoretical and Experimental Determination with Di-4-ANBDQPQ Transmembrane Voltage Sensitive Dye

Optical mapping methods utilize fluorescence dyes to measure dynamic response of cardiac tissue such as changes in transmembrane potential (V m ). For the commonly used V m sensitive dyes, a dye absorption and emission spectra shift as V m changes. Signals relevant to V m are calculated as a relative fluorescence change with respect to the fluorescence baseline. The amplitude of the change depends on the long-pass (LP) filter cut-on wavelength, placed on the sensor side, and the excitation wavelength. An excitation wavelength near the absorption peak, termed the isosbestic point, results in minimal absorption coefficient change as absorption spectra shifts. Consequentially the fluorescence intensity virtually does not change, when fluorescence across the entire emission spectra is measured, irrelevant of V m changes. In this study we experimentally determined the isosbestic point for a near infrared dye Di-4-ANBDQPQ. We then present a theoretical study examining the dye linear or non-linear response as the fractional fluorescence change of V m change, due to emission spectra shift and amplitude change, over a range of excitation wavelengths and LP filters. Linear "optical" response is important to quantify certain aspects of cardiac dynamics such as the action potential (AP) shape and duration, especially when studying drug effects and dynamical substrates for arrhythmia development.