Two-Photon Excitation of di-4-ANEPPS for Optical Recording of Action Potentials in Rabbit Heart

Abstract

Cardiac action potentials have been measured with single-photon excitation (SPE) of transmembrane voltage-sensitive fluorescent dye. Two-photon excitation (TPE) may have advantages for localization and depth of the tissue region from which the action potential is measured. However measurements of action potentials with SPE have not been demonstrated. We sought to develop a method for TPE of di-4-ANEPPS and test whether the method yields voltage-dependent fluorescence in cardiac tissue. We modified our SPE and ratio-metric fluorescence recording system to use a femtosecond pulsed near-infrared laser. Modifications were made to enhance fluorescence collection efficiency and to block infrared laser light from entering the fluorescence collection system. Fluorescence was collected simultaneously in green (510-570 nm) and red (590-700 nm) wavelength bands. Action potentials were observed in the ratio of the green signal to the red signal, but were not observed above the noise level in either of the individual signals. Incorporation of a common-mode noise subtraction method revealed action potentials in green and red signals. We also found that the di-4-ANEPPS fluorescence emission spectrum for TPE at 930 nm was similar to the emission spectrum for SPE at 488 nm. The multiphoton method may be beneficial for highly localized cardiac optical measurements.