Ratiometry of transmembrane voltage-sensitive fluorescent dye emission in hearts

Department of Biomedical Engineering of the School of Engineering, The University of Alabama at Birmingham, Alabama 35294-0019 Transmembrane voltage-sensitive fluorescence measurements are limited by baseline drift that can obscure changes in resting membrane potential and by motion artifacts that can obscure repolarization. Voltage-dependent shift of emission wavelengths may allow reduction of drift and motion artifacts by emission ratiometry. We have tested this for action potentials and potassium-induced changes in resting membrane potential in rabbit hearts stained with di-4-ANEPPS [Pyridinium, 4-(2-(6-(dibutylamino)-2-naphthalenyl) ethenyl)-1-(3-sulfopropyl)-, hydroxide, inner salt] using laser excitation (488 nm) and a two-photomultiplier tube system or spectrofluorometer (resolution of 500-1,000 Hz and