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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 <1 mm). Green and red emissions produced upright
and inverted action potentials, respectively. Ratios of green emission
to red emission followed action potential contours and exhibited larger fractional changes than either emission alone (P < 0.001). The largest changes and signal-to-noise ratio (signal/noise)
were obtained with numerator wavelengths of 525-550 nm and
denominator wavelengths of 650-700 nm. Ratiometry lessened drift
56-66% (P < 0.015) and indicated decreases in
resting membrane potential. Ratiometry lessened motion
artifacts and increased magnitudes of deflections representing
phase-zero depolarizations relative to total deflections by
123-188% in intact hearts (P < 0.02). Durations
of action potentials at different pacing rates, temperatures, and
potassium concentrations were independent of whether they were measured
ratiometrically or with microelectrodes (P 
0.65). The
ratiometric calibration slope was 0.017/100 mV and decreased with time.
Thus emission ratiometry lessens the effects of motion and drift and
indicates resting membrane potential changes and repolarization.
potentiometric dye; Pyridinium, 4-(2-(6-(dibutylamino)-2-naphthalenyl) ethenyl)-1-(3-sulfopropyl)-, hydroxide, inner salt; potassium; action potential
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