Fluorescence lifetime microscopy of the Na+ indicator Sodium Green in HeLacells

This study investigates the usefulness of lifetime measurements of Sodium G reen for evaluating intracellular Na+ concentration ([Na+](i)) in HeLa cell s, Frequency-domain lifetime measurements are performed in HeLa cells and i n different buffer solutions (with and without K+ and bovine serum albumin) . In all cases, the fluorescence decays of Sodium Green are multiexponentia l, with decay times independent of [Na+], Three relaxation times are found in the various buffer solutions. Binding of the indicator to albumin result s in an increase in the long and intermediate decay times. For Sodium Green inside HeLa cells, the intensity decay can be approximated by a biexponent ial, The ratio of the fractional intensity of the long decay time (tau(2) = 2.4 +/- 0.2 ns) to that of the short component (tau(1) = 0.4 +/- 0.1 ns) i ncreases with [Na+](i). The changes in fluorescence decay with [Na+] are si gnificantly less pronounced in cells as compared with the buffer solutions, Similar values for the resting [Na+](i) were estimated from lifetime measu rements of Sodium Green and from ratiometric measurements using SBFI, Alter natively, [Na+](i) can be monitored by measuring only the phase angle at th e modulation frequency of 160 MHz, The usefulness of this latter approach i s demonstrated by following the changes in [Na+](i) induced by reversible i nhibition of the Na+/K+ pump. (C) 2000 Academic Press.