Fluorescence Lifetime Microscopy of the Na+ Indicator Sodium Green in HeLa Cells

• Published in: Analytical biochemistry 2000-06, Vol.281 (2), p.159-175
• Main Authors: Despa, Sanda, Vecer, Jaroslav, Steels, Paul, Ameloot, Marcel
• Format: Article
• Language: English
• Subjects: Animals; Buffers; Calibration; Cattle; Fluorescent Dyes - chemistry; HeLa Cells; Humans; Microscopy, Fluorescence; Organic Chemicals; single-frequency measurements; Sodium Green; Solutions; time-resolved fluorescence microscopy
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.2000.4560

This study investigates the usefulness of lifetime measurements of Sodium Green for evaluating intracellular Na+ concentration ([Na+]i) in HeLa cells. Frequency-domain lifetime measurements are performed in HeLa cells and in different buffer solutions (with and without K+ and bovine serum albumin). In all cases, the fluorescence decays of Sodium Green are multiexponential, with decay times independent of [Na+]. Three relaxation times are found in the various buffer solutions. Binding of the indicator to albumin results in an increase in the long and intermediate decay times. For Sodium Green inside HeLa cells, the intensity decay can be approximated by a biexponential. The ratio of the fractional intensity of the long decay time (τ2 = 2.4 ± 0.2 ns) to that of the short component (τ1 = 0.4 ± 0.1 ns) increases with [Na+]i. The changes in fluorescence decay with [Na+] are significantly less pronounced in cells as compared with the buffer solutions. Similar values for the resting [Na+]i were estimated from lifetime measurements of Sodium Green and from ratiometric measurements using SBFI. Alternatively, [Na+]i can be monitored by measuring only the phase angle at the modulation frequency of 160 MHz. The usefulness of this latter approach is demonstrated by following the changes in [Na+]i induced by reversible inhibition of the Na+/K+ pump.