Fluorescence lifetime microscopy of the Na+ indicator Sodium Green in HeLa cells

Sanda Despa, Jaroslav Vecer, Paul Steels, Marcel Ameloot

Research output: Contribution to journalArticlepeer-review

34 Scopus citations

Abstract

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 and 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. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)159-175
Number of pages17
JournalAnalytical Biochemistry
Volume281
Issue number2
DOIs
StatePublished - Jun 1 2000

Bibliographical note

Funding Information:
This research was supported by the Bilaterale Wetenschappelijke en Technologische Samenwerking tussen Vlaanderen en Roemenië (Project 96/23), the Fonds voor Wetenschappelijk Onderzoek, and the Nationale Loterij. The DWTC is thanked for financial support through UIAP-IV-11. S.D. thanks the Research Fund of the Lim-burgs Universitair Centrum for a fellowship. J.V. was supported by a NATO Collaborative Research grant (No. 971567) and the Research Fund of the Limburgs Universitair Centrum. The authors are grateful to Dr. M. van de Ven for reading the manuscript.

Keywords

  • HeLa cells
  • Single- frequency measurements
  • Sodium Green
  • Time-resolved fluorescence microscopy

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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