Journal of Fluorescence

, Volume 12, Issue 2, pp 139–145 | Cite as

Measurement of Cell Volume Changes by Fluorescence Self-Quenching

  • Steffen HamannEmail author
  • Jens Folke Kiilgaard
  • Thomas Litman
  • Francisco J. Alvarez-Leefmans
  • Benny R. Winther
  • Thomas Zeuthen


At high concentrations, certain fluorophores undergo self-quenching, i.e., fluorescence intensity decreases with increasing fluorophore concentration. Accordingly, the self-quenching properties can be used for measuring water volume changes in lipid vesicles. In cells, quantitative determination of water transport using fluorescence self-quenching has been complicated by the requirement of relatively high (mM) and often toxic loading concentrations. Here we report a simple method that uses low (μM) loading concentrations of calcein-acetoxymethyl ester (calcein-AM) to obtain intracellular concentrations of the fluorophore calcein suitable for measurement of changes in cell water volume by self-quenching. The relationship between calcein fluorescence intensity, when excited at 490 nm (its excitation maximum), and calcein concentration was investigated in vitro and in various cultured cell types. The relationship was bell-shaped, with the negative slope in the concentration range where the fluorophore undergoes fluorescence self-quenching. In cultured retinal pigment epithelial cells, calcein fluorescence and extracellular osmolarity were linearly related. A 25-mOsm hypertonic challenge corresponded to a decrease in calcein fluorescence with high signal-to-noise ratio (>15). Similar results were obtained with the fluorophore BCECF when excited at its isosbestic wavelength (436 nm). The present results demonstrate the usefulness of fluorescence self-quenching to measure rapid changes in cell water volume.

Water transport cell volume calcein self-quenching epithelial cells 


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  1. 1.
    B. Walter (1888) Ann. Phys. (Leipzig) 34, 316–326.Google Scholar
  2. 2.
    B. Walter (1888) Ann. Phys. (Leipzig) 34, 502–517.Google Scholar
  3. 3.
    B. Walter (1888) Ann. Phys. (Leipzig) 34, 518–533.Google Scholar
  4. 4.
    R. F. Chen and J. R. Knutson (1988) Anal. Biochem. 172, 61–77.Google Scholar
  5. 5.
    D. A. Kendall and R. C. MacDonald (1983) Anal. Biochem. 134, 26–33.Google Scholar
  6. 6.
    P. Y. Chen, D. Pearce, and A. S. Verkman (1988) Biochemistry 27, 5713–5718.Google Scholar
  7. 7.
    R. Ye, L.-B. Shi, W. I. Lencer, and A. S. Verkman (1989) J. Gen. Physiol. 93, 885–902.Google Scholar
  8. 8.
    Y. X. Wang, L. B. Shi, and A. S. Verkman (1991) Biochemistry 30, 2888–2894.Google Scholar
  9. 9.
    S. Jayaraman, Y. Song, and A. S. Verkman (2001) J. Gen. Physiol. 117, 423–430.Google Scholar
  10. 10.
    A. S. Verkman (2000) J. Membr. Biol. 173, 73–87.Google Scholar
  11. 11.
    F. J. Alvarez-Leefmans, J. Altamirano, and W. E. Crowe (1995) Meth. Neurosci. 27, 361–391.Google Scholar
  12. 12.
    W. E. Crowe, J. Altamirano, L. Huerto, and F. J. Alvarez-Leefmans (1995) Neuroscience 69, 283–296.Google Scholar
  13. 13.
    J. Farinas, M. Kneen, M. Moore, and A. S. Verkman (1997) J. Gen. Physiol. 110, 283–296.Google Scholar
  14. 14.
    F. Wehner, H. Sauer, and R. K. Kinne (1995) J. Gen. Physiol. 105, 507–535.Google Scholar
  15. 15.
    F. Wehner and H. Tinel (2000) Pflugers Arch. 441, 12–24.Google Scholar
  16. 16.
    F. Wehner and H. Tinel (1998) J. Physiol. 503 (Pt 1), 127–142.Google Scholar
  17. 17.
    R. Greger, D. Heitzmann, M. J. Hug, E. K. Hoffman, and M. Bleich (1999) Pflugers Arch. 438, 165–176.Google Scholar
  18. 18.
    X. M. Wang, P. I. Terasaki, G.W. Rankin, Jr., D. Chia, H. P. Zhong, and S. Hardy (1993) Hum. Immunol. 37, 264–270.Google Scholar
  19. 19.
    S. Muallem, B.-X. Zhang, P. A. Loessberg, and R. A. Star (1992) J. Biol. Chem. 267, 17658–17664.Google Scholar
  20. 20.
    S. P. Srinivas and J. A. Bonanno (1997) Am. J. Physiol. 272, C1405–C1414.Google Scholar

Copyright information

© Plenum Publishing Corporation 2002

Authors and Affiliations

  • Steffen Hamann
    • 1
    Email author
  • Jens Folke Kiilgaard
    • 2
  • Thomas Litman
    • 3
  • Francisco J. Alvarez-Leefmans
    • 4
    • 5
  • Benny R. Winther
    • 6
  • Thomas Zeuthen
    • 3
  1. 1.Department of Medical Physiology, The Panum InstituteUniversity of CopenhagenCopenhagen NDenmark
  2. 2.Eye Pathology InstituteUniversity of CopenhagenCopenhagen ØDenmark
  3. 3.Department of Medical Physiology, The Panum InstituteUniversity of CopenhagenCopenhagen NDenmark
  4. 4.Departmento de Farmacobiologia, Centro de Investigación y de Estudios AvanzadosInstituto Politécnico NacionalMéxico
  5. 5.Departemento de NeurobiologiaInstituto Nacional de PsiquiatriaMéxicoMéxico
  6. 6.Department of Clinical BiochemistryGlostrup HospitalGlostrupDenmark

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