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Related Experiment Videos

Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy

R Sanders1, A Draaijer, H C Gerritsen

  • 1Department of Molecular Biophysics, Debye Institute, University of Utrecht, Buys Ballot Laboratory, The Netherlands.

Analytical Biochemistry
|May 20, 1995
PubMed
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Fluorescence lifetime imaging offers a more convenient method for pH determination compared to ratiometric imaging. This technique simplifies calibration, making quantitative pH imaging in cells more accessible.

Area of Science:

  • Biomedical imaging
  • Analytical chemistry
  • Cell biology

Background:

  • Carboxy SNAFL-1 is a pH-sensitive probe used for cellular imaging.
  • Ratiometric imaging with carboxy SNAFL-1 requires complex calibration for quantitative pH measurement in cells.
  • Fluorescence lifetime imaging (FLIM) offers an alternative approach for pH determination.

Purpose of the Study:

  • To compare the convenience and accuracy of ratiometric imaging versus fluorescence lifetime imaging for pH determination using carboxy SNAFL-1.
  • To investigate the influence of various factors on carboxy SNAFL-1's emission ratio and fluorescence lifetime.
  • To establish FLIM as a more practical method for quantitative intracellular pH imaging.

Main Methods:

  • Comparative study of ratiometric and fluorescence lifetime imaging techniques.

Related Experiment Videos

  • Controlled experiments varying probe concentration, protein concentration, and hydrophobicity.
  • Analysis of carboxy SNAFL-1 behavior in damaged and living cells.
  • Measurement of emission ratio and fluorescence lifetime under different conditions.

    • Main Results:

    • Quantitative pH imaging using the ratiometric method necessitates a time-consuming calibration procedure.
    • Fluorescence lifetime imaging requires straightforward calibration in buffer solutions.
    • Factors such as probe concentration, protein presence, and cell integrity affect carboxy SNAFL-1 signals.
    • FLIM demonstrated greater convenience and practicality for pH determination.

    Conclusions:

    • Fluorescence lifetime imaging is a more user-friendly and efficient technique for quantitative pH determination with carboxy SNAFL-1.
    • FLIM circumvents the complex calibration issues associated with the ratiometric method for intracellular pH imaging.
    • The findings support the adoption of FLIM for routine and accurate pH measurements in biological samples.