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Related Experiment Videos

Quantitative two-photon Ca2+ imaging via fluorescence lifetime analysis.

Christian D Wilms1, Hartmut Schmidt, Jens Eilers

  • 1Leipzig University, Carl-Ludwig-Institute for Physiology, Liebigstr. 27, 04103 Leipzig, Germany. christian.wilms@medizin.uni-leipzig.de

Cell Calcium
|May 13, 2006
PubMed
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This study introduces time-correlated fluorescence lifetime imaging (tcFLIM) for accurate calcium (Ca2+) signal quantification in two-photon microscopy (TPM). This method enables precise measurements in live tissues, overcoming a key limitation of TPM.

Area of Science:

  • Neuroscience
  • Biophysics
  • Optical Imaging

Background:

  • Two-photon microscopy (TPM) enables deep-tissue imaging in vivo but lacks accurate calcium (Ca2+) signal quantification.
  • Quantification of Ca2+ signals is crucial for understanding cellular function and neural activity.

Purpose of the Study:

  • To develop and validate a ratiometric method for quantitative Ca2+ imaging using TPM.
  • To adapt time-correlated fluorescence lifetime imaging (tcFLIM) for Ca2+ signal measurement in TPM.

Main Methods:

  • Utilized time-correlated fluorescence lifetime imaging (tcFLIM) with the Ca2+-indicator dye Oregon Green BAPTA-1 (OGB-1).
  • Recorded fluorescence lifetime of OGB-1 using TPM's ~80 MHz excitation pulses.
  • Performed pixel-wise lifetime recordings via a laser-scanning microscope for quantitative imaging.

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Main Results:

  • Demonstrated Ca2+ dependence of OGB-1's fluorescence lifetime.
  • Achieved quantitative Ca2+ imaging in both full-frame and linescan modes.
  • Validated tcFLIM as a ratiometric quantification method for Ca2+ signals in TPM.

Conclusions:

  • tcFLIM provides an accurate and straightforward method for quantifying Ca2+ signals in TPM.
  • The developed tcFLIM approach is applicable to various Ca2+ dyes and fluorescence indicators.
  • This technique enhances the utility of TPM for studying biological processes requiring precise Ca2+ measurements.