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Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells
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Referencing techniques for the analog mean-delay method in fluorescence lifetime imaging.

Young Jae Won1, Sucbei Moon, Won-Taek Han

  • 1Department of Information and Communications, Gwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712, South Korea.

Journal of the Optical Society of America. A, Optics, Image Science, and Vision
|November 4, 2010
PubMed
Summary

The analog mean-delay (AMD) method for fluorescence lifetime imaging microscopy has accuracy concerns. New electrical and optical referencing techniques significantly reduce measurement errors, improving absolute lifetime accuracy.

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Area of Science:

  • Optics and Photonics
  • Biophysics
  • Analytical Chemistry

Background:

  • The analog mean-delay (AMD) method is a promising technique for high-speed confocal fluorescence lifetime imaging microscopy.
  • While AMD offers comparable photon economy and precision to time-correlated single photon counting, concerns exist regarding its absolute lifetime accuracy.

Purpose of the Study:

  • To investigate and address the accuracy limitations of the analog mean-delay method in determining absolute fluorescence lifetimes.
  • To develop and validate referencing techniques to mitigate errors in AMD measurements.

Main Methods:

  • The study analyzed the impact of temporal waveform drifts and fluctuations on AMD accuracy.
  • Electrical and optical referencing techniques were proposed and implemented to correct for zero-position errors.
  • Measurements were conducted to quantify lifetime precision with and without referencing.

Main Results:

  • Significant drifts (over 2 ns) were observed in temporal waveforms during initial warm-up periods without referencing.
  • Without referencing, the standard deviation of measured lifetimes was as high as 51 ps.
  • The proposed electronic referencing technique reduced this error to 9 ps, while optical referencing further improved it to 4 ps.

Conclusions:

  • Drifts and fluctuations in the absolute zero position are critical error sources in the analog mean-delay method.
  • Electrical and optical referencing techniques effectively suppress these errors, enhancing the accuracy of absolute fluorescence lifetime measurements.
  • The developed referencing methods significantly improve the reliability of AMD for fluorescence lifetime imaging microscopy.