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A fluctuation method to quantify in vivo fluorescence data.

Nitzan Rosenfeld1, Theodore J Perkins, Uri Alon

  • 1Department of Molecular Biology, Weizmann Institute of Science, Rehovot, Israel.

Biophysical Journal
|May 2, 2006
PubMed
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This study introduces a new method to convert fluorescence intensity into molecule numbers. This technique allows for precise quantification of cellular protein levels, crucial for systems biology research.

Area of Science:

  • Cellular biology
  • Systems biology
  • Biophysics

Background:

  • Quantitative in vivo measurements are vital for predictive models of cellular behavior.
  • Accurate quantification of cellular components is a challenge in systems biology.

Purpose of the Study:

  • To develop a technique for converting observed fluorescence intensities into absolute molecule numbers.
  • To establish a quantitative internal calibration for systems biology studies.

Main Methods:

  • Transiently expressing a fluorescently tagged protein and tracking its dilution during cell growth and division.
  • Utilizing asymmetric partitioning of fluorescence between daughter cells to infer molecule numbers.
  • Applying a Bayesian algorithm to determine the fluorescence conversion factor and measurement error.

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Main Results:

  • Demonstrated that asymmetric partitioning of fluorescence reflects actual protein numbers.
  • Developed a robust Bayesian algorithm capable of handling large datasets and missing measurements.
  • Validated the algorithm through extensive simulations and application to experimental data from Escherichia coli.

Conclusions:

  • The presented technique enables accurate conversion of fluorescence to molecule numbers.
  • This method provides a reliable quantitative internal calibration for systems biology.
  • The approach is applicable to both synthetic and endogenous cellular networks.