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Single-molecule fluorescence quantification with a photobleached internal standard.

Jennifer C Gadd1, Bryant S Fujimoto, Sandra M Bajjalieh

  • 1Department of Chemistry, University of Washington, Seattle, Washington 98195-1700, USA.

Analytical Chemistry
|December 6, 2012
PubMed
Summary

This study introduces photobleaching to accurately quantify single molecules in cellular biology. This method overcomes environmental variations affecting fluorescence, enabling precise protein counting in organelles.

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Area of Science:

  • Cellular and Molecular Biology
  • Biophysics
  • Biochemistry

Background:

  • Fluorophores are crucial for tracking and quantifying molecules in cellular processes.
  • Accurate single-molecule quantification is essential for understanding protein variations in subcellular organelles.
  • Environmental factors can significantly alter fluorophore intensity, complicating quantification.

Purpose of the Study:

  • To develop a method for accurate single-molecule quantification that mitigates environmental effects on fluorescence.
  • To establish a direct calibration method using photobleaching for improved protein counting.
  • To validate the technique by quantifying proteins in both environmentally insensitive and sensitive fluorophore systems.

Main Methods:

  • Utilized photobleaching to generate a direct, in-sample calibration intensity distribution.

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  • Applied the photobleaching calibration to single-molecule quantification of proteins.
  • Quantified goat antimouse IgG antibody labeled with Alexa Fluor 488.
  • Quantified SynaptopHluorin in vesicles.

    • Main Results:

    • Photobleaching successfully extracted an accurate single-molecule calibration intensity distribution.
    • Goat antimouse IgG antibody with Alexa Fluor 488 averaged 4.6 single fluorophore equivalents.
    • SynaptopHluorin vesicles averaged 4.4 single green fluorescent proteins per vesicle.

    Conclusions:

    • Photobleaching provides a robust method to overcome environmental influences in single-molecule fluorescence quantification.
    • The technique enables precise counting of proteins within subcellular organelles.
    • This approach enhances the reliability of sensitive molecular biology quantification techniques.