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Related Experiment Video

Updated: May 20, 2026

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes
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Single molecule imaging approach to membrane protein stoichiometry.

Richard Hallworth1, Michael G Nichols

  • 1Department of Biomedical Sciences, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA. hallw@creighton.edu

Microscopy and Microanalysis : the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada
|July 27, 2012
PubMed
Summary

Single molecule imaging reveals the subunit stoichiometry of membrane proteins. This technique, using fluorescent protein tags, analyzes fluorescence steps to determine protein complexes, exemplified by studying prestin in hearing.

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Cell Biology

Background:

  • Single fluorescent molecule imaging allows detailed study of molecular processes.
  • Determining subunit stoichiometry in membrane proteins is crucial for understanding their function.
  • Prestin, a key protein in mammalian hearing, requires accurate stoichiometry assessment.

Purpose of the Study:

  • To review and present a method for determining membrane protein subunit stoichiometry using single fluorescent molecule visualization.
  • To apply this method to investigate the stoichiometry of prestin.

Main Methods:

  • Localization of single fluorescent molecules (coupled to fluorescent proteins) in cell membranes.
  • Continuous low-level excitation to observe discrete fluorescence step decreases (fluorophore bleaching).

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  • Analysis of step counts using a binomial model to deduce stoichiometry.

    • Main Results:

    • Demonstrated direct observation of single fluorophore bleaching events.
    • Successfully applied the method to determine the stoichiometry of prestin.
    • Provided a detailed account of sample preparation, identification, and imaging of single prestin molecules.

    Conclusions:

    • Single molecule fluorescence imaging provides a robust method for determining membrane protein stoichiometry.
    • The approach offers valuable insights into the functional complexes of proteins like prestin.
    • Methodological considerations and comparisons with other techniques are discussed.