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Fluorescence correlation spectroscopy at micromolar concentrations without optical nanoconfinement.

Ted A Laurence1, Sonny Ly, Feliza Bourguet

  • 1Lawrence Livermore National Laboratory , Livermore, California 94550, United States.

The Journal of Physical Chemistry. B
|July 26, 2014
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Summary

This study demonstrates that fluorescence correlation spectroscopy (FCS) can measure biochemical interactions at micromolar concentrations, overcoming previous limitations. Enhanced detectors and laser corrections expand FCS applications to physiological levels.

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Area of Science:

  • Biophysics
  • Biochemistry
  • Analytical Chemistry

Background:

  • Fluorescence correlation spectroscopy (FCS) is a powerful technique for dynamic biochemical interaction studies.
  • Current limitations restrict FCS to nanomolar (nM) concentrations, hindering in vivo and physiological studies.
  • Previous methods to extend FCS concentration range, like optical nanoconfinement, are complex.

Purpose of the Study:

  • To challenge the established belief that FCS is limited to nM concentrations.
  • To demonstrate FCS measurements at significantly higher concentrations (micromolar range).
  • To identify and address the key factors limiting FCS concentration range.

Main Methods:

  • Utilized a high count rate detector system.
  • Implemented laser fluctuation correction methods.
  • Performed FCS measurements across a wide concentration range, including micromolar levels.

Main Results:

  • Successfully conducted FCS measurements up to 38 μM with high signal-to-noise ratio.
  • Demonstrated that detector limits and laser fluctuations, not fundamental principles, restrict FCS concentration range.
  • Showed that optical nanoconfinement is unnecessary with advanced detection and correction techniques.

Conclusions:

  • The concentration limit of FCS is not fundamental and can be overcome with improved technology.
  • Advanced detectors and laser stabilization significantly expand the utility of FCS.
  • This breakthrough allows dynamic biochemical interaction studies at physiologically relevant concentrations.