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Title: Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement

Abstract

Fluorescence correlation spectroscopy (FCS) is an important technique for studying biochemical interactions dynamically that may be used in vitro and in cell-based studies. It is generally claimed that FCS may only be used at nM concentrations. We show that this general consensus is incorrect and that the limitation to nM concentrations is not fundamental but due to detector limits as well as laser fluctuations. With a high count rate detector system and applying laser fluctuation corrections, we demonstrate FCS measurements up to 38 μM with the same signal-to-noise as at lower concentrations. Optical nanoconfinement approaches previously used to increase the concentration range of FCS are not necessary, and further increases above 38 μM may be expected using detectors and detector arrays with higher saturation rates and better laser fluctuation corrections. This approach greatly widens the possibilities of dynamic measurements of biochemical interactions using FCS at physiological concentrations.

Authors:
 [1];  [1];  [1];  [1];  [2]
+ Show Author Affiliations
  1. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
  2. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Univ. of California, Davis, CA (United States). Dept. of Radiation Oncology
Publication Date:
Research Org.:
Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1169272
Report Number(s):
LLNL-JRNL-653521
Journal ID: ISSN 1520-6106
Grant/Contract Number:  
AC52-07NA27344; 11-ERD-037
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Physical Chemistry. B, Condensed Matter, Materials, Surfaces, Interfaces and Biophysical Chemistry
Additional Journal Information:
Journal Volume: 118; Journal Issue: 32; Journal ID: ISSN 1520-6106
Publisher:
American Chemical Society
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; Fluorescence correlation spectroscopy (FCS)

Citation Formats

Laurence, Ted A., Ly, Sonny, Bourguet, Feliza, Fischer, Nicholas O., and Coleman, Matthew A. Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement. United States: N. p., 2014. Web. doi:10.1021/jp505881z.
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Laurence, Ted A., Ly, Sonny, Bourguet, Feliza, Fischer, Nicholas O., & Coleman, Matthew A. Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement. United States. https://doi.org/10.1021/jp505881z
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Laurence, Ted A., Ly, Sonny, Bourguet, Feliza, Fischer, Nicholas O., and Coleman, Matthew A. Thu . "Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement". United States. https://doi.org/10.1021/jp505881z. https://www.osti.gov/servlets/purl/1169272.
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@article{osti_1169272,
title = {Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement},
author = {Laurence, Ted A. and Ly, Sonny and Bourguet, Feliza and Fischer, Nicholas O. and Coleman, Matthew A.},
abstractNote = {Fluorescence correlation spectroscopy (FCS) is an important technique for studying biochemical interactions dynamically that may be used in vitro and in cell-based studies. It is generally claimed that FCS may only be used at nM concentrations. We show that this general consensus is incorrect and that the limitation to nM concentrations is not fundamental but due to detector limits as well as laser fluctuations. With a high count rate detector system and applying laser fluctuation corrections, we demonstrate FCS measurements up to 38 μM with the same signal-to-noise as at lower concentrations. Optical nanoconfinement approaches previously used to increase the concentration range of FCS are not necessary, and further increases above 38 μM may be expected using detectors and detector arrays with higher saturation rates and better laser fluctuation corrections. This approach greatly widens the possibilities of dynamic measurements of biochemical interactions using FCS at physiological concentrations.},
doi = {10.1021/jp505881z},
journal = {Journal of Physical Chemistry. B, Condensed Matter, Materials, Surfaces, Interfaces and Biophysical Chemistry},
number = 32,
volume = 118,
place = {United States},
year = {Thu Aug 14 00:00:00 EDT 2014},
month = {Thu Aug 14 00:00:00 EDT 2014}
}
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Publisher's Version of Record
https://doi.org/10.1021/jp505881z
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    Works referencing / citing this record:

    State-of-the-Art Fluorescence Fluctuation-Based Spectroscopic Techniques for the Study of Protein Aggregation
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    • Kitamura, Akira; Kinjo, Masataka
    • International Journal of Molecular Sciences, Vol. 19, Issue 4
    • DOI: 10.3390/ijms19040964

    High photon count rates improve the quality of super-resolution fluorescence fluctuation spectroscopy
    posted_content, March 2019

    • Schneider, Falk; Hernandez-Varas, Pablo; Lagerholm, B. Christoffer
    • bioRxiv
    • DOI: 10.1101/783639

    Correction to “Strong Plasmon Enhancement of the Saturation Photon Count Rate of Single Molecules”
    journal, April 2020

    • Wang, Yuyang; Horáček, Matěj; Zijlstra, Peter
    • The Journal of Physical Chemistry Letters, Vol. 11, Issue 9
    • DOI: 10.1021/acs.jpclett.0c01034
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