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Measuring, in Solution, Multiple-Fluorophore Labeling by Combining Fluorescence Correlation Spectroscopy and Photobleaching

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Laboratoire de Spectrométrie Physique UMR 5588, Université de Grenoble I/CNRS, BP 87, 38402 Saint Martin d’Hères, France, and Laboratoire de Physiologie Cellulaire et Végétale UMR 5168, Université de Grenoble I/CNRS, BP 53, 38041 Saint Martin d’Hères, France
* To whom correspondence should be addressed. E-mail: [email protected]
†Laboratoire de Spectrométrie Physique UMR 5588, Université de Grenoble I/CNRS.
‡Laboratoire de Physiologie Cellulaire et Végétale UMR 5168, Université de Grenoble I/CNRS.
§Present address: Equipe13, Université de Grenoble I/IAB, 38706 La Tronche Cedex, France.
∥Present address: LaSIM UMR 5579, Université de Lyon I/CNRS, 69622 Villeurbanne Cedex, France.
Cite this: J. Phys. Chem. B 2010, 114, 8, 2988–2996
Publication Date (Web):February 9, 2010
https://doi.org/10.1021/jp910082h
Copyright © 2010 American Chemical Society

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    Abstract

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    Determining the number of fluorescent entities that are coupled to a given molecule (DNA, protein, etc.) is a key point of numerous biological studies, especially those based on a single molecule approach. Reliable methods are important, in this context, not only to characterize the labeling process but also to quantify interactions, for instance within molecular complexes. We combined fluorescence correlation spectroscopy (FCS) and photobleaching experiments to measure the effective number of molecules and the molecular brightness as a function of the total fluorescence count rate on solutions of cDNA (containing a few percent of C bases labeled with Alexa Fluor 647). Here, photobleaching is used as a control parameter to vary the experimental outputs (brightness and number of molecules). Assuming a Poissonian distribution of the number of fluorescent labels per cDNA, the FCS-photobleaching data could be easily fit to yield the mean number of fluorescent labels per cDNA strand (≅2). This number could not be determined solely on the basis of the cDNA brightness, because of both the statistical distribution of the number of fluorescent labels and their unknown brightness when incorporated in cDNA. The statistical distribution of the number of fluorophores labeling cDNA was confirmed by analyzing the photon count distribution (with the cumulant method), which showed clearly that the brightness of cDNA strands varies from one molecule to the other. We also performed complementary continuous photobleaching experiments and found that the photobleaching decay rate of Alexa Fluor 647 in the excited state decreases by about 30% when incorporated into cDNA, while its nonradiative decay rate is increased such that the brightness of individual Alexa labels is decreased by 25% compared to free Alexa dyes.

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    Cited By

    This article is cited by 6 publications.

    1. Julius Sefkow-Werner, Elisa Migliorini, Catherine Picart, Dwiria Wahyuni, Irène Wang, Antoine Delon. Combining Fluorescence Fluctuations and Photobleaching to Quantify Surface Density. Analytical Chemistry 2022, 94 (17) , 6521-6528. https://doi.org/10.1021/acs.analchem.1c05513
    2. Ágnes Batta, Tímea Hajdu, Peter Nagy. Improved estimation of the ratio of detection efficiencies of excited acceptors and donors for FRET measurements. Cytometry Part A 2023, 103 (7) , 563-574. https://doi.org/10.1002/cyto.a.24728
    3. D Wahyuni, M Balland, O Destaing, I Wang, A Delon. Measuring protein surface density on glass substrate using fluorescence fluctuation spectroscopy. Journal of Physics: Conference Series 2020, 1572 (1) , 012044. https://doi.org/10.1088/1742-6596/1572/1/012044
    4. Andreas Nagl, Simon Robert Hemelaar, Romana Schirhagl. Improving surface and defect center chemistry of fluorescent nanodiamonds for imaging purposes—a review. Analytical and Bioanalytical Chemistry 2015, 407 (25) , 7521-7536. https://doi.org/10.1007/s00216-015-8849-1
    5. , , , , , Richard De Mets, Irène Wang, Joseph Gallagher, Olivier Destaing, Martial Balland, Antoine Delon. Determination of protein concentration on substrates using fluorescence fluctuation microscopy. 2014, 895007. https://doi.org/10.1117/12.2040355
    6. Xin Su, Xianjin Xiao, Chen Zhang, Meiping Zhao. Nucleic Acid Fluorescent Probes for Biological Sensing. Applied Spectroscopy 2012, 66 (11) , 1249-1261. https://doi.org/10.1366/12-06803

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