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Elimination of autofluorescence in fluorescence correlation spectroscopy using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time-correlated single-photon counting (TCSPC)

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Abstract

Fluorescence correlation spectroscopy (FCS) is a frequently applied technique that allows for the precise and sensitive analysis of molecular diffusion and interactions. However, the potential of FCS for in vitro or ex vivo studies has not been fully realized due in part to artifacts originating from autofluorescence (fluorescence of inherent components and fixative-induced fluorescence). Here, we propose the azadioxatriangulenium (ADOTA) dye as a solution to this problem. The lifetime of the ADOTA probe, about 19.4 ns, is much longer than most components of autofluorescence. Thus, it can be easily separated by time-correlated single-photon counting methods. Here, we demonstrate the suppression of autofluorescence in FCS using ADOTA-labeled hyaluronan macromolecules (HAs) with Rhodamine 123 added to simulate diffusing fluorescent background components. The emission spectrum and decay rate of Rhodamine 123 overlap with the usual sources of autofluorescence, and its diffusion behavior is well known. We show that the contributions from Rhodamine 123 can be eliminated by time gating or by fluorescence lifetime correlation spectroscopy (FLCS). While the pairing of ADOTA and time gating is an effective strategy for the removal of autofluorescence from fluorescence imaging, the loss of photons leads to erroneous concentration values with FCS. On the other hand, FLCS eliminates autofluorescence without such errors. We then show that both time gating and FLCS may be used successfully with ADOTA-labeled HA to detect the presence of hyaluronidase, the overexpression of which has been observed in many types of cancer.

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Acknowledgments

This work was supported by NIH grants R01EB12003 (Z.G.) and 1RO1HL090786-01A2 (J.B.) and the Danish Council for Independent Research, Technology and Production Sciences grant 10-093546 (T.J.S.). The authors would like to thank Dr. Badri Maliwal for helping with the preparation of ADOTA-NHS.

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Authors and Affiliations

  1. Department of Molecular Biology and Immunology, Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Fort Worth, TX, 76107, USA

    Ryan M. Rich, Zygmunt Gryczynski, Julian Borejdo, Zeno Foldes-Papp, Ignacy Gryczynski & Rafal Fudala

  2. Department of Psychiatry and Behavioral Health, University of North Texas Health Science Center, Fort Worth, TX, 76107, USA

    Mark Mummert

  3. Department of Physics & Astronomy, Texas Christian University, Fort Worth, TX, 76129, USA

    Zygmunt Gryczynski

  4. Department of Cell Biology and Anatomy, University of North Texas Health Science Center, Fort Worth, TX, 76107, USA

    Ignacy Gryczynski

  5. Nano-Science Center & Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100, Copenhagen Ø, Denmark

    Thomas Just Sørensen & Bo W. Laursen

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  1. Ryan M. Rich
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Correspondence to Rafal Fudala.

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Rich, R.M., Mummert, M., Gryczynski, Z. et al. Elimination of autofluorescence in fluorescence correlation spectroscopy using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time-correlated single-photon counting (TCSPC). Anal Bioanal Chem 405, 4887–4894 (2013). https://doi.org/10.1007/s00216-013-6879-0

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