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Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore

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Abstract

Sample autofluorescence (fluorescence of inherent components of tissue and fixative-induced fluorescence) is a significant problem in direct imaging of molecular processes in biological samples. A large variety of naturally occurring fluorescent components in tissue results in broad emission that overlaps the emission of typical fluorescent dyes used for tissue labeling. In addition, autofluorescence is characterized by complex fluorescence intensity decay composed of multiple components whose lifetimes range from sub-nanoseconds to a few nanoseconds. For these reasons, the real fluorescence signal of the probe is difficult to separate from the unwanted autofluorescence. Here we present a method for reducing the autofluorescence problem by utilizing an azadioxatriangulenium (ADOTA) dye with a fluorescence lifetime of approximately 15 ns, much longer than those of most of the components of autofluorescence. A probe with such a long lifetime enables us to use time-gated intensity imaging to separate the signal of the targeting dye from the autofluorescence. We have shown experimentally that by discarding photons detected within the first 20 ns of the excitation pulse, the signal-to-background ratio is improved fivefold. This time-gating eliminates over 96 % of autofluorescence. Analysis using a variable time-gate may enable quantitative determination of the bound probe without the contributions from the background.

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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.).

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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, Badri P. Maliwal, Zygmunt Gryczynski, Julian Borejdo, Ignacy Gryczynski & Rafal Fudala

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

    Dorota L. Stankowska, Raghu R. Krishnamoorthy & Ignacy Gryczynski

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

    Thomas Just Sørensen & Bo W. Laursen

  4. Department of Physics and Astronomy, Texas Christian University, Fort Worth, TX, 76129, USA

    Zygmunt Gryczynski

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  1. Ryan M. Rich
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  2. Dorota L. Stankowska
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Correspondence to Ignacy Gryczynski or Rafal Fudala.

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Rich, R.M., Stankowska, D.L., Maliwal, B.P. et al. Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore. Anal Bioanal Chem 405, 2065–2075 (2013). https://doi.org/10.1007/s00216-012-6623-1

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  • DOI: https://doi.org/10.1007/s00216-012-6623-1

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