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

Ryan M. Rich, Dorota Luiza Stankowska, Badri P. Maliwal, Thomas Just Sørensen, Bo W. Laursen, Raghu Krishnamoorthy, Zygmunt Gryczynski, Julian Borejdo, Ignacy Gryczynski, Rafal Fudala

Research output: Contribution to journalArticle

42 Scopus citations


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.

Original languageEnglish
Pages (from-to)2065-2075
Number of pages11
JournalAnalytical and Bioanalytical Chemistry
Issue number6
StatePublished - 1 Jan 2013



  • Bioanalytical methods
  • Fluorescence
  • Luminescence
  • Spectroscopy instrumentation
  • Spectroscopy theory

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