title: Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore.
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.
authors.affiliation: Department of Molecular Biology and Immunology, Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Rich
authors.forename: Dorota L
authors.initials: DL
authors.lastname: Stankowska
authors.forename: Badri P
authors.initials: BP
authors.lastname: Maliwal
authors.forename: Thomas Just
authors.initials: TJ
authors.lastname: Sørensen
authors.forename: Bo W
authors.initials: BW
authors.lastname: Laursen
authors.forename: Raghu R
authors.initials: RR
authors.lastname: Krishnamoorthy
authors.forename: Zygmunt
authors.initials: Z
authors.lastname: Gryczynski
authors.forename: Julian
authors.initials: J
authors.lastname: Borejdo
authors.forename: Ignacy
authors.initials: I
authors.lastname: Gryczynski
authors.forename: Rafal
authors.initials: R
authors.lastname: Fudala
mesh_terms: D000818:Animals; D016477:Artifacts; D005456:Fluorescent Dyes / Q000032:analysis / Q000138:chemical synthesis*; D006576:Heterocyclic Compounds, 4 or More Rings / Q000032:analysis / Q000138:chemical synthesis*; D008856:Microscopy, Fluorescence; D008867:Microtomy; D057054:Molecular Imaging / Q000379:methods; D009900:Optic Nerve / Q000648:ultrastructure*; D017785:Photons; D051381:Rats; D012160:Retina / Q000648:ultrastructure*; D059629:Signal-To-Noise Ratio; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D006576:Heterocyclic Compounds, 4 or More Rings; C582342:N-(omega-N'-hydroxysuccinimde butanoate)azadioxatriangulenium