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

• Published in: Analytical and bioanalytical chemistry Vol. 405; no. 6; pp. 2065 - 2075
• Main Authors: Rich, Ryan M., Stankowska, Dorota L., Maliwal, Badri P., Sørensen, Thomas Just, Laursen, Bo W., Krishnamoorthy, Raghu R., Gryczynski, Zygmunt, Borejdo, Julian, Gryczynski, Ignacy, Fudala, Rafal
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.02.2013; Springer; Springer Nature B.V
• Subjects: Analytical Chemistry; Animals; Biochemistry; Biological samples; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Dyes; Dyes and dyeing; Emission; Emissions; Fluorescence; Fluorescent Dyes - analysis; Fluorescent Dyes - chemical synthesis; Food Science; Health aspects; Heterocyclic Compounds, 4 or More Rings - analysis; Heterocyclic Compounds, 4 or More Rings - chemical synthesis; Image detection; Imaging; Laboratory Medicine; Lasers; Lifetime; Methods; Microscopy, Fluorescence; Microtomy; Molecular biology; Molecular Imaging - methods; Monitoring/Environmental Analysis; Nanocomposites; Nanomaterials; Nanostructure; Numerical analysis; Optic Nerve - ultrastructure; Original Paper; Photons; Rats; Retina - ultrastructure; Signal-To-Noise Ratio; Spectrometry, Fluorescence - methods; Spectrum analysis; Time Factors; Denmark; Fort Worth Texas; United States > US; Fluorescence; Spectroscopy instrumentation; Bioanalytical methods; Spectroscopy theory; Luminescence
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-012-6623-1

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.