title: Multiple-pulse pumping for enhanced fluorescence detection and molecular imaging in tissue.
abstract: Applications of fluorescence based imaging techniques for detection in cellular and tissue environments are severely limited by autofluorescence of endogenous components of cells, tissue, and the fixatives used in sample processing. To achieve sufficient signal-to-background ratio, a high concentration of the probe needs to be used which is not always feasible. Since typically autofluorescence is in the nanosecond range, long-lived fluorescence probes in combination with time-gated detection can be used for suppression of unwanted autofluorescence. Unfortunately, this requires the sacrifice of the large portion the probe signal in order to sufficiently filter the background. We report a simple and practical approach to achieve a many-fold increase in the intensity of a long-lived probe without increasing the background fluorescence. Using controllable, well separated bursts of closely spaced laser excitation pulses, we are able to highly increase the fluorescence signal of a long-lived marker over the endogenous fluorescent background and scattering, thereby greatly increasing detection sensitivity. Using a commercially available confocal microscopy system equipped with a laser diode and time correlated single photon counting (TCSPC) detection, we are able to enhance the signal of a long-lived Ruthenium (Ru)-based probe by nearly an order of magnitude. We used 80 MHz bursts of pulses (12.5 ns pulse separation) repeated with a 320 kHz repetition rate as needed to adequately image a dye with a 380 ns lifetime. Just using 10 pulses in the burst increases the Ru signal almost 10-fold without any increase in the background signal.
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.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; Department of Cell Biology and Anatomy, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
authors.forename: Ignacy
authors.initials: I
authors.lastname: Gryczynski
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: Rafal
authors.initials: R
authors.lastname: Fudala
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: Julian
authors.initials: J
authors.lastname: Borejdo
authors.affiliation: Department of Cell Biology and Anatomy, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
authors.forename: Dorota L
authors.initials: DL
authors.lastname: Stankowska
authors.affiliation: Department of Cell Biology and Anatomy, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
authors.forename: Raghu R
authors.initials: RR
authors.lastname: Krishnamoorthy
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: Sangram
authors.initials: S
authors.lastname: Raut
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: Badri P
authors.initials: BP
authors.lastname: Maliwal
authors.affiliation: Department of Physics and Astronomy, Texas Christian University, Fort Worth, TX 76129, USA.
authors.forename: Dmytro
authors.initials: D
authors.lastname: Shumilov
authors.affiliation: Department of Physics and Astronomy, Texas Christian University, Fort Worth, TX 76129, USA.
authors.forename: Hung
authors.initials: H
authors.lastname: Doan
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; Department of Physics and Astronomy, Texas Christian University, Fort Worth, TX 76129, USA. Electronic address: zgryczynski@tcu.edu.
authors.forename: Zygmunt
authors.initials: Z
authors.lastname: Gryczynski
mesh_terms: D000818:Animals; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D061848:Optical Imaging / Q000379:methods*; D051381:Rats; D012165:Retinal Ganglion Cells / Q000378:metabolism; D059629:Signal-To-Noise Ratio; D014404:Tubulin / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D014404:Tubulin
keywords: Confocal microscopy; Fluorescence imaging; High contrast imagine; Long-lived probe; Multi-pulse excitation; TCSPC; Time-resolved fluorescence