title: Multi-target spectrally resolved fluorescence lifetime imaging microscopy.
abstract: We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).
authors.affiliation: Department of Biotechnology &Biophysics, Julius Maximilian University of Würzburg, Würzburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Niehörster
authors.affiliation: Department of Biotechnology &Biophysics, Julius Maximilian University of Würzburg, Würzburg, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Löschberger
authors.affiliation: Drittes Physikalisches Institut, Georg-August-Universität, Göttingen, Germany.
authors.forename: Ingo
authors.identifier: http://orcid.org/0000-0002-1775-2159
authors.initials: I
authors.lastname: Gregor
authors.affiliation: PicoQuant GmbH, Berlin, Germany.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Krämer
authors.affiliation: PicoQuant GmbH, Berlin, Germany.
authors.forename: Hans-Jürgen
authors.initials: HJ
authors.lastname: Rahn
authors.affiliation: PicoQuant GmbH, Berlin, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Patting
authors.affiliation: PicoQuant GmbH, Berlin, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Koberling
authors.affiliation: Drittes Physikalisches Institut, Georg-August-Universität, Göttingen, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Enderlein
authors.affiliation: Department of Biotechnology &Biophysics, Julius Maximilian University of Würzburg, Würzburg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Sauer
mesh_terms: D000465:Algorithms; D000818:Animals; D006801:Humans; D007089:Image Enhancement / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D010363:Pattern Recognition, Automated / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity