title: Photobleaching kinetics and time-integrated emission of fluorescent probes in cellular membranes.
abstract: Since the pioneering work of Hirschfeld, it is known that time-integrated emission (TiEm) of a fluorophore is independent of fluorescence quantum yield and illumination intensity. Practical implementation of this important result for determining exact probe distribution in living cells is often hampered by the presence of autofluorescence. Using kinetic modelling of photobleaching combined with pixel-wise bleach rate fitting of decay models with an updated plugin to the ImageJ program, it is shown that the TiEm of a fluorophore in living cells can be determined exactly from the product of bleaching amplitude and time constant. This applies to mono-exponential bleaching from the first excited singlet and/or triplet state and to multi-exponential combinations of such processes. The TiEm can be used to correct for illumination shading and background autofluorescence without the need for fluorescent test layers or separate imaging of non-stained cells. We apply the method to simulated images and to images of cells, whose membranes were labelled with fluorescent sterols and sphingolipids. Our bleaching model can be extended to include a probability density function (PDF) of intrinsic bleach rate constants with a memory kernel. This approach results in a time-dependent bleach rate coefficient and is exemplified for fluorescent sterols in restricted intracellular environments, like lipid droplets. We show that for small deviations from the classical exponential bleaching, the TiEm of decay functions with rate coefficients remains largely independent of fluorescence lifetime and illumination, and thereby represents a faithful measure of probe distribution.
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark. wuestner@bmb.sdu.dk.
authors: Daniel
authors: D
authors: Wüstner
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark. tanjac@bmb.sdu.dk.
authors: Tanja
authors: T
authors: Christensen
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark. lsolanko@gmail.com.
authors: Lukasz M
authors: LM
authors: Solanko
authors: Biomedical Imaging Group, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland. daniel.sage@epfl.ch.
authors: Daniel
authors: D
authors: Sage
mesh_terms: D000465:Algorithms; D002462:Cell Membrane / Q000378:metabolism*; D058948:Cell Tracking; D005347:Fibroblasts / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008962:Models, Theoretical; D038761:Photobleaching; D010777:Photochemistry; D015203:Reproducibility of Results
chemical_list: D005456:Fluorescent Dyes