Next Article in Journal
Mixed Culture Fermentation and Media Optimization by Response Surface Model: Streptomyces and Brachybacterium Species in Bioflocculant Production
Next Article in Special Issue
Secondary Metabolite Localization by Autofluorescence in Living Plant Cells
Previous Article in Journal
Activity of Antifungal Organobismuth(III) Compounds Derived from Alkyl Aryl Ketones against S. cerevisiae: Comparison with a Heterocyclic Bismuth Scaffold Consisting of a Diphenyl Sulfone
Previous Article in Special Issue
Fluorescent Lectins for Local in Vivo Visualization of Peripheral Nerves
Article Menu

Export Article

Open AccessArticle
Molecules 2014, 19(8), 11096-11130;

Photobleaching Kinetics and Time-Integrated Emission of Fluorescent Probes in Cellular Membranes

Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark
Biomedical Imaging Group, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
Author to whom correspondence should be addressed.
Received: 10 April 2014 / Revised: 4 July 2014 / Accepted: 10 July 2014 / Published: 29 July 2014
(This article belongs to the Special Issue Fluorescent Probes)
Full-Text   |   PDF [3686 KB, uploaded 29 July 2014]   |  


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. View Full-Text
Keywords: photophysics; kinetics; autofluorescence correction; cholesterol; BODIPY; transport photophysics; kinetics; autofluorescence correction; cholesterol; BODIPY; transport

Figure 1

This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

Supplementary material


Share & Cite This Article

MDPI and ACS Style

Wüstner, D.; Christensen, T.; Solanko, L.M.; Sage, D. Photobleaching Kinetics and Time-Integrated Emission of Fluorescent Probes in Cellular Membranes. Molecules 2014, 19, 11096-11130.

Show more citation formats Show less citations formats

Related Articles

Article Metrics

Article Access Statistics



[Return to top]
Molecules EISSN 1420-3049 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top