Photobleaching kinetics and time-integrated emission of fluorescent probes in cellular membranes.

Daniel Wüstner, Tanja Christensen, Lukasz Michal Solanko, Daniel Sage

Research output: Contribution to journalJournal articleResearchpeer-review

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.
Original languageEnglish
Article numbermolecules190811096
JournalMolecules
Volume19
Issue number8
Pages (from-to)11096-11130
Number of pages35
ISSN1420-3049
DOIs
Publication statusPublished - 29. Jul 2014

Fingerprint

Photobleaching
Bleaching
Fluorescent Dyes
Membranes
Kinetics
Fluorophores
Lighting
Sterols
Fluorescence
Cells
Sphingolipids
Quantum yield
Cell membranes
Probability density function
Rate constants
Pixels
Lipids
Imaging techniques
Data storage equipment

Keywords

  • photophysics; kinetics; autofluorescence correction; cholesterol; BODIPY; transport

Cite this

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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.",
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Photobleaching kinetics and time-integrated emission of fluorescent probes in cellular membranes. / Wüstner, Daniel; Christensen, Tanja; Solanko, Lukasz Michal; Sage, Daniel.

In: Molecules, Vol. 19, No. 8, molecules190811096, 29.07.2014, p. 11096-11130.

Research output: Contribution to journalJournal articleResearchpeer-review

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T1 - Photobleaching kinetics and time-integrated emission of fluorescent probes in cellular membranes.

AU - Wüstner, Daniel

AU - Christensen, Tanja

AU - Solanko, Lukasz Michal

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PY - 2014/7/29

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AB - 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.

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