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Quantitative Fluorescence Studies of Intracellular Sterol Transport and Distribution

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Fluorescent Methods to Study Biological Membranes

Part of the book series: Springer Series on Fluorescence ((SS FLUOR,volume 13))

Abstract

Unraveling the pathways of intracellular cholesterol transport is of great importance for biomedicine, since disturbed cholesterol trafficking is involved in many metabolic diseases. Most fluorescent probes for cholesterol, however, have physico-chemical properties deviating from the natural sterol. Intrinsically fluorescent sterols like dehydroergosterol (DHE) and the related cholestatrienol (CTL) have great potential for analysis of sterol trafficking due to their close resemblance of ergosterol and cholesterol, respectively. Excitation and emission of both sterols are in the ultraviolet (UV), which, together with high bleaching propensity and low brightness, make fluorescence imaging of DHE and CTL challenging. Here, we present an overview of how UV-sensitive wide field (UV-WF) and multiphoton (MP) microscopy can be applied to image both sterols in living cells and tissues. In addition, we show, for the first time, how applying advanced image denoising can dramatically enhance the signal-to-noise ratio in MP image sequences of DHE. This allowed us to track DHE-containing vesicles and surface protrusions in cells over prolonged time. We also discuss the properties of BODIPY-tagged cholesterol (BChol) compared to DHE and cholesterol and present an overview of fluorescence imaging techniques for analyzing cellular sterol dynamics.

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Acknowledgements

DW acknowledges funding by grants of the Lundbeck Foundation, the Novo Nordisk Foundation, the Danish Research Agency Forskningsstyrelsen, Forskningsrådet for Natur og Univers (FNU) and the Danish Research Agency Forskningsstyrelsen, Forskningsrådet for Sundhed og sygdom (FSS). We are grateful to Florian Luisier (Statistics and Information Sciences Laboratory, Harvard University, Cambridge, USA) and Daniel Sage (Biomedical Imaging Group, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland) for their helpful comments on image denoising using the PURE-LET approach and on an early version of the manuscript.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

    Daniel Wüstner, Frederik W. Lund & Lukasz M. Solanko

  2. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark

    Daniel Wüstner

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  1. Daniel Wüstner
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  2. Frederik W. Lund
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Correspondence to Daniel Wüstner .

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  1. Faculté de Pharmacie, Laboratoire de Biophotonique, Université de Strasbourg, route du Rhin 74, Illkirch, 67401, France

    Yves Mély

  2. Faculté de Pharmacie, Laboratoire de Biophotonique et, Université de Strasbourg, route du Rhin 74, Illkirch, 67401, France

    Guy Duportail

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Wüstner, D., Lund, F.W., Solanko, L.M. (2012). Quantitative Fluorescence Studies of Intracellular Sterol Transport and Distribution. In: Mély, Y., Duportail, G. (eds) Fluorescent Methods to Study Biological Membranes. Springer Series on Fluorescence, vol 13. Springer, Berlin, Heidelberg. https://doi.org/10.1007/4243_2012_57

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