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2013 | OriginalPaper | Chapter

Quantitative Fluorescence Studies of Intracellular Sterol Transport and Distribution

Authors : Daniel Wüstner, Frederik W. Lund, Lukasz M. Solanko

Published in: Fluorescent Methods to Study Biological Membranes

Publisher: Springer Berlin Heidelberg

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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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Title: Quantitative Fluorescence Studies of Intracellular Sterol Transport and Distribution
Authors: Daniel Wüstner
Frederik W. Lund
Lukasz M. Solanko
Publisher: Springer Berlin Heidelberg
Book: Fluorescent Methods to Study Biological Membranes
Print ISBN: 978-3-642-33127-5

Electronic ISBN: 978-3-642-33128-2

Copyright Year: 2013
DOI: https://doi.org/10.1007/4243_2012_57

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