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Potential of Ultraviolet Wide-Field Imaging and Multiphoton Microscopy for Analysis of Dehydroergosterol in Cellular Membranes

D. Wüstner, J.R. Brewer, L. Bagatolli, D. Sage

Microscopy Research and Technique, vol. 74, no. 1, pp. 92-108, January 2011.


Dehydroergosterol (DHE) is an intrinsically fluorescent sterol with absorption/emission in the ultraviolet (UV) region and biophysical properties similar to those of cholesterol. We compared the potential of UV-sensitive low-light-level wide-field (UV-WF) imaging with that of multiphoton (MP) excitation microscopy to monitor DHE in living cells. Significantly reduced photobleaching in MP microscopy of DHE enabled us to acquire three-dimensional z-stacks of DHE-stained cells and to obtain high-resolution maps of DHE in surface ruffles, nanotubes, and the apical membrane of epithelial cells. We found that the lateral resolution of MP microscopy is ∼1.5-fold higher than that of UV-WF deconvolution microscopy, allowing for improved spatiotemporal analysis of plasma membrane sterol distribution. Surface intensity patterns of DHE with a diameter of 0.2 μm persisting over several minutes could be resolved by MP time-lapse microscopy. Diffusion coefficients of 0.25-μm-diameter endocytic vesicles containing DHE were determined by MP spatiotemporal image correlation spectroscopy. The requirement of extremely high laser power for visualization of DHE by MP microscopy made this method less potent for multicolor applications with organelle markers like green fluorescent protein-tagged proteins. The signal-to-noise ratio obtainable by UV-WF imaging could be significantly improved by pixelwise bleach rate fitting and calculation of an amplitude image from the decay model and by frame averaging after pixelwise bleaching correction of the image stacks. We conclude that UV-WF imaging and MP microscopy of DHE provide complementary information regarding membrane distribution and intracellular targeting of sterols.

@ARTICLE(http://bigwww.epfl.ch/publications/wuestner1101.html,
AUTHOR="W{\"{u}}stner, D. and Brewer, J.R. and Bagatolli, L. and Sage,
	D.",
TITLE="Potential of Ultraviolet Wide-Field Imaging and Multiphoton
	Microscopy for Analysis of Dehydroergosterol in Cellular Membranes",
JOURNAL="Microscopy Research and Technique",
YEAR="2011",
volume="74",
number="1",
pages="92--108",
month="January",
note="")

© 2011 Wiley. Personal use of this material is permitted. However, permission to reprint/republish this material for advertising or promotional purposes or for creating new collective works for resale or redistribution to servers or lists, or to reuse any copyrighted component of this work in other works must be obtained from Wiley. This material is presented to ensure timely dissemination of scholarly and technical work. Copyright and all rights therein are retained by authors or by other copyright holders. All persons copying this information are expected to adhere to the terms and constraints invoked by each author's copyright. In most cases, these works may not be reposted without the explicit permission of the copyright holder.
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