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Originally published In Press as doi:10.1194/jlr.D600028-JLR200 on August 29, 2006

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Journal of Lipid Research, Vol. 47, 2589-2594, November 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Methods

Leukocyte lipid bodies: inflammation-related organelles are rapidly detected by wet scanning electron microscopy

Rossana C. N. Melo*,{dagger}, Alon Sabban§ and Peter F. Weller1,{dagger}

* Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil
{dagger} Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
§ QuantomiX, Ltd., Nes-Ziona, Israel

Published, JLR Papers in Press, August 29, 2006.

1 To whom correspondence should be addressed. e-mail: pweller{at}bidmc.harvard.edu

Leukocyte lipid bodies are dynamic, functionally active organelles with central roles in inflammation. Here, we report that leukocyte lipid bodies are facilely detected by a versatile, potent technique, termed wet scanning electron microscopy (SEM), which combines the rapid preparation of light microscopy with the resolution of SEM. Using as leukocyte models resting and agonist-stimulated human eosinophils, cells that generate prominent numbers of lipid bodies in inflammatory conditions, we demonstrated that lipid bodies can be rapidly imaged as bright, highly contrasted structures under wet SEM and scored by computerized image processing. Critical advantages of this approach are that it permits cell observation in a fully hydrated system and facilitates lipid preservation. These attributes are especially important because lipid bodies are degraded during routine dehydration processes. Moreover, this technology is advantageous over lipophilic fluorescent probes because it allows sustained detection of lipid bodies in contrast to short-lived fluorescent labeling of these organelles. The value of wet SEM in enabling rapid and large-scale lipid body imaging and scoring within leukocytes is particularly important because lipid bodies are organelles underlying the heightened functions of inflammatory cells. Wet SEM technology provides new approaches and opportunities for delineations of lipid bodies in inflammatory diseases, including allergic inflammation.

Supplementary key words lipid droplets • lipid imaging • eosinophils • wet samples

Abbreviations: BODIPY, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphate, diammonium salt; BSE, backscattered electron; SEM, scanning electron microscopy; TEM, transmission electron microscopy


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