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Supported Membrane Composition Analysis by Secondary Ion Mass Spectrometry with High Lateral Resolution

Carine Galli Marxer ^*, Mary L. Kraft ^*, Peter K. Weber , Ian D. Hutcheon  and Steven G. Boxer ^*

^* Department of Chemistry, Stanford University, Stanford, California 94305; and Lawrence Livermore National Laboratory, Livermore, California 94551

Correspondence: Address reprint requests to Steven G. Boxer, Tel.: 650-723-4482; Fax: 650-723–4817; E-mail: sboxer{at}stanford.edu.

The lateral organization of lipid components within membranes is usually investigated with fluorescence microscopy, which, though highly sensitive, introduces bulky fluorophores that might alter the behavior of the components they label. Secondary ion mass spectroscopy performed with a NanoSIMS 50 instrument also provides high lateral resolution and sensitivity, and many species can be observed in parallel without the use of bulky labels. A tightly focused beam (100 nm) of Cs ions is scanned across a sample, and up to five of the resulting small negative secondary ions can be simultaneously analyzed by a high-resolution mass spectrometer. Thin layers of ¹⁵N- and ¹⁹F-labeled proteins were microcontact-printed on an oxidized silicon substrate and imaged using the NanoSIMS 50, demonstrating the sensitivity and selectivity of this approach. Supported lipid bilayers were assembled on an oxidized silicon substrate, then flash-frozen and freeze-dried to preserve their lateral organization. Lipid bilayers were analyzed with the NanoSIMS 50, where the identity of each specific lipid was determined through detection of its unique secondary ions, including ¹²C¹H^–, ¹²C²H^–, ¹³C^–, ¹²C¹⁴N^–, and ¹²C¹⁵N^–. Steps toward obtaining quantitative composition analysis of lipid membranes that varied spatially in isotopic composition are presented. This approach has the potential to provide a composition-specific analysis of membrane organization that compliments other imaging modalities.

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