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

Abstract

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 (approximately 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 (15)N- and (19)F-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 (12)C(1)H(-), (12)C(2)H(-), (13)C(-), (12)C(14)N(-), and (12)C(15)N(-). 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.

Colocalization of the Ganglioside GM1 and Cholesterol Detected by Secondary Ion Mass Spectrometry

“…We have advocated the use of multiple-isotope mass spectrometry 35 (MIMS) using a NanoSIMS (Cameca Instruments) as an imaging tool for interrogating membrane composition. 36–39 This technique requires isotopic labeling or atom exchange (e.g. with F) of membrane components but can generate molecule-specific images with high spatial resolution, high sensitivity, and high mass resolving power, and calibration samples can be used to obtain quantitative estimates of the composition of each component.…”

Fluorinated Colloidal Gold Immunolabels for Imaging Select Proteins in Parallel with Lipids Using High-Resolution Secondary Ion Mass Spectrometry

“…21, 23, 28–30 Because this instrument detects monoatomic and diatomic secondary ions, each species of interest must contain a distinct stable isotope so that the secondary ions generated during NanoSIMS analysis can be linked to the parent molecule. 21 The distinct stable isotopes that encode for component identity can be selectively incorporated into cholesterol and specific lipid species with established metabolic labeling techniques.…”

“…Fluorine is an attractive candidate elemental label due to its low abundance in biological materials and high secondary ion yields. 21, 28 Methods to conjugate small fluorine-containing molecules to antibodies have been developed for positron-emission tomography applications. 35, 36 However, we experimentally found that functionalizing antibodies with the high numbers of fluorinated molecules that enable sensitive detection with NanoSIMS compromised antibody binding.…”

Dynamic Reorganization and Correlation among Lipid Raft Components

“…26-29 Briefly, counts for each ion species are summed and then the ratios 19 F − / 12 C 2 − , 13 C 12 C − / 12 C 2 − , 2 H − / 12 C 2 − , and 12 C 15 N − / 12 C 14 N − are calculated. Calibration curves obtained from standard samples are then employed (see Figure S3 and Table S1) to determine the percent molar content for each component of interest.…”