Molecule Specific Imaging of Freeze-Fractured, Frozen-Hydrated Model Membrane Systems Using Mass Spectrometry

Abstract

Imaging time-of-flight secondary ion mass spectrometry is used to chemically resolve the spatial distribution of lipids in submicrometer sections of phospholipid membranes. The results show that it is possible to unravel dynamical events such as chemical fluctuations associated with domain structure in cellular membranes. In this work, a liposome model system has been used to capture the stages of membrane fusion between two merging bilayer systems. Fracturing criteria for preserving chemical distributions are shown to be much more stringent than morphological electron cryomicroscopy studies. Images of membrane heterogeneity, induced via mixing various liposomes followed by fast freezing, demonstrate the necessary sample preparation groundwork to investigate complex, heterogeneous membrane domains. Clear delineation of membrane structure provides direct evidence that specific domains or “rafts” can exist. Moreover, low concentrations of each phospholipid are distributed throughout newly fused liposomes despite the existence of distinct domains. In the liposome model, membrane structure ranges from specific domains to a fluid mosaic of the phospholipids during the fusion event. The availability of mass spectrometric imaging is proposed to facilitate the discovery of functional rafts or substructure in cell membranes before, during, and after events including cell division, exocytosis, endocytosis, intracellular transport, infection of membrane-bound viruses, and receptor clustering. This technology holds the promise to define the biology of cell membranes at the molecular level.

Mass Spectrometric Imaging of Highly Curved Membranes During Tetrahymena Mating

“…TOF-SIMS has been applied to elemental imaging of the distribution of cancer therapy drugs in single cells (14) as well as molecular imaging of tissue (15) (16,21,22) and directly transferred into the analysis chamber. After TOF-SIMS imaging, the samples were further analyzed by scanning ion microscopy and brightfield microscopy to ensure that no substantial sample damage occurred during sample preparation and SIMS analysis.…”

“…TOF-SIMS has been applied to elemental imaging of the distribution of cancer therapy drugs in single cells (14) as well as molecular imaging of tissue (15), liposomes (16,17), paramecia (18), and rat pheochromocytoma (PC12) cells (19,20), but has not yet been demonstrated for subcellular membranes. We incubated conjugating Tetrahymena for 4 hours and then placed a small aliquot of the cell-containing solution between two pieces of silicon for cryogenic freezing.…”

“…We incubated conjugating Tetrahymena for 4 hours and then placed a small aliquot of the cell-containing solution between two pieces of silicon for cryogenic freezing. The frozen sample was then freeze-fractured under ultrahigh vacuum (16,21,22) and directly transferred into the analysis chamber. After TOF-SIMS imaging, the samples were further analyzed by scanning ion microscopy and brightfield microscopy to ensure that no substantial sample damage occurred during sample preparation and SIMS analysis.…”

Mass spectrometry imaging of mating Tetrahymena show that changes in cell morphology regulate lipid domain formation

“…Cells were prepared for analysis in vacuum by using a freeze fracture procedure for SIMS imaging described previously (36). Cells were imaged at three time points; 1, 2, and 3 h following mixing of the two mating types.…”

Preparation of Single Cells for Imaging Mass Spectrometry

“…Currently, the vast majority of cellular MS imaging reports utilize some variation on this method. Improvements to Chandra's technique, most notably maintenance of the sample at cryogenic temperatures throughout the fracturing and analysis [22], have resulted in improvements to the cellular imaging analysis [15].…”