Simultaneous optimization of monolayer formation factors, including temperature, to significantly improve nucleic acid hybridization efficiency on gold substrates.

Past literature investigations have optimized various single factors used in the formation of thiolated, single stranded DNA (ss-DNA) monolayers on gold. In this study a more comprehensive approach is taken, where a design of experiment (DOE) is employed to simultaneously optimize all of the factors involved in construction of the capture monolayer used in a fluorescence-based hybridization assay. Statistical analysis of the fluorescent intensities resulting from the DOE provides empirical evidence for the importance and the optimal levels of traditional and novel factors included in this investigation.

MS/MS methodology to improve subcellular mapping of cholesterol using TOF-SIMS.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) can be utilized to map the distribution of various molecules on a surface with submicrometer resolution. Much of its biological application has been in the study of membrane lipids, such as phospholipids and cholesterol. Cholesterol is a particularly interesting molecule due to its involvement in numerous biological processes.

Time of flight secondary ion mass spectrometry: a powerful high throughput screening tool.

Combinatorial materials libraries are becoming more complicated; successful screening of these libraries requires the development of new high throughput screening methodologies. Time of flight secondary ion mass spectrometry (ToF-SIMS) is a surface analytical technique that is able to detect and image all elements (including hydrogen which is problematic for many other analysis instruments) and molecular fragments, with high mass resolution, during a single measurement. Commercial ToF-SIMS instruments can image 500 microm areas by rastering the primary ion beam over the region of interest.

Secondary ion MS imaging to relatively quantify cholesterol in the membranes of individual cells from differentially treated populations.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a well-established bioanalytical method for directly imaging the chemical distribution across single cells. Here we report a protocol for the use of SIMS imaging to comparatively quantify the relative difference in cholesterol level between the plasma membranes of two cells. It should be possible to apply this procedure to the study of other selected lipids.

Secondary ion MS imaging of lipids in picoliter vials with a buckminsterfullerene ion source.

Investigation of the spatial distribution of lipids in cell membranes can lead to an improved understanding of the role of lipids in biological function and disease. Time-of-flight secondary ion mass spectrometry is capable of molecule-specific imaging of biological molecules across single cells and has demonstrated potential for examining the functional segregation of lipids in cell membranes. In this paper, standard SIMS spectra are analyzed for phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, cholesterol, and sulfatide.

Mass spectrometric imaging of highly curved membranes during Tetrahymena mating.

Biological membrane fusion is crucial to numerous cellular events, including sexual reproduction and exocytosis. Here, mass spectrometry images demonstrate that the low-curvature lipid phosphatidylcholine is diminished in the membrane regions between fusing Tetrahymena, where a multitude of highly curved fusion pores exist. Additionally, mass spectra and principal component analysis indicate that the fusion region contains elevated amounts of 2-aminoethylphosphonolipid, a high-curvature lipid.

Proton transfer in time-of-flight secondary ion mass spectrometry studies of frozen-hydrated dipalmitoylphosphatidylcholine.

A frozen water matrix, as found in freeze-fractured frozen-hydrated cellular samples, enhances the ionization of phosphatidylcholine lipids with static time-of-flight secondary ion mass spectrometry (TOF-SIMS). Isotopic profiles of the phosphocholine ion from deuterated forms of dipalmitoylphosphatidylcholine (DPPC) have been examined under various sample preparation conditions to show that ionization occurs through protonation from the matrix and is enhanced by the water present in freeze-fractured samples. The ionization of DPPC results in positively charged fragment ions, primarily phosphocholine, with a m/z of 184.

Identification of cellular sections with imaging mass spectrometry following freeze fracture.

Freeze-fracture techniques have been used to maintain chemical heterogeneity of frozen-hydrated mammalian cells for static TOF-SIMS imaging. The effects the fracture plane has on scanning electron microscopy and dynamic SIMS images of cells have been studied, but the implications this preparation method has on static SIMS have not been addressed to date. Interestingly, the chemical specificity and surface sensitivity of TOF-SIMS have allowed the identification of unique sections of rat pheochromocytoma cells exposed to the sample surface during freeze fracture.