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. We report on the statistical importance of a novel factor, temperature of the system during monolayer formation of the capture molecule and lateral spacer molecule, and how proper usage of this temperature factor increased the hybridization signal 50%. An initial theory of how the physical factor of heat is mechanistically supplementing the function of the lateral spacer molecule is provided.

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. For many studies, the effectiveness of chemical mapping is limited by low signal intensity from various biomolecules. Because of the high energy nature of the SIMS ionization process, many molecules are identified by detection of characteristic fragments. Commonly, fragments of a molecule are identified using standard samples, and those fragments are used to map the location of the molecule. In this work, MS/MS data obtained from a prototype C60(+)/quadrupole time-of-flight mass spectrometer was used in conjunction with indium LMIG imaging to map previously unrecognized cholesterol fragments in single cells. A model system of J774 macrophages doped with cholesterol was used to show that these fragments are derived from cholesterol in cell imaging experiments. Examination of relative quantification experiments reveals that m/z 147 is the most specific diagnostic fragment and offers a 3-fold signal enhancement. These findings greatly increase the prospects for cholesterol mapping experiments in biological samples, particularly with single cell experiments. In addition, these findings demonstrate the wealth of information that is hidden in the traditional TOF-SIMS spectrum.

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. In this work, we will show that large area analysis can be performed, in one single measurement, by rastering the sample under the ion beam. We show that an entire 70 mm diameter wafer can be imaged in less than 90 min using ToF-SIMS stage (macro)rastering techniques. ToF-SIMS data sets contain a wealth of information since an entire high mass resolution mass spectrum is saved at each pixel in an ion image. Multivariate statistical analysis (MVSA) tools are being used in the ToF-SIMS community to assist with data interpretation; we will demonstrate that MVSA tools provide details that were not obtained using manual (univariate) analysis.

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. This development enables direct comparison of the chemical effects of different drug treatments and incubation conditions in the plasma membrane at the single-cell level. Relative, quantitative TOF-SIMS imaging has been used here to compare macrophage cells treated to contain elevated levels of cholesterol with respect to control cells. In situ fluorescence microscopy with two different membrane dyes was used to discriminate morphologically similar but differentially treated cells prior to SIMS analysis. SIMS images of fluorescently identified cells reveal that the two populations of cells have distinct outer leaflet membrane compositions with the membranes of the cholesterol-treated macrophages containing more than twice the amount of cholesterol of control macrophages. Relative quantification with SIMS to compare the chemical composition of single cells can provide valuable information about normal biological functions, causative agents of diseases, and possible therapies for diseases.

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. Importantly, each of the lipids result in signature mass spectral peaks that allow them to be identified. These signature peaks are also useful for imaging experiments and are utilized here to simultaneously image lipids on a micrometer scale in picoliter vials. Because the low secondary ion signal achieved for lipids from an atomic primary ion source makes cell-imaging experiments challenging, improving signal with cluster primary ion sources is of interest. Here, we compare the secondary ion yield for seven lipids using atomic (Ga+ or In+) ion sources and a buckminsterfullerene (C60+) primary ion source. A 40-1000-fold improvement in signal is found with C60+ relative to the other two ion sources, indicating great promise for future cellular imaging applications using the C60+ probe.

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. This evidence suggests that biological fusion involves and might in fact be driven by a heterogeneous redistribution of lipids at the fusion site.

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. Other ions include the M + H ion (m/z 735) and an ion representing the abstraction of the two palmitoyl fatty acid groups (m/z 224). Freeze-fracture techniques have been used to prepare frozen aqueous samples such as liposomes and cells to expose their membranes for static TOF-SIMS imaging. Due to the importance of surface water during SIMS analyses, sources of gas-phase water resulting from freeze-fracture were examined. Under proper fracturing conditions, water vapor, resulting from water in the sample and water condensed onto the outside of the sample, is released into the vacuum but does not condense back onto the surface. Combining the demonstrated enhancement of phosphatidylcholine lipid signal from water with the freeze-fracture preparation techniques described herein demonstrates potential advantages of studying biological samples in a frozen-hydrated state.

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. Using the extensive chemical information of the fractured surface, cellular sections have been determined using TOF-SIMS images of water, sodium, potassium, hydrocarbons, phosphocholine, and DiI, a fluorescent dye that remains in the outer leaflet of the cell membrane. Higher amounts of potassium have been imaged inside a cell versus the surrounding matrix in a cross-fractured cell. In other fractures exposing the cell membrane, phosphocholine and DiI have been imaged on the outer leaflet of the cell membrane, while phosphocholine alone has been imaged on the inner leaflet. In this paper, we discuss how imaging mass spectrometry isused to uniquely distinguish three possible sections of cells obtained during freeze fracture. The identification of these sections is important in choosing cells with a region of interest, like the cell membrane, exposed to the surface for a more thorough investigation with imaging static TOF-SIMS.