Biomedical subjects

Nicholas Winograd

Publications and source records attributed to Nicholas Winograd.

32 records · Page 2Linked to original sources

PubMed2004

Phosphatidylethanolamine-induced cholesterol domains chemically identified with mass spectrometric imaging.

Coexisting liquid phases of model membrane systems are chemically identified using imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS). The systems studied were Langmuir-Blodgett (LB) model membranes of cholesterol (CH) with two different phospholipids, one a major component in the outer plasma membrane bilayer leaflet (dipalmitoylphosphatidylcholine (PC)) and the other a major component in the inner leaflet (dipalmitoylphosphatidylethanolamine (PE)). Binary mixtures of CH with each of the phospholipids were investigated, as well as a ternary system. A single homogeneous phase is evident for PC/CH, whereas both systems containing PE show lateral heterogeneity with phospholipid-rich and CH-rich regions. The interaction between CH and the two phospholipids differs due to the disparity between the phospholipid headgroups. Imaging TOF-SIMS offers a novel opportunity to chemically identify and differentiate the specific membrane locations of CH and phospholipid in membrane regions without the use of fluorescent dyes. This unique imaging method has been used to demonstrate the formation of micrometer-size CH domains in phosphatidylethanolamine-rich systems and is further evidence suggesting that CH may facilitate transport and signaling across the two leaflets of the plasma membrane.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2004

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.

Animals↗

PubMed2004

Influence of molecular environment on the analysis of phospholipids by time-of-flight secondary ion mass spectrometry.

Understanding the influence of molecular environment on phospholipids is important in time-of-flight secondary ion mass spectrometry (TOF-SIMS) studies of complex systems such as cellular membranes. Varying the molecular environment of model membrane Langmuir-Blodgett (LB) films is shown to affect the TOF-SIMS signal of the phospholipids in the films. The molecular environment of a LB film of dipalmitoylphosphatidylcholine (DPPC) is changed by varying the film density, varying the sample substrate, and the addition of cholesterol. An increase in film density results in a decrease in the headgroup fragment ion signal at a mass-to-charge ratio of 184 (phosphocholine). Varying the sample substrate increases the secondary ion yield of phosphocholine as does the addition of proton-donating molecules such as cholesterol to the DPPC LB film. Switching from a model system of DPPC and cholesterol to one of dipalmitoylphosphatidylethanolamine (DPPE) and cholesterol demonstrates the ability of cholesterol to also mask the phospholipid headgroup ion signal. TOF-SIMS studies of simplistic phospholipid LB model membrane systems demonstrate the potential use of these systems in TOF-SIMS analysis of cells.

Cholesterol↗

PubMed2004

Molecule-specific imaging with mass spectrometry and a buckminsterfullerene probe: application to characterizing solid-phase synthesized combinatorial libraries.

We employ a newly developed buckminsterfullerene (C(60)) primary ion beam with time-of-flight secondary ion mass spectrometry to create molecule-specific images of resin particles employed in the solid-phase synthesis of peptide combinatorial libraries. This new cluster ion source, when operated at an incident energy of 20 keV, is remarkably effective at desorbing small peptides directly from a polymer surface and opens new possibilities for characterizing large arrays of diverse sets of molecules. In addition, the C(60) ion beam may be focused to a spot of 1.5 microm in diameter, enabling molecule-specific images of single 100 microm resin particles to be acquired. We report three significant aspects associated with utilizing the C(60) projectile that show how this technology can be taken to a more advanced level, especially when compared to results obtained with more conventional atomic primary ions. First, the useful yield of molecular ions is generally observed to be enhanced by at least 3 orders of magnitude over those previously possible. Second, the energy dissipation process associated with the C(60) impact is most efficient at desorbing molecules on soft substrates such as polymer surfaces rather than harder substrates such as metals or semiconductors. Third, there is a greatly reduced tendency for insulating surfaces to build up excess charge, obviating the need for charge compensation. Using a small five-member peptide library as a model, we show that by utilizing the focusing properties of the C(60) beam, it is possible to assay the surface composition of 100-microm polymer beads at a rate of up to 10 particles/s. Moreover, even at the picomole level, there are enough sequence ions in the mass spectrum to determine a unique composition. The results illustrate the ability to quickly assay large libraries without the use of tags and suggest the strategy may be applicable to a range of high-throughput experiments.

Combinatorial Chemistry Techniques↗

PubMed2004

The dynamics of noble metal atom penetration through methoxy-terminated alkanethiolate monolayers.

We have studied the interaction of vapor-deposited Al, Cu, Ag, and Au atoms on a methoxy-terminated self-assembled monolayer (SAM) of HS(CH(2))(16)OCH(3) on polycrystalline Au[111]. Time-of-flight secondary ion mass spectrometry, infrared reflection spectroscopy, and X-ray photoelectron spectroscopy measurements at increasing coverages of metal show that for Cu and Ag deposition at all coverages the metal atoms continuously partition into competitive pathways: penetration through the SAM to the S/substrate interface and solvation-like interaction with the -OCH(3) terminal groups. Deposited Au atoms, however, undergo only continuous penetration, even at high coverages, leaving the SAM "floating" on the Au surface. These results contrast with earlier investigations of Al deposition on a methyl-terminated SAM where metal atom penetration to the Au/S interface ceases abruptly after a approximately 1:1 Al/Au layer has been attained. These observations are interpreted in terms of a thermally activated penetration mechanism involving dynamic formation of diffusion channels in the SAM via hopping of alkanethiolate-metal (RSM-) moieties across the surface. Using supporting quantum chemical calculations, we rationalized the results in terms of the relative heights of the hopping barriers, RSAl > RSAg, RSCu > RSAu, and the magnitudes of the metal-OCH(3) solvation energies.

Journal Article↗

PubMed2003

Applicability of imaging time-of flight secondary ion MS to the characterization of solid-phase synthesized combinatorial libraries.

We employ imaging time-of-flight secondary ion mass spectrometry to perform high-throughput analysis of solid-phase synthesized combinatorial libraries by acquiring mass spectra from arrays of polymer resin particles. To generalize this procedure to various types of resins and their associated chemical linkers, it is necessary to understand the dynamics associated with the analyte molecules during chemical pretreatment steps. Using stearic acid as a model compound, we examine the influence of three classes of linkers-acid or base labile linkers, a thermally labile linker, and a photochemically cleavable linker- all of which are used to anchor one end of the analyte to the polymer resin. With data obtained using secondary ion mass spectrometry, scanning electron microscopy, and X-ray photoelectron spectroscopy, we conclude that an effective treatment of the resin needs to include cleaving the linker and extracting the unbound analyte to the resin surface. We also demonstrate that the hydrophilicity of the polymeric constituents of a resin particle affects the experiments by changing the location of the analyte molecules during resin treatment. With this information, it is possible to utilize imaging TOF-SIMS to assay a range of material supports with assurance that high-quality spectra can be acquired.

Combinatorial Chemistry Techniques↗

PubMed2003

Laser desorption and imaging of proteins from ice via UV femtosecond laser pulses.

We have employed 200-fs, 400-nm laser pulses to desorb intact protein molecular ions directly from a frozen aqueous matrix. The resulting spectra obtained using a variety of proteins varying in molecular weight from 1060 (bradykinin) to 5778 Da (insulin) are compatible with those obtained with traditional matrix-assisted laser desorption/ionization experiments. High-quality spectra could be generated using a fluence of 4.0-9.0 J/cm2 to desorb proteins from an aqueous solution frozen onto metal substrates with a sensitivity in the femtomole range. Although the mechanism behind this effect is still not clear, we speculate that it involves explosive boiling of the ice layer due to rapid heating of the substrate. Imaging experiments conducted on the ice layer suggest that the yield of protein is approximately independent of the film thickness and is very reproducible from shot to shot. The results are particularly significant since they open the possibility of examining a range of biomaterials directly from the in vivo aqueous environment.

Ice↗

PubMed2003

Enhancement of sputtering yields due to C60 versus Ga bombardment of Ag[111] as explored by molecular dynamics simulations.

The mechanism of enhanced desorption initiated by 15-keV C60 cluster ion bombardment of a Ag single crystal surface is examined using molecular dynamics computer simulations. The size of the model microcrystallite of 165,000 atoms and the sophistication of the interaction potential function yields data that should be directly comparable with experiment. The C60 model was chosen since this source is now being used in secondary ion mass spectrometry experiments in many laboratories. The results show that a crater is formed on the Ag surface that is approximately 10 nm in diameter, a result very similar to that found for Au3 bombardment of Au. The yield of Ag atoms is approximately 16 times larger than for corresponding atomic bombardment with 15-keV Ga atoms, and the yield of Ag3 is enhanced by a factor of 35. The essential mechanistic reasons for these differences is that the C60 kinetic energy is deposited closer to the surface, with the deeply penetrating energy propagation occurring via a nondestructive pressure wave. The numbers predicted by the model are testable by experiment, and the approach is extendable to include the study of organic overlayers on metals, a situation of growing importance to the SIMS community.

Journal Article↗

PubMed2003

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.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2002

Imaging of freeze-fractured cells with in situ fluorescence and time-of-flight secondary ion mass spectrometry.

Bioanalytical imaging techniques have been employed to investigate cellular composition at the single-cell and subcellular regimes. Four imaging modes have been performed sequentially in situ to demonstrate the utility of a more integrated approach to imaging cells. The combination of bright-field, scanning ion, and fluorescence microscopy complements TOF-SIMS imaging of native biomolecules. Bright-field microscopy provides a blurred visualization of cells in frozen-hydrated samples, while scanning ion imaging provides a morphological view of freeze-fractured cells after TOF-SIMS analysis is completed. With the use of selective fluorescent labels, fluorescence microscopy allows single mammalian cells to be located in the complex ice matrix of freeze-fractured samples, a task that has not been routine with either bright-field or TOF-SIMS. A fluorescent label, DiI (m/z 834), that does not interfere with the mass spectra of membrane phosphatidylcholine, has been chosen for fluorescence and TOF-SIMS imaging of membrane phospholipids. In this paper, in situ fluorescence microscopy allows the distinction of single cells from ice and other sample debris, previously not possible with bright-field or scanning ion imaging. Once cells are located, TOF-SIMS imaging reveals the localization of membrane lipids, even in the membrane of a single 15-microm rat pheochromocytoma cell. The utility of mapping lipids in the membranes of single cells using this integrated approach will provide more understanding of the functional role of specific lipids in functions of cellular membranes.

Adrenal Gland Neoplasms↗

PubMed2002

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.

Adrenal Gland Neoplasms↗

PubMed2002

Bond insertion, complexation, and penetration pathways of vapor-deposited aluminum atoms with HO- and CH(3)O-terminated organic monolayers.

The interaction of vapor-deposited Al atoms with self-assembled monolayers (SAMs) of HS-(CH(2))(16)-X (X = -OH and -OCH(3)) chemisorbed at polycrystalline Au[111] surfaces was studied using time-of-flight secondary-ion mass spectrometry, X-ray photoelectron spectroscopy, and infrared reflectance spectroscopy. Whereas quantum chemical theory calculations show that Al insertion into the C-C, C-H, C-O, and O-H bonds is favorable energetically, it is observed that deposited Al inserts only with the OH SAM to form an -O-Al-H product. This reaction appears to cease prior to complete -OH consumption, and is followed by formation of a few overlayers of a nonmetallic type of phase and finally deposition of a metallic film. In contrast, for the OCH(3) SAM, the deposited Al atoms partition along two parallel paths: nucleation and growth of an overlayer metal film, and penetration through the OCH(3) SAM to the monolayer/Au interface region. By considering a previous observation that a CH(3) terminal group favors penetration as the dominant initial process, and using theory calculations of Al-molecule interaction energies, we suggest that the competition between the penetration and overlayer film nucleation channels is regulated by small differences in the Al-SAM terminal group interaction energies. These results demonstrate the highly subtle effects of surface structure and composition on the nucleation and growth of metal films on organic surfaces and point to a new perspective on organometallic and metal-solvent interactions.

Alcohols↗

PubMedDate not supplied

Osteoblast adhesion on poly(L-lactic acid)/polystyrene demixed thin film blends: effect of nanotopography, surface chemistry, and wettability.

Biomaterial surface characteristics are critical cues that regulate cell function. We produced a novel series of poly(l-lactic acid) (PLLA) and polystyrene demixed nanotopographic films to provide nonbiological cell-stimulating cues. The increase in PLLA weight fraction (phi) in blend solutions resulted in topography changes in spin-cast films from pit-dominant to island-dominant morphologies having nanoscale depth or height (3-29 nm). Lower molecular weight PLLA segregated to the top surface of demixed films, as observed by X-ray photoelectron spectroscopy and secondary ion mass spectroscopy (SIMS). For phi > or = 0.5, the topmost film layer was predominantly filled with PLLA (>96% by SIMS at 20-A depth). Nanotextured substrata stimulated osteoblastic cell adhesion to a greater degree than did flat PLLA (phi = 1), and this effect was more pronounced for nanoisland (phi = 0.7 and 0.9) relative to nanopit topographies (phi = 0.5). Demixed films having relatively lower water contact angles generally enhanced cell adhesion and spreading. Our results reveal that cell adhesion is affected by surface chemistry, topography, and wettability simultaneously and that nanotextured surfaces may be utilized in regulating cell adhesion.

Adhesiveness↗

PubMedDate not supplied

Spectroscopically encoded resins for high throughput imaging time-of-flight secondary ion mass spectrometry.

Spectroscopic barcoding was recently introduced as a new pre-encoding strategy wherein the resin beads are not just carriers for solid phase synthesis, but are, in addition, the repository of the synthetic scheme to which they were subjected. To expand the repertoire of spectroscopically barcoded resins (BCRs), here we introduce a new family of halogenated polystyrene-based polymers designed for high-throughput combinatorial analysis using not only infrared and Raman spectroscopy but also imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, we have established that (a) the halogen content of these new resins can be used as an encoding element in quantitative imaging ToF-SIMS and (b) the number of styrene monomers used to generate unique vibrational fingerprints can be significantly reduced by using monomers in different molar ratios. The combination of quantitative imaging ToF-SIMS and vibrational spectroscopy is anticipated to dramatically increase the repertoire of possible BCRs from a few hundreds to several thousands.

Combinatorial Chemistry Techniques↗