Characterizing in situ Glycerophospholipids with SIMS and MALDI Methodologies.

The dual functionality of a C(60) (+)-Q-Star hybrid instrument allows for the examination of complex lipid profiles with both MALDI and SIMS methodologies.([1]) The difficulties associated with characterizing lipids in situ - isobaric interference and salt contamination - are discussed. The inherent advantages of both methodologies were used to deconvolute complex lipid spectra and establish characteristic fragmentation pathways. The high lateral resolution of SIMS allows for single cell images of aplysia californica neurons, while the established protocols were utilized to identify a major lipid component.

Investigating the Fundamentals of Molecular Depth Profiling Using Strong-field Photoionization of Sputtered Neutrals.

Molecular depth profiles of model organic thin films were performed using a 40 keV C60+ cluster ion source in concert with TOF-SIMS. Strong-field photoionization of intact neutral molecules sputtered by 40 keV C60+ primary ions was used to analyze changes in the chemical environment of the guanine thin films as a function of ion fluence. Direct comparison of the secondary ion and neutral components of the molecular depth profiles yields valuable information about chemical damage accumulation as well as changes in the molecular ionization probability. An analytical protocol based on the erosion dynamics model is developed and evaluated using guanine and trehalose molecular secondary ion signals with and without comparable laser photoionization data.

Ionization effects in molecular depth profiling of trehalose films using buckminsterfullerene (C60) cluster ions.

Salts play a mysterious role in desorption mass spectrometry, especially in biological samples.[1] We used trehalose films doped with a peptide as a well defined model system to investigate the ionization effects in organic molecular depth profiling. Sodium salts at 1% level were added into the solution used to produce the trehalose films, and depth profiles were obtained with a C60 ion source. The results show that the protonated molecular ion signal from the peptide and the quasimolecular ion signal of trehalose are significantly suppressed by the addition of salts, whereas the signals representing salt clusters and salt adducts of trehalose are formed in both positive and negative modes. The formation of protonated molecular ions is found to correlate with the ratio between protonated and bare water ions, suggesting that the latter can be used as an indicator for the accumulation of protons liberated by the ion bombardment. In experiments where no salt was added, it is shown that the surface variation of the protonated molecular ion signal strongly depends upon the water content of the trehalose film.

Fundamental studies of molecular depth profiling using organic delta layers as model systems.

Alternating Langmuir-Blodgett multilayers of barium arachidate (AA) and barium dimyristoyl phosphatidate (DMPA) were used to elucidate the factors that control depth resolution in molecular depth profiling experiments. More specifically, thin (4.4 nm) layers of DMPA were embedded in relatively thick (~50 nm) multilayer stacks of AA, resulting in a well-defined delta-layer model system closely resembling a biological membrane. This system was subjected to a three-dimensional imaging depth profile analysis using a focused buckminsterfullerene (C60) cluster ion beam. The depth response function measured in these experiments exhibits similar features as those determined in inorganic depth profiling: namely, an asymmetric shape with quasi-exponential leading and trailing edges and a central Gaussian peak. The magnitude of the corresponding characteristic rise and decay lengths is found to be 5 and 16 nm, respectively, while the total half width of the response function characterizing the apparent depth resolution was about 29 nm. Ion-induced mixing is proposed to be largely responsible for the broadening, rather than topography, as determined by atomic force microscopy.

Temperature effects in the sputtering of a molecular solid by energetic atomic and cluster projectiles.

Temperature effects in the sputtering of an organic molecule were investigated by subjecting a well defined film of coronene to Au1 and C60 primary ions at 100 and 300 K. Strong field photoionization of the sputtered neutral flux was employed to monitor the change in flight time and kinetic energy distributions of intact and fragmented species.

Strong-field Photoionization of Sputtered Neutral Molecules for Molecular Depth Profiling.

Molecular depth profiles of an organic thin film of guanine vapor deposited onto a Ag substrate are obtained using a 40 keV C(60) cluster ion beam in conjunction with time-of-flight secondary ion mass spectrometric (ToF-SIMS) detection. Strong-field, femtosecond photoionization of intact guanine molecules is used to probe the neutral component of the profile for direct comparison with the secondary ion component. The ability to simultaneously acquire secondary ions and photoionized neutral molecules reveals new fundamental information about the factors that influence the properties of the depth profile. Results show that there is an increased ionization probability for protonated molecular ions within the first 10 nm due to the generation of free protons within the sample. Moreover, there is a 50% increase in fragment ion signal relative to steady state values 25 nm before reaching the guanine/Ag interface as a result of interfacial chemical damage accumulation. An altered layer thickness of 20 nm is observed as a consequence of ion beam induced chemical mixing. In general, we show that the neutral component of a molecular depth profile using the strong-field photoionization technique can be used to elucidate the effects of variations in ionization probability on the yield of molecular ions as well as to aid in obtaining accurate information about depth dependent chemical composition that cannot be extracted from TOF-SIMS data alone.

Three-dimensional depth profiling of molecular structures.

Molecular time of flight secondary ion mass spectrometry (ToF-SIMS) imaging and cluster ion beam erosion are combined to perform a three-dimensional chemical analysis of molecular films. The resulting dataset allows a number of artifacts inherent in sputter depth profiling to be assessed. These artifacts arise from lateral inhomogeneities of either the erosion rate or the sample itself. Using a test structure based on a trehalose film deposited on Si, we demonstrate that the "local" depth resolution may approach values which are close to the physical limit introduced by the information depth of the (static) ToF-SIMS method itself.

Molecular dynamics simulations of sputtering of Langmuir-Blodgett multilayers by keV C(60) projectiles.

Coarse-grained molecular dynamics computer simulations are applied to investigate fundamental processes induced by an impact of keV C(60) projectile at an organic overlayer composed of long, well-organized linear molecules. The energy transfer pathways, sputtering yields, and the damage induced in the irradiated system, represented by a Langmuir-Blodgett (LB) multilayers composed from molecules of bariated arachidic acid, are investigated as a function of the kinetic energy and impact angle of the projectile and the thickness of the organic system. In particular, the unique challenges of depth profiling through a LB film vs. a more isotropic solid are discussed.The results indicate that the trajectories of projectile fragments and, consequently, the primary energy can be channeled by the geometrical structure of the overlayer. Although, a similar process is known from sputtering of single crystals by atomic projectiles, it has not been anticipated to occur during C(60) bombardment due to the large size of the projectile. An open and ordered molecular structure of LB films is responsible for such behavior. Both the extent of damage and the efficiency of sputtering depend on the kinetic energy, the impact angle, and the layer thickness. The results indicate that the best depth profiling conditions can be achieved with low-energy cluster projectiles irradiating the organic overlayer at large off-normal angles.

Strong-field ionization of sputtered molecules for biomolecular imaging.

Photoionization of molecules sputtered from molecular thin films has been achieved using high field 125 fs pulses in the mid-IR spectral range. Using several model systems, we show that it is possible to significantly reduce molecular fragmentation induced by the laser field by increasing the photoionization wavelength. By examining the photoionization spectra as a function of wavelength, it is apparent that the photoionization mechanism is changing from a non-adiabatic multi-electron excitation process to a process that involves tunnel ionization. The results of these observations are discussed in terms of their significance for bioimaging with focused ion beams and mass-spectrometry.

Investigating lipid-lipid and lipid-protein interactions in model membranes by ToF-SIMS.

With the chemical imaging capability of ToF-SIMS, biological molecules are identified and localized in membranes without any chemical labels. We have developed a model membrane system made with supported Langmuir-Blodgett (LB) monolayers. This simplified model can be used with different combinations of molecules to form a membrane, and thus represents a bottom-up approach to study individual lipid-lipid or lipid-protein interactions. We have used ternary mixtures of sphingomyelin (SM), phosphatidylcholine (PC), and cholesterol (CH) in the model membrane to study the mechanism of domain formation and interactions between phospholipids and cholesterol. Domain structures are observed only when the acyl chain saturation is different for SM and PC in the mixture. The saturated lipid, whether it is SM or PC, is found to be localized with cholesterol, while the unsaturated one is excluded from the domain area. More complicated model membranes which involve a functional membrane protein glycophorin are also investigated and different membrane properties are observed compared to the systems without glycophorin.

Imaging macrophages in trehalose with SIMS.

Phagocytosis is a major component of the animal immune system where apoptotic cellular material, metabolites, and waste are safely processed. Further, efficient phagocytosis by macrophages is key to maintaining healthy vascular systems and preventing atherosclerosis. Single-cell images of macrophage phagocytosis of red blood cells, RBCs, and polystyrene microspheres have been chemically mapped with TOF-SIMS. We demonstrate here cholesterol and phosphocholine localizations as relative to time and activity.

Chemical pathways in the interactions of reactive metal atoms with organic surfaces: vapor deposition of Ca and Ti on a methoxy-terminated alkanethiolate monolayer on Au.

In situ time-of-flight secondary ion mass spectrometry, infrared spectroscopy, and X-ray photoelectron spectroscopy measurements have been used to characterize the interfacial chemistry that occurs upon physical vapor deposition of Ti and Ca atoms onto a -OCH(3) terminated alkanethiolate self-assembled monolayer (SAM) on Au{111}. While the final result for both metals is near-exhaustive degradation of the methoxy terminal group and partial degradation of the alkyl chains to inorganic products such as carbides, hydrides, and oxides, the reaction mechanisms differ significantly. Titanium reacts in parallel with the -OCH(3) and -CH(2)- units, extensively degrading the latter until a metallic overlayer forms preventing further degradation. At this point, there is a cessation of the Ti-SAM reactions. In contrast, Ca is initially consumed by the -OCH(3) terminal group via a reaction mechanism involving two -OCH(3) groups; subsequent depositions lead to alkyl chain degradation, but at a rate slower than that for Ti deposition. These results demonstrate the subtle differences in chemistry that can arise in the vapor deposition of reactive metals, and have important implications for the behavior of electrical interfaces in organic and molecular devices made with Ti or Ca top contacts.

Modification and stability of aromatic self-assembled monolayers upon irradiation with energetic particles.

We have studied ion and electron irradiation of self-assembled monolayers (SAMs) of 2-(4'-methyl-biphenyl-4yl)-ethanethiol (BP2, CH3-C6H4C6H4CH2CH2-SH), phenyl mercaptan (PEM, C6H5CH2CH2-SH), and 4'-methyl-biphenyl-4-thiol (BP0, CH3-C6H4C6H4-SH) deposited on Au(111) substrates. Desorption of neutral particles from PEM/Au and BP2/Au was investigated using laser ionization in combination with mass spectrometry. The ion-induced damage of both BP2 and PEM SAMs is very efficient and interaction with a single ion leads to the modification of tens of molecules. This feature is the result of a desorption process caused by a chemical reaction initiated by an ion impact. Both for ions and electrons, experiments indicate that the possibility for scission of the Au-S bond strongly depends on the chemical nature of the SAM system. We attribute the possible origin of this effect to the orientation of the Au-S-C angle or adsorption sites of molecules. The analysis of electron-irradiated PEM/Au and BP2/Au, using ion-initiated laser probing, enabled measurements of the cross section for the electron-induced damage of the intact molecule or specific fragment. Analysis of electron-irradiated BP0/Au by using time-of-flight secondary ion mass spectrometry (TOF-SIMS) provides direct evidence for the quasi-polymerization process induced by electron irradiation.

Quantitative chemical analysis of single cells.

A fundamental perspective can be achieved by targeting single cells for analysis with the goal of deconvoluting complex biological functions. However, single-cell studies have their own difficulties, such as minute volumes and sample amounts. Quantitative chemical analysis of single cells has emerged as a powerful new area in recent years due to several technological advancements. The development of microelectrodes has allowed the measurement of redox-active species as a function of cellular dynamics. This miniaturization trend is also evident in the separation sciences with the application of small column separations to single cells. Desorption ionization methods with mass spectrometric detection have shown single-cell capability owing to numerous technological developments. Finally, fluorescence imaging has also progressed to the point where single-cell dynamics can be probed by native fluorescence utilizing either single or multiple photon excitation. The results of these studies are reviewed with an emphasis on the quantitation of single-cell dynamics.

Spatially resolved detection of attomole quantities of organic molecules localized in picoliter vials using time-of-flight secondary ion mass spectrometry.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been utilized to detect femtomole and attomole quantities of organic species from within silicon nanovials. By using high-density arrays (10,000 nanovials/cm2) it is possible to chemically characterize diverse sample sets within a single chemical image. Molecular sensitivities, for the compounds investigated, very between 85 attomoles and 25 femtomoles, and typical acquisition times are approximately 100 ms per nanovial. These vials are fabricated using photolithography and KOH etching of Si[001] wafers to create wells, with a pyramidal cross section, ranging in size from 25 to 5625 micron 2. The volume ranges from 30 femtoliters to 100 picoliters, respectively. A drawn glass microinjector and solenoid-driven dispenser are utilized to array picoliter volumes of organic compounds into individual silicon nanovials. Solution concentrations typically range from 1 x 10(-2) to 1 x 10(-4) M allowing femtomole and even attomole quantities of material to be dispensed into each vial.

Photoionization of gas-phase versus ion-beam-desorbed dopamine with femtosecond laser pulses.

We have investigated the photoionization of gas-phase and ion-beam desorbed dopamine using femtosecond laser pulses at wavelengths of 800, 400, 267, and 200 nm. Photoionization of gas-phase dopamine is found to produce the molecular ion, and three fragment ions at all four wavelengths, with the branching ratios strongly wavelength dependent. Photoionization at 400 and 267 nm yields the highest molecular ion signal, while that at 800 and 200 nm produces very little molecular ion signal. An excited-state lifetime of approximately 10 ps following 267-nm excitation has been measured for dopamine using time-resolved pump-probe techniques. The short-lived excited state suggests that internal conversion, intersystem crossing, and/or dissociation is a concern when ionizing at this wavelength using longer laser pulses. Photoionization of ion-beam-desorbed dopamine exhibits a large degree of fragmentation at all four wavelengths, though 267-nm photoionization produces the highest yield of dopamine fragment ions. Power dependence studies show a high degree of internal excitation. A direct comparison of ion yields obtained for photoionization of ion-beam-desorbed dopamine at 267 nm to that for SIMS shows a 20-fold increase in signal.

Static time-of-flight secondary ion mass spectrometry imaging of freeze-fractured, frozen-hydrated biological membranes.

The study of cell membrane lipid and steroid composition and distribution is important for the understanding of membrane dynamics and function. Here we present efforts to chemically image phospholipid distributions on a submicron scale on freeze-fractured and frozen-hydrated liposomes and red blood cells using time-of-flight secondary ion mass spectrometry. Sample preparation by freeze fracturing of membranes is described. Fragments representative of phospholipid headgroups are found to be localized on both liposomes and red blood cells. In addition, the cholesterol molecular ion [M + H] is localized on liposome surfaces.

Vacuum ultraviolet single photon versus femtosecond multiphoton ionization of sputtered germanium clusters.

Neutral atoms and clusters desorbed from a solid germanium surface by ion bombardment are detected by laser postionization and time-of-flight mass spectrometry. Two different photoionization schemes are compared which are generally believed to be candidates for the 'soft' ionization of polyatomic species without significant photon induced fragmentation. First, a single photon ionization process is employed using an F2 laser as an intense VUV source with a photon energy in excess of all relevant ionization potentials. It is shown that the available laser pulse energy is sufficient to saturate the ionization of Ge atoms and all detected Ge(n) clusters. The resulting mass spectra are compared to those obtained with a non-resonant multiphoton ionization process using a high intensity laser delivering pulses of 250 femtoseconds duration at a wavelength of 267 nm. Also in this case, the ionization process can apparently be driven into saturation. The mass spectra measured under these conditions are found to be almost identical to those obtained using single photon ionization. We take this as an indication that the results obtained with both postionization techniques closely reflect the true cluster sputtering yields and, in particular, are not dominated by photon induced fragmentation.

Ejection of neutral molecules from ion-bombarded organic surfaces.

Time-of-flight distributions of neutral molecules ejected from various organic surfaces have been measured subsequent to 8 keV Ar+ and H2+ ion bombardment. The distributions show that depending on the physical and chemical nature of the substrate, the neutral molecules have strikingly different desorption profiles. For C6H6/Ag¿111¿, at low coverage the C6H6 molecules eject with energies in the range 0.25-1 eV while at high coverage most of the molecules desorb with thermal kinetic energies (approximately 0.04 eV). At intermediate coverage two peaks are present in the time-of-flight distribution indicating that two different mechanisms contribute to the desorption process. For self-assembled monolayers of phenylethanethiol on Au, while a minor ejection is observed at higher kinetic energy (approximately 1 eV) most of the molecules desorb with thermal kinetic energies (approximately 0.03 eV). Pyrenebutyric acid molecules ejected from monolayer and multilayer samples have kinetic energies close to 0.2 eV. One ejection mechanism is observed in this case. For tryptophan, most molecules eject with kinetic energies close to 0.1 eV. In addition, a feature unique to this case is the continuous emission of molecules from the surface that extends beyond 200 microseconds after ion impact. For all the multilayer samples investigated, a molecular collision cascade in the solid leads to ejection of molecules with kinetic energies in the range 0.1-0.3 eV.