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Determination of the Sputtering Yield of Cholesterol Using Arn + and C 60 +(+) Cluster Ions
Abstract
The sputtering yield of cholesterol films on silicon wafers is measured using Arn(+) and C60(+(+)) ions in popular energy (E) and cluster size (n) ranges. It is shown that the C60(+(+)) ions form a surface layer that stabilizes the film so that a well-behaved profile is obtained. On the other hand, the Arn(+) gas clusters leave the material very clean but, at room temperature, the layer readily restructures into molecular bilayers, so that, although a useful measure may be made of the sputtering yield, the profiles become much more complex. This restructuring does not occur at room temperature normally but results from the actions of the beams in the sputtering process for profiling in secondary ion mass spectrometry. Better profiles may be made by reducing the sample temperature to -100 °C. This is likely to be necessary for many lower molecular weight materials (below 1000 Da) to avoid the movement of molecules. Measurements for cholesterol films on 37 nm of amiodarone on silicon are even better behaved and show the same sputtering yields at room temperature as those films directly on silicon at -100 °C. The yields for both C60(+(+)) and Arn(+) fit the Universal Equation to a standard deviation of 11%.
Gas cluster ion beams bring new opportunities for diagnostics and modification of materials surfaces. In this work impact of argon clusters on silicon dioxide has been studied by molecular dynamics simulations and experimentally. We have obtained dependencies of crater size and the SiO2 sputtering yield on cluster size and specific energy. High reactive selectivity of sputtered products has been revealed for a high specific energy of clusters. It can cause modification of the target surface layer composition in case of long time irradiation. Peculiarities of experimental and computational data matching have been discussed.
Cholesterol has been shown to promote cell proliferation/migration in many cells; however the mechanism(s) have not yet been fully identified. Here we demonstrate that cholesterol increases Ca(2+) entry via the TRPM7 channel, which promoted proliferation of prostate cells by inducing activation of the AKT and/or the ERK pathway. Additionally, cholesterol mediated Ca(2+) entry induced calpain activity that showed a decrease in E-cadherin expression, which together could lead to migration of prostate cancer cells. Overexpression of TRPM7 significantly facilitated cholesterol dependent Ca(2+) entry, cell proliferation and tumor growth. Whereas, TRPM7 silencing or inhibition of cholesterol synthesis by statin showed a significant decrease in cholesterol-mediated activation of TRPM7, cell proliferation, and migration of prostate cancer cells. Consistent with these results, statin intake was inversely correlated with prostate cancer patients and increase in TRPM7 expression was observed in samples obtained from prostate cancer patients. Altogether, we provide evidence that cholesterol-mediated activation of TRPM7 is important for prostate cancer and have identified that TRPM7 could be essential for initiation and/or progression of prostate cancer.
Argon cluster sputtering of an organic multilayer reference material consisting of two organic components, 4,4′-bis[N-(1-naphthyl-1-)-N-phenyl- amino]-biphenyl (NPB) and aluminium tris-(8-hydroxyquinolate) (Alq3), materials commonly used in organic light-emitting diodes industry, was carried out using time-of-flight SIMS in dual beam mode. The sample used in this study consists of a ~400-nm-thick NPB matrix with 3-nm marker layers of Alq3 at depth of ~50, 100, 200 and 300 nm. Argon cluster sputtering provides a constant sputter yield throughout the depth profiles, and the sputter yield volumes and depth resolution are presented for Ar-cluster sizes of 630, 820, 1000, 1250 and 1660 atoms at a kinetic energy of 2.5 keV. The effect of cluster size in this material and over this range is shown to be negligible. © 2014 The Authors. Surface and Interface Analysis published by John Wiley & Sons Ltd.
The thermotropic phase behavior of cholesterol monohydrate in water was investigated by differential scanning calorimetry, polarizing light microscopy, and x-ray diffraction. In contrast to anhydrous cholesterol which undergoes a polymorphic crystalline transition at 39 degrees C and a crystalline to liquid transition at 151 degrees C, the closed system of cholesterol monohydrate and water exhibited three reversible endothermic transitions at 86, 123, and 157 degrees C. At 86 degrees C, cholesterol monohydrate loses its water of hydration, forming the high temperature polymorph of anhydrous cholesterol. At least 24 hours were required for re-hydration of cholesterol and the rate of hydration was dependent on the polymorphic crystalline form of anhydrous cholesterol. At 123 degrees C, anhydrous crystalline cholesterol in the presence of excess water undergoes a sharp transition to a birefringent liquid crystalline phase of smectic texture. The x-ray diffraction pattern obtained from this phase contained two sharp low-angle reflections at 37.4 and 18.7 A and a diffuse wide-angle reflection centered at 5.7 A, indicating a layered smectic type of liquid crystalline structure with each layer being two cholesterol molecules thick. The liquid crystalline phase is stable over the temperature range of 123 to 157 degrees C before melting to a liquid dispersed in water. The observation of a smectic liquid crystalline phase for hydrated cholesterol correlates with its high surface activity and helps to explain its ability to exist in high concentrations in biological membranes.
Gray matter, white matter, and myelin were isolated from the frontal lobes of humans aged 10 months, 6 yr, 9 yr, and 55 yr and the lipid compositions of each were determined. Myelin had a much higher lipid content (78–81% of the dry weight) than white matter (49–66%) or gray matter (36–40%). Myelin contained much higher molar percentages of cerebroside and cerebroside sulfate, slightly higher molar percentages of cholesterol, and lower molar percentages of ethanolamine glycerophosphatides and choline glycerophosphatides than gray matter. The molar percentages of serine glycerophosphatides and sphingomyelin were about the same in each tissue.
The aldehyde content of glycerophosphatides, expressed as molar percentage of the total lipoidal residues in each lipid, were as follows: ethanolamine glycerophosphatides from myelin 40–50%; ethanolamine glycerophosphatides from gray matter 21–27%; serine glycerophosphatides from myelin 21–36%; serine glycerophosphatides from gray matter 0.3–3.8%. Choline glycerophosphatides from either tissue contained only traces of aldehydes.
The extra-myelin portion of white matter had a lipid composition that was very similar to that of myelin, but quite different from that of gray matter.
Assuming a molecular weight of 28,000 for myelin protein(s), it was calculated that for each protein molecule in human myelin there are 186 lipid molecules, 111 of which are polar lipids and 75 of which consist of cholesterol. The over-all molar ratios of the polar lipids are phosphatidal ethanolamine:serine glycerophosphatides:choline glycerophosphatides:sphingomyelin:cerebroside:cerebroside sulfate:ceramide:uncharacterized lipids 25:9:20:9:29:7:3:9. It was calculated that the molar ratio of protein amino acids to polar lipids in human myelin is 2.38 to 1.
An analysis is presented of the effect of experimental parameters such as energy, angle and cluster size on the depth resolution in depth profiling organic materials using Ar gas cluster ions. The first results are presented of the incident ion angle dependence of the depth resolution, obtained at the Irganox 1010 to silicon interface, from profiles by X-ray photoelectron spectrometry (XPS). By analysis of all relevant published depth profile data, it is shown that such data, from delta layers in secondary ion mass spectrometry (SIMS), correlate with the XPS data from interfaces if it is assumed that the monolayers of the Irganox 1010 adjacent to the wafer substrate surface have an enhanced sputtering rate. SIMS data confirm this enhancement. These results show that the traditional relation for the depth resolution, FWHM = 2.1Y(1/3) or slightly better, FWHM = PXY(1/3)/n(0.2), where n is the argon gas cluster size, and PX is a parameter for each material are valid both at the 45° incidence angle of the argon gas cluster sputtering ions used in most studies and at all angles from 0° to 80°. This implies that, for optimal depth profile resolution, 0° or >75° incidence may be significantly better than the 45° traditionally used, especially for the low energy per atom settings required for the best resolved profiles in organic materials. A detailed analysis, however, shows that the FWHM requires a constant contribution added in quadrature to the above such that there are minimal improvements at 0° or greater than 75°. A critical test at 75° confirms the presence of this constant contribution.
This chapter seeks to provide a brief overview of the present capabilities and the future possibilities of time-of-flight secondary ion mass spectrometry (ToF-SIMS) using cluster primary ions in the study of biological cells and tissue. There are basically two types of ToF-SIMS instrumentation available at present. By far, the majority of instruments utilize a pulsed primary beam and a reflectron time of flight mass spectrometer. Very recently a new concept of instrument has appeared that uses a DC primary beam and a hybrid two-stage mass spectrometer arrangement. First, the chapter summarizes the benefits common to both instruments and then outlines those specific to the new ToF-SIMS platforms. The challenges facing ToF-SIMS in biological studies is then discussed, followed by some illustrative examples of the present status of ToF-SIMS in biological analyses.
Detecting metabolites and parent compound within a cell type is now a priority for pharmaceutical development. In this context, three-dimensional secondary ion mass spectrometry (SIMS) imaging was used to investigate the cellular uptake of the antiarrhythmic agent amiodarone, a phospholipidosis-inducing pharmaceutical compound. The high lateral resolution and 3D imaging capabilities of SIMS combined with the multiplex capabilities of ToF mass spectrometric detection allows for the visualization of pharmaceutical compound and metabolites in single cells. The intact, unlabeled drug compound was successfully detected at therapeutic dosages in macrophages (cell line: NR8383). Chemical information from endogenous biomolecules was used to correlate drug distributions with morphological features. From this spatial analysis, amiodarone was detected throughout the cell with the majority of the compound found in the membrane and subsurface regions and absent in the nuclear regions. Similar results were obtained when the macrophages were doped with amiodarone metabolite, desethylamiodarone. The FWHM lateral resolution measured across an intracellular interface in a high lateral resolution ion images was approximately 550 nm. Overall, this approach provides the basis for studying cellular uptake of pharmaceutical compounds and their metabolites on the single cell level.
Imaging mass spectrometry has shown to be a valuable method in medical research and can be performed using different instrumentation and sample preparation methods, each one with specific advantages and drawbacks. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has the advantage of high spatial resolution imaging but is often restricted to low mass molecular signals and can be very sensitive to sample preparation artefacts. In this report we demonstrate the advantages of using gas cluster ion beams (GCIBs) in combination with trifluoracetic acid (TFA) vapour exposure for the imaging of lipids in mouse brain sections. There is an optimum exposure to TFA that is beneficial for increasing high mass signal as well as producing signal from previously unobserved species in the mass spectrum. Cholesterol enrichment and crystallisation on the sample surface is removed by TFA exposure uncovering a wider range of lipid species in the white matter regions of the tissue greatly expanding the chemical coverage and the potential application of ToF-SIMS imaging in neurological studies. 40 keV Ar4000 + in combination with TFA treatment facilitates high resolution, high mass imaging closing the gap between ToF-SIMS and MALDI.
With the advent of large argon cluster beams, organic materials can be sputtered without the accumulation of radiation damage. The dual beam mode with a Bi cluster analysis beam is successfully applied to depth profiling and 3D analysis of organic materials providing both high-depth resolution and high-lateral resolution. For the analysis of very small sample volumes, it is desirable that a rather large fraction of the material is consumed by the analysis beam and contributes to the analytical signal. However, at higher analysis beam sputter rates, the radiation damage by the Bi clusters can become quite severe leading to a molecular ion signal decay with depth. In this paper, we investigate the conditions for the optimum use of the sample material in the dual beam mode using the Irganox delta layer structure introduced by NPL (National Physical Laboratory (UK)). A model that describes the dual beam mode and allows calculating the intact area fraction in the steady state is presented. Yield volumes and damage volumes are determined for Bi3+ and Bi5++ as well as the useful sample fraction that contributes to the molecular ion signal. We will compare the results to the depth profiling with C60 and Ar clusters in a single DC beam mode. Copyright
An analysis is provided of the data of Cristaudo et al. for the sputtering yields of polystyrene and polymethylmethacrylate by argon cluster ion beams as a function of their molecular weight. This analysis is made using the universal sputtering equation of Seah to evaluate the effect of the end-group density on the parameter A. This parameter is thought to be related to the average energy per atom (excluding hydrogen) to liberate fragments and it is shown that A decreases as the end-group density rises in the manner expected. Equations are provided relating A to the molecular weight or the end-group density that are obeyed for both materials. Copyright © 2014 Crown Copyright.
Dual beam depth profiling was applied in order to investigate the possibilities and limitations of C60 and Ar cluster ion sputtering for depth profiling of polymer materials. Stability and intensity of characteristic high mass molecular ion signals as well as sputter yields will be compared. For this purpose, different beam energies resulting in 2–10 eV/atom for Arn and 167–667 eV/atom for C60 sputtering were applied to various polymer samples. From our experiments, we can conclude that most of the limitations C60 sputtering suffers from could be successfully overcome and that the Ar gas cluster ion beam seems to be a more universal tool for sputtering of organic materials. Copyright © 2012 John Wiley & Sons, Ltd.
Ar cluster sputtering of organic multilayers such as organic light-emitting diode model structures and Irganox delta layers is studied with time-of-flight secondary ion mass spectroscopy in the dual beam mode. Results for sputtering yield volumes and depth resolution are presented for Ar clusters with sizes from 500 to 5000 atoms in the energy range from 2.5 to 20 keV. The sputtering yield volume shows a linear dependence on the energy per atom for all materials in this study with a material-dependent threshold below 1 eV/atom. The sputtering yield volume at a given energy per atom increases with the cluster size. At constant beam energies, the sputtering yield volume decreases slightly with increasing cluster size. The depth resolution is investigated for the two model systems as a function of energy and cluster size, and it will be shown that the depth resolution depends mainly on the sample roughening. The depth resolution is approximately proportional to the depth of the impact crater at a given cluster size and energy. The optimum depth resolution achieved is in the range of 4–5 nm and is fairly constant with depth. At very low energies per atom close to the threshold energy, ripple formation is observed that leads to a fast degradation of the depth resolution with depth. This can be completely eliminated by fast sample rotation. Finally, the perspective of 3D analysis of organic devices with high depth resolution in the dual beam mode will be discussed. Copyright © 2012 John Wiley & Sons, Ltd.
A study has been made of the fragmentation of three organic molecules, fluorenylmethyloxycarbonyl-l-pentafluorophenylalanine, tris(8-hydroxyquinolinato)aluminum, and Irganox 1010 under bombardment by argon cluster ions with sizes, n, between 500 and 10000 and for 10 and 20 keV energies, E. It is shown that the intensities of the fragments are governed by the same relation as that for the total volume sputtering yield but that, importantly, the parameter AM that governs the transition in behavior between low and high values of E/n is thought to be related to the energy required to remove that particular fragment from the ensemble of molecules. This causes a great reduction in intensity for the more strongly bound fragments as n is increased, such that, at n = 10000, the intensities of the molecular group peaks, which are weakly bound to the substrate, then dominate the spectrum. For studies involving weakly bound molecules, the low E/n values give the most intense molecular group peaks, but high E/n values give a greater number of small, characteristic fragments. For molecules more strongly bound to the bulk or substrate, it is not as likely that the low E/n data will be so helpful.
An analysis is made of the sputtering yields of materials for argon gas cluster ion beams used in SIMS and XPS as a function of the beam energy, E, and the cluster size, n. The analysis is based on the yield data for the elements Si and Au, the inorganic compound SiO2, and the organic materials Irganox 1010, the OLED HTM-1, poly(styrene), poly(carbonate), and poly(methyl methacrylate). The argon primary ions have cluster sizes, n, in the range 100–16 000 and beam energies, E, from 2.5 to 80 keV. It is found that the elemental and compound data expressed as the yields, Y, of atoms sputtered per primary ion may all be described by a simple universal equation: Y/n = (E/An)q/[1 + (E/An)q−1] where the parameters A and q are established by fitting. The sputtering yields of the three organic materials are given as yield volumes expressed in nm3. For these, an extra parameter B is included multiplying the right-hand side of the equation where B is found by fitting to be of the order (0.18 nm)3 to (0.26 nm)3. This universal equation exhibits no threshold energy, and no deviation was observed from the equation, indicating that any threshold energy would have to be significantly below E/n = 1 eV per atom. The equation also shows that doubling the cluster size at the same energy per atom simply doubles the sputtering yield so that in this sense, and probably this sense alone, the sputtering effects are linearly additive. The parameter A is related, inversely, to the mean sputtered fragment size, and the low A values for organic materials are indicative of high yield volumes. For materials with low A values, the universal equation is close to a linear dependence on energy, and if that linear dependence is assumed, an apparent threshold energy is predicted and observed experimentally.
Altered lipid metabolism is increasingly recognized as a signature of cancer cells. Enabled by label-free Raman spectromicroscopy, we performed quantitative analysis of lipogenesis at single-cell level in human patient cancerous tissues. Our imaging data revealed an unexpected, aberrant accumulation of esterified cholesterol in lipid droplets of high-grade prostate cancer and metastases. Biochemical study showed that such cholesteryl ester accumulation was a consequence of loss of tumor suppressor PTEN and subsequent activation of PI3K/AKT pathway in prostate cancer cells. Furthermore, we found that such accumulation arose from significantly enhanced uptake of exogenous lipoproteins and required cholesterol esterification. Depletion of cholesteryl ester storage significantly reduced cancer proliferation, impaired cancer invasion capability, and suppressed tumor growth in mouse xenograft models with negligible toxicity. These findings open opportunities for diagnosing and treating prostate cancer by targeting the altered cholesterol metabolism.
Citation Format: Hyeon Jeong Lee, Shuhua Yue, Junjie Li, Seung-Young Lee, Tian Shao, Bing Song, Liang Cheng, Timothy A. Masterson, Xiaoqi Liu, Timothy L. Ratliff, Ji-Xin Cheng. Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A06.
This fundamental contribution on time-of-flight secondary ion mass spectrometry polymer depth-profiling by massive argon clusters is focused on the investigation of the influence of the Ar cluster size and the molecular weight of the investigated polymers on the efficiency of the sputtering process. For this purpose, 100-nm thin films of polymethylmethacrylate and polystyrene, with a range of molecular weights (103–1.5 × 105 amu) spin-coated onto Si wafers, are sputtered by 10 keV Arn+ cluster ions with selected sizes (1500, 3000 and 5000 atoms/cluster) employing Bi5+ as an analysis source. The experiments show that the sputtering yield volume (nm3/primary ion), Y, decreases with the increase of the molecular weight, Mw, for a selected cluster size, and at constant molecular weight, Y decreases with the increase in cluster size. The experimental results are in good agreement with molecular dynamics simulations and previous studies for high molecular weight PS. Furthermore, the trend of Y versus Mw seems to be related to the variation of the glass transition temperature (Tg) of the investigated polymers. Copyright © 2014 John Wiley & Sons, Ltd.
Molecular depth profiling of organic thin films by erosion with energetic cluster ion beams is a unique aspect of secondary ion mass spectrometry (SIMS) experiments. Although depth profiles of complex multilayer organic structures can be acquired with little damage accumulation and with depth resolution of <10 nm using either C60+ or Arx+ with x = 500-5000, hybrid materials consisting of both organic and inorganic layers often yield poor results. To unravel the factors that lead to this difficulty, we developed a model system composed of a thin gold layer of 1.4 to 3.5 nm deposited either on top of or sandwiched within a cholesterol thin film matrix which is several hundred nanometers thick. For these systems, the results show that by erosion with a 40 keV C60+ beam, reliable depth profiles can always be acquired as indicated by the presence of a steady state molecular ion signal. During the erosion process, however, gold atoms from the gold overlayer are implanted into the cholesterol matrix beneath it, resulting in a reduced sputter yield, an increase in the amount of cholesterol fragmentation and an increase in the thickness of the cluster ion-induced altered layer. The results also show that the effects of the metal film on the organic substrate are independent of the gold film thickness once the film thickness exceeds 1.4 nm. In general, this model study provides mechanistic insight into the depth profiling of heterogeneous thin film structures and offers a possible path for improving the quality of the depth profiles by employing low energy atomic ion sputtering in the region of the metal layer.
Single-cell imaging mass spectrometry (IMS) is a powerful technique used to map the distributions of endogenous biomolecules with subcellular resolution. Currently, secondary ion mass spectrometry is the predominant technique for single-cell IMS, thanks to its submicron lateral resolution and surface sensitivity. However, recent methodological and technological developments aimed at improving the spatial resolution of matrix assisted laser desorption ionization (MALDI) have made this technique a potential platform of single-cell IMS. MALDI opens the field of single-cell IMS to new possibilities, including single cell proteomic imaging and atmospheric pressure analyses; however, sensitivity is a challenge. In this report, we estimate the availability of proteins and lipids in a single cell and discuss strategies employed to improve sensitivity at the single-cell level.
Argon cluster ion sources for sputtering and secondary ion mass spectrometry use projectiles consisting of several hundreds of atoms, accelerated to 10-20 keV, and deposit their kinetic energy within the top few nanometers of the surface. For organic materials, the sputtering yield is high removing material to similar depth. Consequently, the exposed new surface is relatively damage free. It has thus been demonstrated on model samples that it is now really possible to perform dual beam depth profiling experiments in organic materials with this new kind of ion source. Here, this possibility has been tested directly on tissue samples, 14 µm thick rat brain sections, allowing primary ion doses much larger than the so-called static SIMS limit and demonstrating the possibility to enhance the sensitivity of TOF-SIMS biological imaging. However, the depth analyses have also shown some variations of the chemical composition as a function of depth, particularly for cholesterol, as well as some possible matrix effects due to the presence or absence of this compound.
Imaging time-of-flight-secondary ion mass spectrometry (TOF-SIMS) was used to analyse the lateral distributions of lipids on the surface of freeze-dried mouse brain sections. Tissue sections (14μm thick) were prepared by cryosectioning, placed on glass or Si substrates and desalinated by submersion in NH3HCOO solution. Immediately prior to analysis, the samples were freeze-dried by thawing the sample in vacuum. TOF-SIMS analysis was carried out using 25keV Au3+ or Bi3+ primary ions, always keeping the accumulated ion dose below 4×1012ions/cm2. Positive and negative ion images over the entire mouse brain section and of analysis areas down to 100μm×100μm show characteristic distributions of various lipids. The signals from cholesterol and sulfatides are primarily located to white matter regions, while the phosphocholine and phosphatidylinositol signals are strongest in grey matter regions. By using two different staining methods, structures observed in the TOF-SIMS images could be identified as ribosome-rich regions and cell nuclei, respectively. Analysis of freeze-dried mouse brain sections at varying sample temperatures between −130 and 60°C showed an abrupt increase in the cholesterol signal at T>0°C, indicating extensive migration of cholesterol to the tissue surface under vacuum conditions.
With the aim of evaluating the potential of SIMS to provide molecular information from small molecules within biological systems, here we investigate the effect of different biological compounds as they act as matrices. The results highlight the fact that the chemical environment of a molecule can have a significant effect on its limit of detection. This has implications for the imaging of drugs and xenobiotics in tissue sections and other biological matrices.A 1:1 mixture of the organic acid 2,4,6-trihydroxyacetophenone and the dipeptide valine–valine demonstrates that almost complete suppression of the [M+H]+ ion of one compound can be caused by the presence of a compound of higher proton affinity. The significance of this is highlighted when two similar drug molecules, atropine (a neutral molecule) and ipratropium bromide (a quaternary nitrogen containing salt) are mixed with brain homogenate. The atropine [M+H]+ ion shows significant suppression whilst the [M−Br]+ of ipratopium bromide is detected at an intensity that can be rationalised by its decreased surface concentration.By investigating the effect of two abundant tissue lipids, cholesterol and dipalmitoylphosphatidyl choline (DPPC), on the atropine [M+H]+ signal detected in mixtures with these lipids we see that the DPPC has a strong suppressing effect, which may be attributed to gas phase proton transfer.
A study is presented of the effects of the different positive ion beam species: Ar+, Ga+, Xe+, Cs+ and SF5+ and of their energies from 4 to 25 keV, on the fragmentation behaviour in static Secondary Ion Mass Spectrometry (SIMS) spectra for samples of the polymers: polytetrafluoroethylene (PTFE), polystyrene (PS) and polycarbonate (PC). The overall effect of energy is found to be weak over the entire mass spectrum. However, large differences are observed in restricted mass ranges amongst fragmentation groups. The fragmentation is quantified in terms of the partition functions of the fragments from a plasma with effective temperature, Tp. It is found that fragmentation is least for high mass projectiles at low energies, but that the trend is different for polyatomic ions. A methodology is developed, which unifies all of the fragmentation behaviour to a single plot — the Unified Cascade Gradient plot. An equivalence of mass and energy is shown and that the chemistry of the bombarding ion is unimportant. By extrapolation of the data to low Tp, a new spectroscopy, known as gentle-SIMS or G-SIMS is formed. The G-SIMS spectrum is in the static regime. Significant peaks in the G-SIMS spectra are those peaks, which would be emitted from a surface plasma of very low temperature and thus have little post-emission rearrangement or fragmentation. Those peaks are, thus, directly characteristic of the material without rearrangement and provide a direct interpretation and identification. In the tests of the method described, this is supported and indicates that the G-SIMS analysis will be significantly less ambiguous than static SIMS so that interpretation will be possible in the absence of a relevant reference spectrum.
Time-of-flight secondary ion mass spectrometry (TOF-SIMS) was utilized to address the issue of cholesterol localization in rat cerebellum, a subject not previously investigated.Rat cerebellum was prepared by three different procedures: (1) fixation in formaldehyde, freeze-protection by sucrose, freezing in liquid nitrogen and sectioning by cryoultramicrotomy and drying at room temperature or (2) freezing in liquid nitrogen, cryostat sectioning at −40°C and drying at room temperature or (3) high-pressure freezing, freeze-fracturing and freeze-drying.The samples were analyzed in an imaging TOF-SIMS instrument equipped with a Bi1–7+-source. The cholesterol signal (m/z 369 and 385), showed high intensity in the glial cells in white matter and lower intensity in Purkinje cells and in nuclei of granular layer cells. Specimen treated by procedure 1 showed some signs of diffusion of cholesterol in the tissue. Specimen treated by procedure 2 showed freeze-damage of the cells. Specimen treated by procedure 3 showed distinct localization of cholesterol in well preserved tissue. Thus, high-pressure freezing and freeze-fracturing was used for further characterization of the distribution of cholesterol in rat cerebellum.
Atomic force microscopy (AFM) and chemical force microscopy (CFM) techniques have been used to characterize the chemical functionality of cholesterol monohydrate single-crystal surfaces in different solution environments. Both synthetic and natural crystals adopt a platelike habit in which the largest face is (001). Under aqueous and organic solution conditions, in situ contact mode topographical images reveal that the plate face is primarily terminated by bilayer molecular structures and therefore largely homogeneous. Adhesion force measurements obtained with chemically modified tips demonstrate that the functionality of the crystal surface can be altered by changes in the solution composition. The 3-hydroxyl end of cholesterol molecules is presented on the plate face in aqueous media, while alkyl tail groups terminate the surface in organic solutions. Contact angle measurements on (001) surfaces exposed to solvents of different hydrophilicity also show similar trends, providing additional support to these AFM assignments. These studies link molecular-level and macroscopic adhesion properties and demonstrate that solution environment can exert a strong influence on the surface properties of this important biomaterial.
Although cholesterol has been involved in the pathophysiology of Alzheimer disease (AD), its distribution in the cerebral cortex over the course of AD is unknown. We describe an original method to quantify cholesterol distribution using time-of-flight secondary ion mass spectrometry imaging. Cholesterol was unevenly distributed along the cortical thickness, being more abundant close to the white matter, in both control and AD cases. However, the mean cholesterol signal was significantly higher in the lower half of the cortex in AD samples compared to controls. This increase, when converted into cortical layers, was statistically significant for layers III and IV and did not reach significance in layers V + VI, the variability being too high at the interface between grey and white matter. The density of neurofibrillary tangles and of senile plaques was not statistically linked to the abundance of cholesterol. Cholesterol overload thus appears a new and independent alteration of AD cerebral cortex. The structure in which cholesterol accumulates and the mechanism of this accumulation remain to be elucidated.
The depth profiling of organic materials with argon cluster ion sputtering has recently become widely available with several manufacturers of surface analytical instrumentation producing sources suitable for surface analysis. In this work, we assess the performance of argon cluster sources in an interlaboratory study under the auspices of VAMAS (Versailles Project on Advanced Materials and Standards). The results are compared to a previous study that focused on C(60)(q+) cluster sources using similar reference materials. Four laboratories participated using time-of-flight secondary-ion mass spectrometry for analysis, three of them using argon cluster sputtering sources and one using a C(60)(+) cluster source. The samples used for the study were organic multilayer reference materials consisting of a ∼400-nm-thick Irganox 1010 matrix with ∼1 nm marker layers of Irganox 3114 at depths of ∼50, 100, 200, and 300 nm. In accordance with a previous report, argon cluster sputtering is shown to provide effectively constant sputtering yields through these reference materials. The work additionally demonstrates that molecular secondary ions may be used to monitor the depth profile and depth resolutions approaching a full width at half maximum (fwhm) of 5 nm can be achieved. The participants employed energies of 2.5 and 5 keV for the argon clusters, and both the sputtering yields and depth resolutions are similar to those extrapolated from C(60)(+) cluster sputtering data. In contrast to C(60)(+) cluster sputtering, however, a negligible variation in sputtering yield with depth was observed and the repeatability of the sputtering yields obtained by two participants was better than 1%. We observe that, with argon cluster sputtering, the position of the marker layers may change by up to 3 nm, depending on which secondary ion is used to monitor the material in these layers, which is an effect not previously visible with C(60)(+) cluster sputtering. We also note that electron irradiation, used for charge compensation, can induce molecular damage to areas of the reference samples well beyond the analyzed region that significantly affects molecular secondary-ion intensities in the initial stages of a depth profile in these materials.
An analysis is made of the molecular secondary ion yield (MSIY) variation with primary ion source mass and number, n, of constituent particles. The theory is based on the linear cascade model, extended into the nonlinear regime for higher deposition energy densities using Sigmund and Claussen's thermal spike model. The analysis is generalised to remove sample specificity and is evaluated for light, organic materials. It provides the MSIY dependence for this material as a function of the primary ion particle parameters. This is applied to the data of Kersting et al. and of Kollmer for the (M − H)− de-protonated secondary ion yields, Y(M − H)−, from Irganox 1010 for Ga+, Cs+, SF5+, Au+, Au2+, Au3+ and C60+ primary ions. The previously postulated dependence of Y(M − H)− on the sputtering yield squared is validated. This formulation permits the prediction of Y(M − H)− for new primary ions such as Bin+ and C70+. It is shown that further MSIY gains of possibly up to an order of magnitude are attainable. For analysis, raising the MSIY is extremely helpful but it is the efficiency (the ratio of the MSIY to the disappearance cross-section) that is critical. The distinction between the damage and disappearance cross-sections is clarified and a description involving both terms and correlating with data for all ions is given. It is shown that the efficiency increases with Y(M − H)−, initially linearly and finally with a square root dependence. The chemical nature of the primary ion species is shown to be relatively unimportant in these processes. © Crown copyright 2007. Reproduced with the permission of Her Majesty's Stationery Office. Published by John Wiley & Sons, Ltd.
An interlaboratory study involving 32 time-of-flight static SIMS instruments from 13 countries has been conducted. In Part I of the analysis of data, we showed that 84% of instruments have excellent repeatabilities of better than 1.9% and that a relative instrument spectral response (RISR) can be used to evaluate variations between different generic types of instrument. Use of the RISR improves comparability between instruments by a factor of 33. Here, in Part II, we study the accuracy of the mass scale calibration in TOF-SIMS and evaluate instrument compatibility with G-SIMS. We show that the accuracy of calibration of the mass scale is much poorer than generally expected (−60 ppm for peaks <200 u and −150 ppm for a large molecular peak at 647 u). This is a major issue for analysts. Elsewhere, we have developed a detailed study of the factors affecting the mass calibration and have developed a generic protocol that improves accuracy by a factor of 5. Here, this framework of understanding is used to interpret the results presented. Furthermore, we show that eight out of the ten participants submitting data for G-SIMS could use operating conditions that generated G-SIMS spectra of the PC reference material. This demonstrates that G-SIMS may be conducted with a wide variety of instrument designs. © Crown Copyright 2007. Reproduced by permission of the Controller of HMSO. Published by John Wiley & Sons, Ltd.
Cholesterol and its relatives possessing the 1,2- cyclopentanoperhydrophenanthrene ring system form the sterolome, which comprises a chemical library of more than 1000 natural products found in all forms of eukaryotes and some prokaryotes that serve a myriad of biological functions. Central to the advances of the past two decades is the development of molecular genetic approaches that have witnessed the cloning, primary amino acid sequences, and functional characterization of a large number of enzymes that act on sterol and revealed unexpected inborn errors of cholesterol metabolism. The relevant committed step that distinguishes sterol from isoprenoid-triterpenoid biosynthesis occurs at the cyclization of oxidosqualene. There is still much to learn about sterol biosynthesis and the enzymes involved in the pathway.
G-SIMS (gentle-SIMS) is a powerful method that considerably simplifies complex static secondary ion mass spectrometry (SSIMS) analysis of organics at surfaces. G-SIMS uses two primary ion beams that generate high and low fragmentation conditions at the surface. This allows an extrapolation to equivalent experimental conditions with very low fragmentation. Consequently, the spectra are less complex, contain more structural information and are simpler to interpret. In general, G-SIMS spectra more closely resemble electron ionisation mass spectra than SSIMS spectra. A barrier for the wider uptake of G-SIMS is the requirement for two ion beams producing suitably different fragmentation conditions and the need for their spatial registration (spatial alignment) at the surface, which is especially important for heterogeneous samples. The most popular source is the liquid metal ion source (LMIS), which is now sold with almost every new time-of-flight (TOF)-SIMS instrument. Here, we have developed a novel bismuth-manganese emitter (the 'G-tip') for the popular LMISs. This simplifies the alignment and gives excellent G-SIMS imaging and spectroscopy without significantly compromising the bismuth cluster ion beam performance.
Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an established technique for the characterization of solid sample surfaces. The introduction of polyatomic ion beams, such as C(60), has provided the associated ability to perform molecular depth-profiling and 3D molecular imaging. However, not all samples perform equally under C(60) bombardment, and it is probably naïve to think that there will be an ion beam that will be applicable in all situations. It is therefore important to explore the potential of other candidates. A systematic study of the suitability of argon gas cluster ion beams (Ar-GCIBs) of general composition Ar(n)(+), where n = 60-3000, as primary particles in TOF-SIMS analysis has been performed. We have assessed the potential of the Ar-GCIBs for molecular depth-profiling in terms of damage accumulation and sputter rate and also as analysis beams where spectral quality and secondary ion yields are considered. We present results with direct comparison with C(60) ions on the same sample in the same instrument on polymer, polymer additive, and biomolecular samples, including lipids and small peptides. Large argon clusters show reduced damage accumulation compared with C(60) with an approximately constant sputter rate as a function of Ar cluster size. Further, on some samples, large argon clusters produce changes in the mass spectra indicative of a more gentle ejection mechanism. However, there also appears to be a reduction in the ionization of secondary species as the size of the Ar cluster increases.
Secondary ion emission with large gas cluster ion is reviewed from the point of view of secondary ion mass spectroscopy (SIMS). We have proposed to use large cluster ions to realize fragment-free ionization for SIMS analysis for various organic materials, such as amino acids and peptides. When large cluster ions with optimized size and energy were incident on biomolecular samples, the relative yields of the fragment ions decreased drastically with the velocity of incident cluster ions. Molecular depth profiling capability is also demonstrated by using large gas cluster ions as the primary ion for SIMS.
The present theoretical study explores the interaction of various energetic molecular projectiles and clusters with a model polymeric surface, with direct implications for surface analysis by mass spectrometry. The projectile sizes (up to 23 kDa) are intermediate between the polyatomic ions (SF(5), C(60)) used in secondary ion mass spectrometry and the large organic microdroplets generated, for example, in desorption electrospray ionization. The target is a model of amorphous polyethylene, already used in a previous study [Delcorte, A.; Garrison, B. J. J. Phys. Chem. C 2007, 111, 15312]. The chosen method relies on classical molecular dynamics (MD) simulations, using a coarse-grained description of polymeric samples for high energy or long time calculations (20-50 ps) and a full atomistic description for low energy or short time calculations (<1 ps). Two regions of sputtering or desorption are observed depending on the projectile energy per nucleon (i.e., effectively the velocity). The transition, occurring around 1 eV/nucleon, is identified by a change of slope in the curve of the sputtering yield per nucleon vs energy per nucleon. Beyond 1 eV/nucleon, the sputtering yield depends only on the total projectile energy and not on the projectile nuclearity. Below 1 eV/nucleon, i.e., around the sputtering threshold for small projectiles, yields are influenced by both the projectile energy and nuclearity. Deposition of intact molecular clusters is also observed at the lowest energies per nucleon. The transition in the sputtering curve is connected to a change of energy deposition mechanisms, from atomistic and mesoscopic processes to hydrodynamic flow. It also corresponds to a change in terms of fragmentation. Below 1 eV/nucleon, the projectiles are not able to induce bond scissions in the sample. This region of molecular emission with minimal fragmentation offers new analytical perspectives, out of reach of smaller molecular clusters such as fullerenes.
The effect of incident angle on the quality of SIMS molecular depth profiling using C(60) (+) was investigated. Cholesterol films of ~300 nm thickness on Si were employed as a model and were eroded using 40 keV C(60) (+) at an incident angle of 40° and 73° with respect to the surface normal. The erosion process was characterized by determining at each angle the relative amount of chemical damage, the total sputtering yield of cholesterol molecules, and the interface width between the film and the Si substrate. The results show that there is less molecule damage at an angle of incidence of 73° and that the total sputtering yield is largest at an angle of incidence of 40°. The measurements suggest reduced damage is not necessarily dependent upon enhanced yields and that depositing the incident energy nearer the surface by using glancing angles is most important. The interface width parameter supports this idea by indicating that at the 73° incident angle, C(60) (+) produces a smaller altered layer depth. Overall, the results show that 73° incidence is the better angle for molecular depth profiling using 40 keV C(60) (+).
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.
Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/62532/1/267287a0.pdf
Crystalline cholesterol undergoes a phase transition a few degrees below human body temperature. The high-temperature form
has an unusually complex structure with 16 independent molecules. In the transition two molecules change side chain conformation,
four reorient about their long axes, and ten remain unchanged. The transition mechanism implies relatively nonspecific intermolecular
interactions, qualitatively consistent with the behavior of cholesterol in biomembranes. The transition preserves a remarkably
closely obeyed pseudosymmetry present in the structure.
Experimental data that define conditions under which cholesterol crystallites form in cholesterol/phospholipid model membranes are reviewed. Structural features of the phospholipids that determine cholesterol crystallization include the length and degree of unsaturation of the acyl chains, the presence of charge on the headgroups and interheadgroup hydrogen bonds.
Here, we show the localization of a whole organic molecule in biological tissue using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Rat kidneys were sectioned by cryoultramicrotomy and dried at room temperature. The samples were covered with a thin silver layer and analyzed in an imaging TOF-SIMS instrument equipped with a Ga(+)-source. The cholesterol signal showed a high concentration in the nuclear areas of the epithelial cells and a lower concentration over areas representing the basal lamina of renal tubules. A more diffuse distribution of cholesterol was also found over areas representing the cytoplasm or plasma membrane of the epithelial cells.
Membrane lipids are essential for biological functions ranging from membrane trafficking to signal transduction. The composition of lipid membranes influences their organization and properties, so it is not surprising that disorders in lipid metabolism and transport have a role in human disease. Significant recent progress has enhanced our understanding of the molecular and cellular basis of lipid-associated disorders such as Tangier disease, Niemann-Pick disease type C and atherosclerosis. These insights have also led to improved understanding of normal physiology.
In this paper, the effect of prolonged C60(+) primary ion bombardment on the chemical information available from a section of rat brain is discussed. Initial attempts demonstrate the rapid loss of molecular signal from the bombarded area with both C60(+) and Au(+) used as a monatomic comparison. However, the nature of this signal disappearance is shown to be different. Analysis of the C60(+) data indicates a correlation between signal loss and the appearance of sodium and potassium adducts of phosphate and protein fragments; this is supported by model systems. By using an ammonium formate wash to reduce the salt levels within the tissue this effect is removed, allowing the chemistry of the tissue section to be better probed. Results collected from multiple sections suggest that at room temperature under vacuum conditions there is a migration of lipids to the surface of the tissue. Three-dimensional (3D) imaging is used to demonstrate that once these lipids are removed other species, such as proteins, are uncovered. By depth profiling the sample in a frozen state, the degree and importance of lipid migration to the observed localization of native compounds is assessed. This investigation into the behavior of biological tissue under high C60(+) fluxes not only allows an evaluation of the potential accuracy of 3D SIMS mapping of important biological molecules but also demonstrates the possibility of using ion doses beyond the traditional "static limit" to provide higher secondary ion yields that could lead to greater detection limits and smaller useful lateral resolution within such analyses.
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