Biological tissue imaging with time-of-flight secondary ion mass spectrometry and cluster ion sources

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) using liquid metal ion guns (LMIGs) is now sensitive enough to produce molecular-ion images directly from biological tissue samples. Primary cluster ions strike a spot on the sample to produce a mass spectrum. An image of this sample is achieved by rastering the irradiated point over the sample surface. The use of secondary ion mass spectrometry for mapping biological tissue surfaces provides unique analytical capabilities; in particular, it enables in a single acquisition a large variety of biological compounds to be localised on a micrometer scale and scrutinised for colocalisations. Without any treatment of the sample, this method is fully compatible with subsequent and complementary analyses like fluorescence microscopy, histochemical staining, or even matrix-assisted laser desorption/ionisation imaging. Basic physical concepts, required instrumentation (ion source and mass analyzer), sample preparation methods, image acquisition, image processing, and emerging biological applications will be described and discussed.     

Charge compensation for imaging large insulating samples by using secondary ion tandem mass spectrometry

A charge compensation technique has been developed for secondary ion mass spectrometry and imaging of insulating samples as large as 1 cm² using a triple quadrupole-based microprobe. The microprobe secondary ion extraction field is synchronized with a periodic primary Cs⁺ beam to allow a sheetlike beam of 5-eV electrons to pass over the sample surface when the extraction field is zeroed. Electrons are attracted to, and neutralize, any points on the sample that have accumulated positive charge. Positive secondary ion images from Teflon®, a well-known insulator, illustrate the effectiveness of charge compensation. Locating and identifying analytes on dry filter paper by using tandem mass spectrometry are also demonstrated.     

Why are electron capture negative ion mass spectra not reproducible? An ion source problem

Differences in the electron capture negative ion mass spectra of environmentally related organic compounds acquired on a VG 30-250 triple quadruple mass spectrometer and on an HP 5985B gas chromatography/mass spectrometry system were investigated with respect to the ion formation process. Neither ion source temperature nor pressure was responsible for the differences. The populations of thermal electrons in both ion sources were experimentally determined and found to be similar, suggesting that electron capturing reactions should proceed with comparable efficiencies in both ion sources. The ion extraction efficiencies of the two instruments were examined by monitoring the transmission profiles of low- and high-mass ions as a function of lens potentials. Results indicated that the HP 5985B extraction lens significantly suppressed low-mass ions. Further, theoretical evaluation of ion trajectories using SIMION suggested that on the HP 5985B, low-mass ions entered the mass analyzer as a defocused beam, but high-mass ions entered the analyzer as a well-collimated beam. On the VG 30–250, low- and high-mass ions were transmitted to the analyzer with equal efficiency by the ion extraction system.     

Radial stratification of ions as a function of mass to charge ratio in collisional cooling radio frequency multipoles used as ion guides or ion traps

Collisional cooling in radio frequency (RF) ion guides has been used in mass spectrometry as an intermediate step during the transport of ions from high pressure regions of an ion source into high vacuum regions of a mass analyzer. Such collisional cooling devices are also increasingly used as 'linear', two-dimensional (2D) ion traps for ion storage and accumulation to achieve improved sensitivity and dynamic range. We have used the effective potential approach to study m/z dependent distribution of ions in the devices. Relationships obtained for the ideal 2D multipole demonstrate that after cooling the ion cloud forms concentric cylindrical layers, each of them composed of ions having the same m/z ratio; the higher the m/z, the larger is the radial position occupied by the ions. This behavior results from the fact that the effective RF focusing is stronger for ions of lower m/z, pushing these ions closer to the axis. Radial boundaries of the layers are more distinct for multiply charged ions, compared to singly charged ions having the same m/z and charge density. In the case of sufficiently high ion density and low ion kinetic energy, we show that each m/z layer is separated from its nearest neighbor by a radial gap of low ion density. The radial gaps of low ion population between the layers are formed due to the space charge repulsion. Conditions for establishing the m/z stratified structure include sufficiently high charge density and adequate collisional relaxation. These conditions are likely to occur in collisional RF multipoles operated as ion guides or 2D ion traps for external ion accumulation. When linear ion density increases, the maximum ion cloud radius also increases, and outer layers of high m/z ions approach the multipole rods and may be ejected. This 'overfilling' of the multipole capacity results in a strong discrimination against high m/z ions. A relationship is reported for the maximum linear ion density of a multipole that is not overfilled.     

The Orbitrap mass analyzer with direct ion injection interfaced to a laser desorption/ionization ion source

The results of a study on interfacing an Orbitrap mass analyzer with direct ion injection to a surface assisted laser desorption/ionization (SALDI) ion source are presented. Osmium complexes with 8-mercaptoquinoline were studied. Titanium oxide thin films prepared by electron beam evaporation were found an effective emitter of the ions of the test complexes. It was demonstrated that interfacing the Orbitrap mass analyzer to a SALDI source can significantly improve the analytical performance of this method in comparison to a typical combination of SALDI/time-of-flight mass spectrometer.     

Topography and field effects in secondary ion mass spectrometry Part II: insulating samples

A study is conducted on the effects of sample topography on the secondary ion mass spectrometry (SIMS) analysis of insulating samples, using poly(ethylene terephthalate) fibres (100 µm diameter) as a model system and simulations of the ion extraction field using finite element analysis. We focus on two significant issues: topographic field effects caused by the penetration of the extraction field into the sample, and the effect of charge compensation on the secondary ion images. Guidance is provided for setting the reflector voltage correctly for insulating fibres in reflectron SIMS instruments. The presence of the topographic sample distorts the extraction field, causing the secondary ions to be deflected laterally. This results in the severe loss of ion signals from the sides of the fibres because of the limited angular acceptance of the analyser. Strategies to reduce topographic field effects, including alternative sample mounting methods, are discussed. We also find that, in general, insulating samples are charged by the flood gun electrons resulting in a negative surface potential. This causes large variations in the SIMS images depending on the electron current, electron energy, raster mode and secondary ion polarity. Recommendations are given for analysts to obtain more reproducible images and reduce the effect of differential electron charging, for example by using a lower electron flood beam energy. © 2011 Crown copyright.     

Ga‐focused ion beam time‐of‐flight secondary ion mass spectrometer analysis of the grain boundary segregation/precipitation of boron in steel

Ga‐focused ion beam time‐of‐flight secondary ion mass spectrometry (FIB‐TOF‐SIMS) analysis was performed to investigate the grain boundary segregation/precipitation of boron in steel. To overcome the low secondary ion yield from the primary Ga⁺ source and the sensitivity using a high‐resolution Ga‐FIB source, a low energy oxygen ion beam was used prior to the Ga‐FIB‐TOF‐SIMS analysis. As a result, it was found that Ga‐FIB‐TOF‐SIMS is a very powerful tool for mapping boron segregation and/or precipitation in steel with a spatial resolution of ~200 nm. In addition, the results were strongly dependent on the surface composition. Copyright © 2014 John Wiley & Sons, Ltd.     

Experimental apparatus for investigation of sputtering and secondary ion emission induced by energetic ion beams

The construction of an experimental apparatus, for investigation of implantation, secondary ion emission and sputtering processes, during irradiation of samples with an ion beam of up to 70 keV energy, is described. The basis of the apparatus is an electromagnetic mass separator equipped with a quadrupole mass spectrometer located in the collector chamber. The computer data acquisition control system makes it possible to perform the experimental measurements with high accuracy and precision. Preliminary results of secondary ion mass spectral measurements, obtained for C, Al, Si and Cu targets bombarded with Ar(+) and Kr(+) ions, are presented.     

Depth profiling of Irganox‐3114 nanoscale delta layers in a matrix of Irganox‐1010 using conventional Cs+ and O2+ ion beams

Depth profiling of an organic reference sample consisting of Irganox 3114 layers of 3 nm thickness at depths of 51.5, 104.5, 207.6 and 310.7 nm inside a 412 nm thick Irganox 1010 matrix evaporated on a Si substrate has been studied using the conventional Cs⁺ and O₂⁺ as sputter ion beams and Bi⁺ as the primary ion for analysis in a dual beam time‐of‐flight secondary ion mass spectrometer. The work is an extension of the Versailles Project on Advanced Materials and Standards project on depth profiling of organic multilayer materials. Cs⁺ ions were used at energies of 500 eV, 1.0 keV and 2.0 keV and the O₂⁺ ions were used at energies of 500 eV and 1.0 keV. All four Irganox 3114 layers were identified clearly in the depth profile using low mass secondary ions. The depth profile data were fitted to the empirical expression of Dowsett function and these fits are reported along with the full width at half maxima to represent the useful resolution for all the four delta layers detected. The data show that, of the conditions used in these experiments, an energy of 500 eV for both Cs⁺ beam and O₂⁺ beam provides the most useful depth profiles. The sputter yield volume per ion calculated from the slope of depth versus ion dose matches well with earlier reported data. Copyright © 2013 John Wiley & Sons, Ltd.     

A secondary ion microprobe ion trap mass spectrometer

An ion trap mass analyzer has been attached to an organic secondary ion microprobe. A pressure differential >100 can be maintained between the ion trap and microprobe. The well-focused secondary ion beam can transit a small (2 mm) diameter tube, but gas flow from ion trap to microprobe is impeded. This pressure differential allows the microprobe to retain imaging capability. Ion trap and microprobe data systems are integrated by taking advantage of the highly reproducible periodicity of the ion trap operating in resonant ejection mode and asynchronous signal and data acquisition afforded by commercially available interface cards. Secondary ion mass spectra and images obtained indicate an approximately 10-fold improvement in sensitivity, although preliminary evidence indicates low (<1%) trapping efficiency. Image data acquisition using the ion trap for mass analysis requires at least 10 times as much time compared to using a quadrupole mass filter because the mass-selected instability mode is used for mass analysis, i.e., mass resolution in the ion trap is not continuous as it is in the quadrupole.     

Influence of experimental conditions on the liquid secondary ion mass spectra of sulfonated azo dyes

Two monosulfonated and eight disulfonated azo dyes of varying relative molecular mass were examined by liquid secondary ion mass spectrometry (LSIMS). The effects of matrix, concentration, primary beam energy, and mode of operation were addressed in order to optimize sample ionization, whilst minimizing interference from matrix ions. Seven matrices were investigated: glycerol, thioglycerol, 3-nitrobenzyl alcohol, diethanolamine, 2-hydroxyethyl disulfide, a 1:1 (v/v) mixture of 2-hydroxyethyl disulfide and thioglycerol, and a 1 : 3 (v/v) mixture of dithioerythritol and dithiothreitol. Of these matrices, 3-nitrobenzyl alcohol produced LSIMS spectra that exhibited the most intense sample ions and the least inteiference from matrix ions. Minimum concentrations of 0.4 μg/μl and 4 μg/μl (dye in matrix) were necessary to produce useful full-scan spectra for monosulfonated azo dyes and disulfonated azo dyes, respectively; maximum sample ion intensities were obtained with concentrations ranging from 20 μg/μl to 60 μg/μl. A primary ion beam (cesium) of 10 to 15 kV produced the greatest secondary ionization efficiency, and a negative-ion analysis mode produced more useful spectra than those obtained in the positive-ion mode.     

Negative and positive secondary ion mass spectra of organic acids

Organic compounds can be ionized by sputtering the solid sample. The resulting negative and positive secondary ions provide mass spectra which characterize both the molecular weights and the structures of the compounds. Ionization occurs either by direct ejection of charged species from the solid into vacuum or by electron or proton transfer. The sputtered secondary ions dissociate unimolecularly to give fragment ions. These reactions are identical to those which occur when the secondary ions are independently generated by chemical ionization, selected by mass and dissociated in a high energy gas phase collision. The negative ion SIMS spectra show molecular ions (M^?.) or (M-H)^? ions as the dominant high mass species together with fragments due to decarboxylation, dehydration and losses of other simple molecules. Stronger acids show larger (M-H)^?/M^?.abundance ratios. The positive ion spectra are complementary and also useful in characterizing molecular structures. Attachment of cations to organic molecules (cationization) occurs much more readily than anion attachment and this makes negative SIMS spectra simpler than these positive ion counterparts.     

宽离子能量检测范围垂直引入反射式飞行时间质谱仪的研制

本实验室研制了国内首台宽离子能量检测范围飞行时间质谱仪。仪器采用紧凑式电子轰击源设计,配合离子透镜系统有效的调制离子流,飞行时间质量分析器采用了离子垂直引入式,双场加速和双场反射以及大尺寸MCP检测装置设计。仪器单离子信号半峰宽约2 ns,仪器分辨率优于1600FWHM,检测实际样品质量范围为1~127 amu(仪器理论质量检测上限优于800 amu),可检测离子能量范围优于2个数量级(3~140 eV)。若该TOF质量分析器与短瞬高压脉冲放电离子源耦合联用,可广泛应用于高能离子束的快速检测,如真空阴极放电对制备薄膜、离子注入材料的表征,导电材料的离子电荷态分布以及离子扩散速度的测定等。     

Improvement of biological time-of-flight-secondary ion mass spectrometry imaging with a bismuth cluster ion source

A new liquid metal ion gun (LMIG) filled with bismuth has been fitted to a time-of-flight-secondary ion mass spectrometer (TOF-SIMS). This source provides beams of Bi(n)q+ clusters with n = 1-7 and q = 1 and 2. The appropriate clusters have much better intensities and efficiencies than the Au3+ gold clusters recently used in TOF-SIMS imaging, and allow better lateral and mass resolution. The different beams delivered by this ion source have been tested for biological imaging of rat brain sections. The results show a great improvement of the imaging capabilities in terms of accessible mass range and useful lateral resolution. Secondary ion yields Y, disappearance cross sections sigma, efficiencies E = Y/sigma , and useful lateral resolutions deltaL have been compared using the different bismuth clusters, directly onto the surface of rat brain sections and for several positive and negative secondary ions with m/z ranging from 23 up to more than 750. The efficiency and the imaging capabilities of the different primary ions are compared by taking into account the primary ion current for reasonable acquisition times. The two best primary ions are Bi3+ and Bi5(2+). The Bi3+ ion beam has a current at least five times larger than Au3+ and therefore is an excellent beam for large-area imaging. Bi5(2+) ions exhibit large secondary ions yields and a reasonable intensity making them suitable for small-area images with an excellent sensitivity and a possible useful lateral resolution <400 nm.     

The distribution of ion acceptance in atmospheric pressure ion sources: Spatially resolved APLI measurements

It is demonstrated that spatially resolved mass selected analysis using atmospheric pressure laser ionization mass spectrometry (APLI MS) represents a new powerful tool for mechanistic studies of ion-molecule chemistry occurring within atmospheric pressure (AP) ion sources as well as for evaluation and optimization of ion source performance. A focused low-energy UV laser beam is positioned computer controlled orthogonally on a two-dimensional grid in the ion source enclosure. Resonance enhanced multiphoton ionization (REMPI) of selected analytes occurs only within the confined volume of the laser beam. Depending on the experimental conditions and the reactivity of the primary photo-generated ions, specific signal patterns become visible after data treatment, as visualized in, e.g., contour or pseudo-color plots. The resulting spatial dependence of sensitivity is defined in this context as the distribution of ion acceptance (DIA) of the source/analyzer combination. This approach provides a much more detailed analysis of the diverse processes occurring in AP ion sources compared with conventional bulk signal response measurements.     

Integral cross sections for ion—molecule reactions. I. The guided beam technique

A technique is described, that allows the measurement of integral cross sections for ion-molecule reactions and electron-transfer processes in the energy range from typically 0.1 to 20 eV (lab). Basically similar to the tandem mass spectrometer method, it uses inhomogeneous oscillatory electric fields for the storage and guidance of the primary ions and for the collection of the secondary ions. By these means a reduction of the number of excited ions in the primary beam and a good definition of the kinetic energy are obtained, together with a collection and detection probability for the secondary ions, that approaches unity for all scattering angles in a broad energy band. Tire ion beam intensity (10⁵ to 10⁷ ions per second) is only weakly dependent on the kinetic energy down to typically 0.15 eV (lab). The distribution of the collision energies is mainly determined by the thermal motion of the reactant gas in the scattering chamber (T ≈ 300 K). Measurements are reported for the reactions Ar⁺ + D₂ → ArD⁺ + D and Ne⁺ + CO → C⁺ Ne+O.     

A comparison of negative and positive ion mass spectrometry

Negative ion mass spectrometry using a conventional mass spectrometer with a special ion source and a sample pressure of approximately 2 × 10^?5 Torr is shown to be an excellent method for the qualitative analysis of lower mass alcohols, mercaptans, ketones, aldehydes, aliphatic carboxylic acids and esters, the spectra of which are characterized by intense [M – H] ^? ions. The method may be termed a ‘selective’ low energy ionization technique, being suitable for the above organic compounds, but not for nitriles, nitro compounds, hydrocarbons, ethers, amines, amides, nitrogen heterocycles and chlorinated compounds. This method can be looked upon as a complementary method, to positive ion mass spectrometry.     

Development of resonance‐enhanced multiphoton ionization SNMS for state‐selective detection of sputtered atoms under low‐energy ion irradiation

An instrument for a sputtered neutral mass spectrometry with a quadrupole mass spectrometer (QMS) by resonance‐enhanced multiphton ionization method is developed to study sputtered neutrals emission phenomena under ion irradiation in a low‐energy region. We have prepared a pulsed primary ion beam and an ion counting system, and have optimized the operation parameter including a sample bias, energy analyzer voltages, pulsed timing of laser and ion beam, etc. A yield ratio of the lowest‐lying excited state a⁵S₂ to the ground state a⁷S₃ for sputtered Cr atoms has been measured as a function of incident energy of Ar⁺ and O₂⁺ down to 600 eV using a polycrystalline Cr sample. The yield ratio has become a constant value for the Ar⁺ incidence, while it has exponentially increased below 1 keV for the O₂⁺ incidence. It is found that the internal energy distribution of sputtered Cr atoms has been significantly influenced by oxygen density at the surface. Copyright © 2011 John Wiley & Sons, Ltd.     

Combining low and high mass ion accumulation for enhancing shotgun proteome analysis by accurate mass measurement

A simple procedure is described that increases sensitivity and dynamic range for the analysis of a proteome batch digest by FT-ICR mass spectrometry. Ions at the low and high mass ranges are preferentially collected using two different sets of tuning conditions. By combing data collected using tuning conditions that favor low mass (m/z < 2000) and high mass (m/z > 2000) ions, 277 proteins are identified for a whole cell lysate of Methanococcus maripaludis in a single HPLC-MALDI FT-ICR mass spectrometry experiment, a 70% improvement compared with previous analyses using a wide mass range acquisition. This procedure improves the detection of low abundance ions and thereby increases the range of proteins that are observed. Because the observed mass range is effectively narrower for each spectrum, mass calibration is more accurate than for the standard method that provides a wide range of masses. The trap plate potential on the analyzer cell may be set to a higher value than used for wide mass range measurements, increasing the ion capacity of the analyzer cell and extending the dynamic range, while still maintaining mass accuracy.     

Design of a high-sensitivity negative ion source time-of-flight mass analyzer assembly created by cylindrical electrodes with a common axis

The new design incorporates the negative ion source and the mass analyzer, both constructed from cylindrical electrodes. The ion source is formed by three gridded cylindrical electrodes: a pulsed grid, the intermediate grid and the final accelerating grid. During a first time lapse, the electrons penetrate through the pulsed grid into the retarding field between this grid and the intermediate grid. The electrons are turning at some depth inside this intergrid space, where the attachment to neutral molecules most probably occurs. Next, the pulsed grid becoming strongly negative and ions are extracted towards the final acceleration grid. The ions from the cylindrical surface where they were created concentrate on the common axis of the electrodes (lateral focusing). The source lateral and time focus are coincident. A cylindrical electrostatic mirror is fitted to the source. The design, with a single stage, ensures also lateral focusing of the ions diverging from the common axis of the electrodes. The mirror electric and geometric parameters were selected to ensure both lateral and time focusing on the final detector with subsequent high luminosity. The basic parameters of the specific negative ion source time-of-flight mass analyzer design proposed here, are ion source final acceleration, intermediate, pulsed cylindrical grid radii 10, 20 and 30 mm, respectively, electrostatic mirror earthed grid and ion turning points surface radii 0.6 and 0.8 m, respectively. Ion packet smearing by the ion energy spread (resulting from the initial electron energy spread as electrons are turning at different depths inside the ionization region, from the moment when ions were created, being accelerated towards the pulsed grid during ionization) and by the turnaround time inside the cylindrical field was accounted for. Maintaining very high sensitivity, a resolution of the order of 100 is expected.