Structural effects in the analysis of supported lipid bilayers by time-of-flight secondary ion mass spectrometry

We contribute to the rapidly emerging interest in the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) for chemical analysis of biological materials by presenting a careful TOF-SIMS investigation of structurally different SiO2-supported phospholipid assemblies. Freeze-dried supported 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (POPC) bilayers, Langmuir-Blodgett POPC monolayers, and disordered thick POPC films were investigated. Compared with the two latter structures, the supported bilayer showed a strong (5-10 times) enhancement in the yield of both the molecular and the dimer ion peaks of POPC, suggesting that the molecular peak may be used as a sensitive indicator for changes in the membrane structure and, in particular, an indicator for the presence of bilayer structures in, e.g., cell and tissue samples. The detection efficiency and the useful lateral resolution indicate that a lateral resolution of around 100 nm can be obtained on all structures by imaging the phosphocholine ion at 184 u using Bi3+ primary ions. For the chemically specific molecular peak at 760 u, the measured detection efficiencies correspond to a useful lateral resolution of around 2 microm for the bilayer structure. The results are discussed in relation to recent dynamic SIMS (nano-SIMS) analysis of freeze-dried supported lipid bilayers, displaying similar or higher lateral resolution, but which in contrast to TOF-SIMS requires isotopic labeling of the analyzed lipids.     

Time-of-flight secondary ion mass spectrometric analysis of the interface between bone and titanium implants

Implant healing into bone tissue is a process where the mature bone grows towards and eventually fuses with the implant. In this study we investigated implant healing during 4 weeks with focus on the implant-tissue interface. Our main interest was to study the mineralization process around the implant. Titanium discs were implanted in rat tibia for 2 and 4 weeks. After implantation cross sections of bone and implant were made using a low-speed saw equipped with a diamond wafering blade. One section from each sample was stained with basic fuchsin and micrographed by light microscopy (LM). The other section was analyzed with imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) using a Bi(3)(+) cluster ion source. This ion source has recently been shown to enable identification of high-mass hydroxyapatite (HA) fragment ions (m/z 291-653) in bone samples. The LM images were used to identify areas suitable for TOF-SIMS analysis. Three areas were selected for mass spectral analysis, corresponding to interface region, bone and soft tissue, from which positive ion spectra were recorded. In the areas identified as bone, high-mass HA fragments ions were found after both 2 and 4 weeks. In the soft tissue area, no high-mass ions were found after 4 weeks. However, after 2 weeks HA-related ions were identified in mineralized spots in areas defined as soft tissue. After 4 but not after 2 weeks, high-mass HA fragment ions were found in the interface region. In conclusion, differences were observed regarding mineralization between 2 and 4 weeks of implantation and between different regions surrounding the implants. Imaging TOF-SIMS analysis using a Bi(3)(+) cluster as ion source enables identification of high-mass HA fragment ions at implant-tissue interfaces in bone. This technique might therefore be useful for biocompatibility assessment and for studying the mineralization process at implant surfaces.     

Chemical Analysis of Inorganic and Organic Surfaces and Thin Films by Static Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS)

By using mass spectrometry to analyze the atomic and molecular secondary ions that are emitted from a solid surface when bombarded with ions, one obtains detailed information about the chemical composition of the surface. A time-of-flight mass spectrometer is especially suitable for the analysis of secondary ions because of its high transmission, high mass resolution, and ability to detect ions of different masses simultaneously. By using a finely focused primary ion beam it is also possible to analyze microareas and generate surface images with a lateral resolution of 0.1 μm or less. Static time-of-flight secondary ion mass spectrometry (TOF-SIMS) allows monolayer imaging and local analysis of monolayers with high sensitivity, a wide mass range, high mass resolution, and high lateral resolution. Besides information on elements and isotopes, the technique yields direct information on the molecular level and can also be used to analyze surface species of high molecular mass that are thermally unstable and cannot be vaporized. The method can be applied to practically all types of materials and sample forms, including insulators in particular. In this article the basic principles of TOF-SIMS are explained, and its analytical capabilities for both large area and imaging applications are illustrated by examples. These include silicon surfaces (both uniform and structured), thermally unstable organic molecules on surfaces, synthetic polymers, and synthetically prepared molecular surface films, particles, and fibers. Emitted neutral particles can also be analyzed by postionization with a laser, and the possibilities of this technique are discussed.     

Effect of cocrystallization due to bile acid on molecular-ion sensitivity of biological phospholipids in Bi cluster time-of-flight secondary ion mass spectrometry measurements

Bi cluster time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a useful method for evaluating organic surfaces. However, its ability to detect large molecules is limited. One of the problems is that the sensitivities of macromolecules are lower than those of small molecules because larger molecules tend to exhibit lower ionization efficiencies and/or higher probabilities of fragmentation. Matrix-enhanced (ME)-SIMS is a sensitivity enhancement technique for intact molecular ions. The crystal structure of a mixed substance composed of an analyte and a matrix is known to affect the sensitivity of the analysis target. In this study, the effect of cocrystallization, which occurs due to the presence of bile acid, on the molecular-ion sensitivity was investigated using Bi cluster TOF-SIMS. Biological phospholipids and bile acids, which exhibit surfactant behaviors, were selected as the evaluated molecules and additives, respectively. The mass spectra indicated that the secondary-ion yields of phospholipids with bile acid were substantially greater than those of the pristine lipid. Specifically, samples with an analyte/bile acid ratio of 1:100 achieved approximately 60–100-fold sensitivity enhancement of [M + H]⁺ and [2M + H]⁺ molecular ions than the sensitivity achieved with the pristine samples. In the evaluation of molecular distribution, higher signal counts of intact ions were obtained from the cocrystallization area, although less-fragmented ions were emitted from these regions. Consequently, the results indicate that the cocrystallization due to the presence of bile acid provides an effective crystal structure for facilitating emission of larger molecules.     

Ion yield improvement for static secondary ion mass spectrometry by use of polyatomic primary ions

Static secondary ion mass spectrometry (S-SIMS) is one of the potentially most powerful and versatile tools for the analysis of surface components at the monolayer level. Current improvements in detection limit (LOD) and molecular specificity rely on the optimisation of the desorption-ionisation (DI) process. As an alternative to monoatomic projectiles, polyatomic primary ion (P.I.) bombardment increases ion yields non-linearly. Common P.I. sources are Ga+ (liquid metal ion gun (LMIG), SF5+ (electron ionisation) and the newer Au(n)+, Bi(n)q+ (both LMIG) and C60+ (electron ionisation) sources. In this study the ion yield improvement obtained by using the newly developed ion sources is assessed. Two dyes (zwitterionic and/or thermolabile polar functionalities on a largely conjugated backbone) were analysed as a thin layer using Ga+, SF5+, C60+, Bi+, Bi3(2+) and Bi5(2+) projectiles under static conditions. The study aims at evaluating the improvement in LOD, useful and characteristic yield and molecular specificity. The corrected total ion count values for the different P.I. sources are compared for different instruments to obtain a rough estimate of the improvements. Furthermore, tentative ionisation and fragmentation schemes are provided to describe the generation of radical and adduct ions. Characteristic ion yields are discussed for the different P.I. sources. An overview of the general appearances of the mass spectra obtained with the different P.I. sources is given to stress the major improvement provided by polyatomic P.I.s in yielding information at higher m/z values.     

Attempts for molecular depth profiling directly on a rat brain tissue section using fullerene and bismuth cluster ion beams

The capabilities of time of flight secondary ion mass spectrometry (TOF-SIMS) have been recently greatly improved with the arrival in this field of polyatomic ion sources. This technique is now able to map at the micron scale intact organic molecules in a range of a thousand Daltons or more, at the surface of tissue samples. Nevertheless, this remains a surface analysis technique, and three-dimensional information on the molecular composition of the sample could not be obtained due to the damage undergone by the organic molecules during their irradiation. The situation changed slightly with the low damage and low penetration depth of the C₆₀ fullerene ion beams. Recent promising studies have shown the possibility of organic molecular depth profiling using this kind of beams onto model samples. This possibility has been tried out directly onto a rat brain tissue section, which is the most commonly used biological tissue model in TOF-SIMS imaging method developments. The tissue surface has been sputtered with a 10 keV energy fullerene ion beam, and surface analyses were done with a 25 keV Bi₃⁺ ion beam at regular time intervals. The total depth which was analysed was more than two microns, with total primary ion doses of more than 10¹⁶ ions cm⁻². Although not in contradiction with results previously published but with much lower doses, it is found that the molecular damage remains too large, thus making molecular imaging very difficult. In addition, most of the lipids, which are usually the main observable molecules in TOF-SIMS, are concentrated close to the sample surface in the first hundreds of nanometers.     

Structures of mixed gold-silver cluster cations (Ag(m)Au(n)+, m+n<6): ion mobility measurements and density-functional calculations

The collision cross sections of Ag(m)Au(n)+ (m+n)<6 cluster ions were determined. For bimetallic clusters, we observe a significant intracluster charge transfer leaving most of the ions positive charge on the silver atoms. The mixed trimeric ions Ag2Au+ and AgAu2+ are triangular like the pure gold and silver trimers. Most of the tetrameric clusters are rhombus shaped, with the exception of Ag3Au+, which has a Y structure with the gold atom in the center. Among the pentamers we find distorted X structures for all systems. For Ag2Au3+ we find an additional isomer which is a trigonal bipyramid. These findings are in line with predictions based on density-functional theory calculations, i.e., all these structures either represent the global minima or are within less than 0.1 eV of the predicted global minimum.     

Chemical modification of fluorinated self-assembled monolayer surfaces by low energy reactive ion bombardment

Reactive collisions of low energy (<100-eV) mass-selected ions are used to chemically modify fluorinated self-assembled monolayer surfaces comprised of alkanethiolate chains CF₃(CF₂)₁₁(CH₂)₂S— bound to Au. Typical experiments were done by using 1-nA/cm² beams and submonolayer doses of reactant ions. Characterization of the modified surface was achieved by in situ chemical sputtering (60-eV Xe^+·) and by independent high mass resolution time-of-flight-secondary ionization mass spectrometry (TOF-SIMS) (15–25-keV, Ga⁺) experiments. Treatment with Si³⁵C1 ₄ ^+· produced a surface from which Xe⁺ sputtering liberated CF₂ ³⁵C1⁺ ions, which suggested Cl-for-F halogen exchange at the surface. Isotopic labeling studies that used Si³⁵Cl₂ ³⁷Cl ₂ ^+· ; and experiments with bromine-containing and iodine-containing projectiles, confirmed this reaction. High mass resolution TOF-SIMS spectra, as well as high spatial resolution images, provided further evidence as to the existence of halogen-exchanged species at the bombarded surface. Analogous Cl-for-F halogen substitution was observed in a model gas-phase reaction. The ion-surface reaction is suggested to proceed through an intermediate fluoronium ion in which the projectile is bonded to the target molecule. The most significant conclusion of the study is that selective chemical modification of monolayer surfaces can be achieved by using reactive ion beams, which lead to new covalent bonds at the surface and in the scattered ions.     

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.     

A high-resolution scanning microprobe matrix-assisted laser desorption/ionization ion source for imaging analysis on an ion trap/Fourier transform ion cyclotron resonance mass spectrometer

A new scanning microprobe matrix-assisted laser desorption/ionization (SMALDI) ion source for high spatial resolution has been developed for linear ion trap and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The source is fully compatible with commercial ion trap flanges (such as the LTQ series, Thermo Fisher Scientific). The source is designed for atmospheric pressure (AP) operation but is also suitable for mid-pressure operation. The AP mode is especially useful for investigating volatile compounds. The source can be interchanged with other ion sources within a minute when operated in the AP mode. Combining high-lateral resolution MALDI imaging with high mass resolution and high mass accuracy mass spectrometry, available in the FT-ICR mode, provides a new quality of analytical information, e.g. from biological samples. First results obtained with the new ion source demonstrate a maximum lateral resolution of 0.6 by 0.5 microm. Depending on the limit of detection of the chosen mass analyzer, however, the size of the focus had to be enlarged to a diameter of up to 8 microm in the FT-ICR mode, in order to create enough ions for detection. Mass spectra acquired for analytical imaging were obtained from single laser pulses per pixel in all the experiments. This mode allows us to investigate biological thin sections with desorption focus diameters in the micrometer range, known to cause complete evaporation of material under the laser focus with a very limited number of laser pulses. As a first example, peptide samples deposited in microstructures were investigated with the new setup. A high quality and validity of the acquired images were obtained in the ion trap mode due to the low limit of detection. High mass resolution and accuracy but poorer image quality were obtained in the ICR mode due to the lower detection sensitivity of the ICR detector.     

Analysis of bone minerals by time-of-flight secondary ion mass spectrometry: a comparative study using monoatomic and cluster ions sources

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an important tool for the analysis of bone minerals at implant surfaces. Most studies have been performed with monoatomic primary ion sources such as Ga(+) with poor secondary molecular ion production efficiency and only elemental distributions and minor fragments of bone minerals have been reported. By using cluster ion sources, such as Au(1-3) (+) and Bi(1-3) (+), identification of larger hydroxyapatite species at m/z 485, 541, 597 and 653, identified as Ca(5)P(3)O(12), Ca(6)P(3)O(13), Ca(7)P(3)O(14) and Ca(8)P(3)O(15), respectively, became possible. The ions appear to be fragments of the hydroxyapatite unit cell Ca(10)(PO(4))(6)(OH)(2). Each ion in the series is separated by 55.9 m/z units, corresponding to CaO, and this separation might reflect the columnar nature of the unit cell.     

Fluorescence, XPS, and TOF-SIMS surface chemical state image analysis of DNA microarrays

Performance improvements in DNA-modified surfaces required for microarray and biosensor applications rely on improved capabilities to accurately characterize the chemistry and structure of immobilized DNA molecules on micropatterned surfaces. Recent innovations in imaging X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) now permit more detailed studies of micropatterned surfaces. We have exploited the complementary information provided by imaging XPS and imaging TOF-SIMS to detail the chemical composition, spatial distribution, and hybridization efficiency of amine-terminated single-stranded DNA (ssDNA) bound to commercial polyacrylamide-based, amine-reactive microarray slides, immobilized in both macrospot and microarray diagnostic formats. Combinations of XPS imaging and small spot analysis were used to identify micropatterned DNA spots within printed DNA arrays on slide surfaces and quantify DNA elements within individual microarray spots for determination of probe immobilization and hybridization efficiencies. This represents the first report of imaging XPS of DNA immobilization and hybridization efficiencies for arrays fabricated on commercial microarray slides. Imaging TOF-SIMS provided distinct analytical data on the lateral distribution of DNA within single array microspots before and after target hybridization. Principal component analysis (PCA) applied to TOF-SIMS imaging datasets demonstrated that the combination of these two techniques provides information not readily observable in TOF-SIMS images alone, particularly in identifying species associated with array spot nonuniformities (e.g., "halo" or "donut" effects often observed in fluorescence images). Chemically specific spot images were compared to conventional fluorescence scanned images in microarrays to provide new information on spot-to-spot DNA variations that affect current diagnostic reliability, assay variance, and sensitivity.     

An ion trap-ion mobility-time of flight mass spectrometer with three ion sources for ion/ion reactions

This instrument combines the capabilities of ion/ion reactions with ion mobility (IM) and time-of-flight (TOF) measurements for conformation studies and top-down analysis of large biomolecules. Ubiquitin ions from either of two electrospray ionization (ESI) sources are stored in a three dimensional (3D) ion trap (IT) and reacted with negative ions from atmospheric sampling glow discharge ionization (ASGDI). The proton transfer reaction products are then separated by IM and analyzed via a TOF mass analyzer. In this way, ubiquitin +7 ions are converted to lower charge states down to +1; the ions in lower charge states tend to be in compact conformations with cross sections down to ～880 Ų. The duration and magnitude of the ion ejection pulse on the IT exit and the entrance voltage on the IM drift tube can affect the measured distribution of conformers for ubiquitin +7 and +6. Alternatively, protein ions are fragmented by collision-induced dissociation (CID) in the IT, followed by ion/ion reactions to reduce the charge states of the CID product ions, thus simplifying assignment of charge states and fragments using the mobility-resolved tandem mass spectrum. Instrument characteristics and the use of a new ion trap controller and software modifications to control the entire instrument are described.     

Infrared ion spectroscopy inside a mass‐selective cryogenic 2D linear ion trap

We demonstrate operation of the first cryogenic 2D linear ion trap (LIT) with mass‐selective capabilities. This trap presents a number of advantages for infrared ion “action” spectroscopy studies, particularly those employing the “tagging/messenger” spectroscopy approach. The high trapping efficiencies, trapping capacities, and low detection limits make 2D LITs a highly suitable choice for low‐concentration analytes from scarce biological samples. In our trap, ions can be cooled down to cryogenic temperatures to achieve higher‐resolution infrared spectra, and individual ions can be mass selected prior to irradiation for a background‐free photodissociation scheme. Conveniently, multiple tagged analyte ions can be mass isolated and efficiently irradiated in the same experiment, allowing their infrared spectra to be recorded in parallel. This multiplexed approach is critical in terms of increasing the duty cycle of infrared ion spectroscopy, which is currently a key weakness of the technique. The compact design of this instrument, coupled with powerful mass selection capabilities, set the stage for making cryogenic infrared ion spectroscopy viable as a bioanalytical tool in small molecule identification.     

Identification of Different Copper Green Pigments in Renaissance Paintings by Cluster-TOF-SIMS Imaging Analysis

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging using cluster primary ion beams is used for the identification of a green painting layer on the scene The Angels Concert from the Issenheim Altarpiece (painted in 1516) from a German Renaissance painter, Matthias Grünewald. Copper carboxylate clusters inside a basic copper chloride (atacamite) layer have been identified and located in the copper green layer. The mechanisms of transformation of atacamite into copper carboxylates may be initiated by an aging of the paint layers. The combination of the high mass resolution of the technique together with a micrometer spatial resolution and the possibility to simultaneously identify both minerals and organics, has proven to be the method of choice for the study of the stratigraphy of a paint cross-section.     

Structural analysis of secondary ions by post-source decay in time-of-flight secondary ion mass spectrometry

Tandem mass spectrometry measurements have been achieved using time-of-flight secondary ion mass spectrometry (TOF-SIMS) and a post source decay (PSD)-like method. The performance of the method has been demonstrated on model molecules with well-known fragmentation pathways. Several lipids have been fragmented including the phosphocholine ion, phosphatidylcholines, cholesterol and vitamin E. Pure samples were analyzed, and the results compared with those obtained with the same compounds on a quadrupole-TOF hybrid mass spectrometer. Then, the structures of some lipids which are currently observed in the TOF-SIMS imaging of mammalian tissue sections were verified.     

Introduction to time-of-flight secondary ion mass spectrometry application in chromatographic analysis

New on-line analytical system coupling thin layer chromatography (TLC) and high selective identification unit-time of flight secondary ion mass spectrometry (TOF-SIMS) is introduced in this article. Chromatographic mixture separation and analyte surface deposition followed with surface TOF-SIMS analysis on-line allows to identify the analytes at trace and ultratrace levels. The selected analytes with different detectability and identification possibility were analysed in this hyphenated unit (Methyl Red indicator, Terpinolen and Giberrelic acid). Here, the chromatographic thin layer plays a universal role: separation unit, analyte depositing surface and TOF-SIMS interface, finally. Two depositing substrates and TOF-SIMS compatible interfaces were tested in above-mentioned interfacing unit: modified aluminium backed chromatographic thin layer and monolithic silica thin layer. The sets of positive and negative ions TOF-SIMS spectra obtained from different SIMS modes of analysis were used for analyte identification purposes. SIMS enables analyte detection with high mass resolution at the concentration level that is not achieved by other methods.     

An investigation of secondary ion yield enhancement using Bi n 2+ (n=1, 3, 5) primary ions

We have investigated secondary ion yield enhancement using Bin2+ (n=1, 3, 5) primary ions impacting phenylalanine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), cholesterol, Irganox 1010, and polymer films adsorbed on silicon and aluminum. Secondary ion yields are increased using Bi2+and Bi3(2+) primary ions for the molecular layers and polymers that can undergo allyl cation rearrangements. For Irganox 1010, the deprotonated molecular ion yields (m/z 1175; [M-H]-) are one to two times larger for Bi2+ and Bi(3)2+ primary ions than for Bi+ and Bi3+ at the same primary ion velocities. In the positive ion mode, the largest fragment ion yield (m/z 899) is 1.5 times larger for Bi2+ ions than for Bi+. For Bi3(2+) the largest fragment ion yield is only 70% of the ion yield using Bi3+, but the secondary ion yields of the fragment ions at m/z 57 and 219 are enhanced. For polymers that can undergo allyl cation rearrangement reactions the secondary ion yield enhancements of the monomer ions range from 1.3 to 4.3. For Bi(5)2+ primary ions, secondary ion yields were the same or slightly larger than for Bi5+ in the negative ion mass spectra for Irganox 1010, but lower in the positive ion mode. No secondary ion yield enhancements were measured on polymer samples for Bi5(2+). For all polymer films studied, secondary ion intensities from the oligomer regions are substantially decreased using Bin2+ (n=1, 3, 5). We discuss differences in the ionization mechanisms for doubly and singly-charged Bi primary ion bombardment.     

Peptide structural analysis using continuous Ar cluster and C60 ion beams

A novel application of time-of-flight secondary ion mass spectrometry (ToF-SIMS) with continuous Ar cluster beams to peptide analysis was investigated. In order to evaluate peptide structures, it is necessary to detect fragment ions related to multiple neighbouring amino acid residues. It is, however, difficult to detect these using conventional ToF-SIMS primary ion beams such as Bi cluster beams. Recently, C₆₀ and Ar cluster ion beams have been introduced to ToF-SIMS as primary ion beams and are expected to generate larger secondary ions than conventional ones. In this study, two sets of model peptides have been studied: (des-Tyr)-Leu-enkephalin and (des-Tyr)-Met-enkephalin (molecular weights are approximately 400 Da), and [Asn¹ Val⁵]-angiotensin II and [Val⁵]-angiotensin I (molecular weights are approximately 1,000 Da) in order to evaluate the usefulness of the large cluster ion beams for peptide structural analysis. As a result, by using the Ar cluster beams, peptide molecular ions and large fragment ions, which are not easily detected using conventional ToF-SIMS primary ion beams such as Bi₃ ⁺, are clearly detected. Since the large fragment ions indicating amino acid sequences of the peptides are detected by the large cluster beams, it is suggested that the Ar cluster and C₆₀ ion beams are useful for peptide structural analysis.     

Matrix-free high-resolution imaging mass spectrometry with high-energy ion projectiles

The importance of imaging mass spectrometry (MS) for visualizing the spatial distribution of molecular species in biological tissues and cells is growing. We have developed a new system for imaging MS using MeV ion beams, termed MeV-secondary ion mass spectrometry (MeV-SIMS) here, and demonstrated more than 1000-fold increase in molecular ion yield from a peptide sample (1154 Da), compared to keV ion irradiation. This significant enhancement of the molecular ion yield is attributed to electronic excitation induced in the near-surface region by the impact of high energy ions. In addition, the secondary ion efficiency for biologically important compounds (>1 kDa) increased to more than 10(10) cm(-2), demonstrating that the current technique could, in principle, achieve micrometer lateral resolution. In addition to MeV-SIMS, peptide compounds were also analyzed with cluster-SIMS and the results indicated that in the former method the molecular ion yields increased substantially compared to the latter. To assess the capability of MeV-SIMS to acquire heavy-ion images, we have prepared a micropatterned peptide surface and successfully obtained mass spectrometric imaging of the deprotonated peptides (m/z 1153) without any matrix enhancement. The results obtained in this study indicate that the MeV-SIMS technique can be a powerful tool for high-resolution imaging in the mass range from 100 to over 1000 Da.