Cyclotron Resonance Mass Spectrometry Imaging Research Articles

Patterns of Carbon-Bound Exogenous Compounds Impact Disease Pathophysiology in Lung Cancer Subtypes in Different Ways.

Carbon-bound exogenous compounds, such as polycyclic aromatic hydrocarbons (PAHs), tobacco-specific nitrosamines, aromatic amines, and organohalogens, are known to affect both tumor characteristics and patient outcomes in lung squamous cell carcinoma (LUSC); however, the roles of these compounds in lung adenocarcinoma (LUAD) remain unclear. We analyzed 11 carbon-bound exogenous compounds in LUAD and LUSC samples using in situ high mass-resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging and performed a cluster analysis to compare the patterns of carbon-bound exogenous compounds between these two lung cancer subtypes. Correlation analyses were conducted to investigate associations among exogenous compounds, endogenous metabolites, and clinical data, including patient survival outcomes and smoking behaviors. Additionally, we examined differences in exogenous compound patterns between normal and tumor tissues. Our analyses revealed that PAHs, aromatic amines, and organohalogens were more abundant in LUAD than in LUSC, whereas the tobacco-specific nitrosamine nicotine-derived nitrosamine ketone was more abundant in LUSC. Patients with LUAD and LUSC could be separated according to carbon-bound exogenous compound patterns detected in the tumor compartment. The same compounds had differential impacts on patient outcomes, depending on the cancer subtype. Correlation and network analyses indicated substantial differences between LUAD and LUSC metabolomes, associated with substantial differences in the patterns of the carbon-bound exogenous compounds. These data suggest that the contributions of these carcinogenic compounds to cancer biology may differ according to the cancer subtypes.

Metabolic heterogeneity in adrenocortical carcinoma impacts patient outcomes.

Spatially resolved metabolomics enables the investigation of tumoral metabolites in situ. Inter- and intratumor heterogeneity are key factors associated with patient outcomes. Adrenocortical carcinoma (ACC) is an exceedingly rare tumor associated with poor survival. Its clinical prognosis is highly variable, but the contributions of tumor metabolic heterogeneity have not been investigated thus far to our knowledge. An in-depth understanding of tumor heterogeneity requires molecular feature-based identification of tumor subpopulations associated with tumor aggressiveness. Here, using spatial metabolomics by high-mass resolution MALDI Fourier transform ion cyclotron resonance mass spectrometry imaging, we assessed metabolic heterogeneity by de novo discovery of metabolic subpopulations and Simpson's diversity index. After identification of tumor subpopulations in 72 patients with ACC, we additionally performed a comparison with 25 tissue sections of normal adrenal cortex to identify their common and unique metabolic subpopulations. We observed variability of ACC tumor heterogeneity and correlation of high metabolic heterogeneity with worse clinical outcome. Moreover, we identified tumor subpopulations that served as independent prognostic factors and, furthermore, discovered 4 associated anticancer drug action pathways. Our research may facilitate comprehensive understanding of the biological implications of tumor subpopulations in ACC and showed that metabolic heterogeneity might impact chemotherapy.

Spatial metabolomics reveals upregulation of several pyrophosphate-producing pathways in cortical bone of Hyp mice.

Patients with the renal phosphate-wasting disease X-linked hypophosphatemia (XLH) and Hyp mice, the murine homolog of XLH, are characterized by loss-of-function mutations in phosphate-regulating endopeptidase homolog X-linked (PHEX), leading to excessive secretion of the bone-derived phosphotropic hormone FGF23. The mineralization defect in patients with XLH and Hyp mice is caused by a combination of hypophosphatemia and local accumulation of mineralization-inhibiting molecules in bone. However, the mechanism by which PHEX deficiency regulates bone cell metabolism remains elusive. Here, we used spatial metabolomics by employing matrix-assisted laser desorption/ionization (MALDI) Fourier-transform ion cyclotron resonance mass spectrometry imaging (MSI) of undecalcified bone cryosections to characterize in situ metabolic changes in bones of Hyp mice in a holistic, unbiased manner. We found complex changes in Hyp bone metabolism, including perturbations in pentose phosphate, purine, pyrimidine, and phospholipid metabolism. Importantly, our study identified an upregulation of several biochemical pathways involved in intra- and extracellular production of the mineralization inhibitor pyrophosphate in the bone matrix of Hyp mice. Our data emphasize the utility of MSI-based spatial metabolomics in bone research and provide holistic in situ insights as to how Phex deficiency-induced changes in biochemical pathways in bone cells are linked to impaired bone mineralization.

Integration of Mass Spectrometry Imaging and Machine Learning Visualizes Region-Specific Age-Induced and Drug-Target Metabolic Perturbations in the Brain

Detailed metabolic imaging of specific brain regions in early aging may expose pathophysiological mechanisms and indicate effective neuropharmacological targets in the onset of cognitive decline. Comprehensive imaging of brain aging and drug-target effects is restricted using conventional methodology. We simultaneously visualized multiple metabolic alterations induced by normal aging in specific regions of mouse brains by integrating Fourier-transform ion cyclotron resonance mass spectrometry imaging and combined supervised and unsupervised machine learning models. We examined the interplay between aging and the response to tacrine-induced acetylcholinesterase inhibition, a well-characterized therapeutic treatment against dementia. The dipeptide carnosine (β-alanyl-l-histidine) and the vitamin α-tocopherol were significantly elevated by aging in different brain regions. l-Carnitine and acetylcholine metabolism were found to be major pathways affected by aging and tacrine administration in a brain region-specific manner, indicating altered mitochondrial function and neurotransmission. The highly interconnected hippocampus and retrosplenial cortex displayed different age-induced alterations in lipids and acylcarnitines, reflecting diverse region-specific metabolic effects. The subregional differences observed in the hippocampal formation of several lipid metabolites demonstrate the unique potential of the technique compared to standard mass spectrometry approaches. An age-induced increase of endogenous antioxidants, such as α-tocopherol, in the hippocampus was detected, suggesting an augmentation of neuroprotective mechanisms in early aging. Our comprehensive imaging approach visualized heterogeneous age-induced metabolic perturbations in mitochondrial function, neurotransmission, and lipid signaling, not always attenuated by acetylcholinesterase inhibition.

Desorption ionization using through-hole alumina membrane offers higher reproducibility than 2,5-dihydroxybenzoic acid, a widely used matrix in Fourier transform ion cyclotron resonance mass spectrometry imaging analysis.

DIUTHAME (desorption ionization using through-hole alumina membrane), a recently developed matrix-free ionization-assisting substrate, was examined for reproducibility in terms of mass accuracy and intensity using standard lipid and mouse brain sections. The impregnation property of DIUTHAME significantly improved the reproducibility of mass accuracy and intensity compared with 2,5-dihydroxybenzoic acid (DHB). Frozen tissue sections were mounted on indium tin oxide-coated glass slides. DIUTHAME and DHB were applied to individual sections. Subsequently, a solution of a phosphatidylcholine standard, PC(18:2/18:2), was poured onto the DIUTHAME and matrix. Finally, the samples were subjected to laser desorption ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry. The reproducibility was tested by calculating the mean ± standard deviation values of mass errors and intensities of individual ion species. Analysis of the PC(18:2/18:2) standard showed significantly (p < 0.01) lower mass error for DIUTHAME-MS than for MALDI-MS. Endogenous PC(36:4) analysis in mouse brain section also showed significantly (p < 0.05) lower mass errors for DIUTHAME-MS. Furthermore, we investigated the mass error of some abundant lipid ions in brain sections and observed similar results. DIUTHAME-MS displayed lower signal intensity in standard PC analysis. Interestingly, it offered higher signal intensities for all the endogenous lipid ions. Lower fluctuations of both mass accuracies and signal intensities were observed in DIUTHAME-MS. Our results demonstrated that DIUTHAME-MS offers higher reproducibility for mass accuracies and intensities than MALDI-MS in both standard lipid and mouse brain tissue analyses. It can potentially be used instead of conventional MALDI-MS and mass spectrometry imaging analyses to achieve highly reproducible data for mass accuracy and intensity.

Mass spectrometry imaging of blast overpressure induced modulation of GABA/glutamate levels in the central auditory neuraxis of Chinchilla

Acoustic trauma damages inner ear neural structures including cochlear hair cells which result in hearing loss and neurotransmitter imbalances within the synapses of the central auditory pathway. Disruption of GABA/glutamate levels underlies, tinnitus, a phantom perception of sound that persists post-exposure to blast noise which may manifest in tandem with acute/chronic loss of hearing. Many putative theories explain tinnitus physiology based on indirect and direct assays in animal models and humans, although there is no comprehensive evidence to explain the phenomenon. Here, GABA/glutamate levels were imaged and quantified in a blast overpressure model of chinchillas using Fourier transform ion cyclotron resonance mass spectrometry imaging. The direct measurement from whole-brain sections identified the relative levels of GABA/glutamate in the central auditory neuraxis centers including the cochlear nucleus, inferior colliculus, and auditory cortex. These preliminary results provide insight on the homeostasis of GABA/glutamate within whole-brain sections of chinchilla for investigation of the pathomechanism of blast-induced tinnitus.

Epilipidomics of Senescent Dermal Fibroblasts Identify Lysophosphatidylcholines as Pleiotropic Senescence-Associated Secretory Phenotype (SASP) Factors

During aging, skin accumulates senescent cells. The transient presence of senescent cells, followed by their clearance by the immune system, is important in tissue repair and homeostasis. The persistence of senescent cells that evade clearance contributes to the age-related deterioration of the skin. The senescence-associated secretory phenotype of these cells contains immunomodulatory molecules that facilitate clearance but also promote chronic damage. Here, we investigated the epilipidome-the oxidative modifications of phospholipids-of senescent dermal fibroblasts, because these molecules are among the bioactive lipids that were recently identified as senescence-associated secretory phenotype factors. Using replicative- and stress- induced senescence protocols, we identified lysophosphatidylcholines as universally elevated in senescent fibroblasts, whereas other oxidized lipids displayed a pattern that was characteristic for the used senescence protocol. When we tested the lysophosphatidylcholines for senescence-associated secretory phenotype activity, we found that they elicit chemokine release in nonsenescent fibroblasts but also interfere with toll-like receptor 2 and 6/CD36 signaling and phagocytic capacity in macrophages. Using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry imaging, we localized two lysophosphatidylcholine species in aged skin. This suggests that lysophospholipids may facilitate immune evasion and low-grade chronic inflammation in skin aging.

Rapid discrimination of pediatric brain tumors by mass spectrometry imaging

PurposeMedulloblastoma, the most common primary pediatric malignant brain tumor, originates in the posterior fossa of the brain. Pineoblastoma, which originates within the pineal gland, is a rarer malignancy that also presents in the pediatric population. Medulloblastoma and pineoblastoma exhibit overlapping clinical features and have similar histopathological characteristics. Histopathological similarities confound rapid diagnoses of these two tumor types. We have conducted a pilot feasibility study analyzing the molecular profile of archived frozen human tumor specimens using mass spectrometry imaging (MSI) to identify potential biomarkers capable of classifying and distinguishing between medulloblastoma and pineoblastoma.MethodsWe performed matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry imaging on eight medulloblastoma biopsy specimens and three pineoblastoma biopsy specimens. Multivariate statistical analyses were performed on the MSI dataset to generate classifiers that distinguish the two tumor types. Lastly, the molecules that were discriminative of tumor type were queried against the Lipid Maps database and identified.ResultsIn this pilot study we show that medulloblastoma and pineoblastoma can be discriminated using molecular profiles determined by MSI. The highest-ranking discriminating classifiers of medulloblastoma and pineoblastoma were glycerophosphoglycerols and sphingolipids, respectively.ConclusionWe demonstrate proof-of-concept that medulloblastoma and pineoblastoma can be rapidly distinguished by using MSI lipid profiles. We identified biomarker candidates capable of distinguishing these two histopathologically similar tumor types. This work expands the current molecular knowledge of medulloblastoma and pineoblastoma by characterizing their lipidomic profiles, which may be useful for developing novel diagnostic, prognostic and therapeutic strategies.

Abstract A068: Metabolic signature in lethal vs. nonlethal prostate cancer

Abstract Introduction and Aim: Prostate cancer (PCa) is the most frequent malignancy detected in males and the second leading cause of death from cancer in males. Despite the current diagnostic standards, mainly based on PSA detection, histologic grading, and tumor-lymph nodes-metastases (TNM) staging, there is still an urgent need for the identification of new prognostic markers to help in the definition of an individualized and personalized treatment for patients. Metabolites reflect gene expression and protein function and perturbed metabolism is considered a novel “hallmark of cancer.” Therefore, the analysis of the metabolomic profiling of high-risk and lethal PCa might help in the characterization of the molecular characteristics of this subgroup of patients. In this study, we aimed to identify the metabolic signature for lethal prostate cancer, in order to characterize those patients at highest risk of metastatic progression. Experimental Procedure: We employed high-resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR MSI) to detect and visualize metabolites in formalin-fixed, paraffin-embedded (FFPE) PCa tissue microarrays (TMAs) in a cohort of 211 PCa patients who underwent radical prostatectomy between 1994 and 2001. To calculate the prognostic power of measured metabolites, we separated the patients’ cohort into good and poor survivor groups by the application of intensity cut-offs optimized to the clinical endpoint (e.g., PSA-free survival). Statistical differences in patient survival were measured using the Kaplan-Meier log-rank test and multivariate survival analysis performed using Cox proportional hazards regression models. Results: We employed a metabolic profiling approach to distinguish lethal from nonlethal disease in a subset of lethal PCa patients (PCa-related death within 5 years following radical prostatectomy) vs. nonlethal PCa patients (survival &amp;gt; 10 years following radical prostatectomy, without evidence of recurrence). By virtual microdissection molecular signatures were selected from tumor regions and subsequently statistically compared, leading to 172 small molecules significantly different between lethal and nonlethal PCa patients (p&amp;lt;0.05). Visualization of these m/z species displayed clear differences in the intensity patterns between lethal vs. non-lethal disease. Next, we used metabolite MSI data to address patient survival outcome between lethal and nonlethal PCa cases. Statistical analysis showed that different m/z species significantly correlated with patients’ outcome (e.g., PSA-free survival and disease-free survival). Preliminary pathway enrichment analysis displayed a significant upregulation of purine and pyrimidine metabolism KEGG pathway in lethal vs. nonlethal disease. Strikingly, these networks have been previously associated with PCa progression in RNA-based studies. Conclusion: In this study we identified a metabolic pattern able to discriminate between lethal and nonlethal disease. Further validation of an independent cohort is needed to validate the novel findings of metabolites and might lead to the identification of novel biologic markers involved in PCa progression. Citation Format: Eugenio Zoni, Achim Buck, Annette Feuchtinger, Martin Spahn, Axel Walch, Marianna Kruithof-de Julio. Metabolic signature in lethal vs. nonlethal prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A068.

Pharmacometabolic response to pirfenidone in pulmonary fibrosis detected by MALDI-FTICR-MSI.

Idiopathic pulmonary fibrosis (IPF) is a fatal condition that reduces life expectancy and shows a limited response to available therapies. Pirfenidone has been approved for treatment of IPF, but little is known about the distinct metabolic changes that occur in the lung upon pirfenidone administration.Here, we performed a proof-of-concept study using high-resolution quantitative matrix-assisted laser desorption/ionisation Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FTICR-MSI) to simultaneously detect, visualise and quantify in situ endogenous and exogenous metabolites in lungs of mice subjected to experimental fibrosis and human patients with IPF, and to assess the effect of pirfenidone treatment on metabolite levels.Metabolic pathway analysis and endogenous metabolite quantification revealed that pirfenidone treatment restores redox imbalance and glycolysis in IPF tissues, and downregulates ascorbate and aldarate metabolism, thereby likely contributing to in situ modulation of collagen processing. As such, we detected specific alterations in metabolite pathways in fibrosis and, importantly, metabolic recalibration following pirfenidone treatment.Together, these results highlight the suitability of high-resolution MALDI-FTICR-MSI for deciphering the therapeutic effects of pirfenidone and provide a preliminary analysis of the metabolic changes that occur during pirfenidone treatment in vivo These data may therefore contribute to improvement of currently available therapies for IPF.

CCL5-CCR5 interactions modulate metabolic events during tumor onset to promote tumorigenesis

BackgroundIn earlier studies we have shown that CCL5 activation of CCR5 induces the proliferation and survival of breast cancer cells in a mechanistic target of rapamycin (mTOR)-dependent manner and that this is in part due to CCR5-mediated increases in glycolytic metabolism.MethodsUsing the MDA-MB-231 triple negative human breast cancer cell line and mouse mammary tumor virus – polyomavirus middle T-antigen (MMTV-PyMT) mouse primary breast cancer cells, we conducted in vivo tumor transplant experiments to examine the effects of CCL5-CCR5 interactions in the context of regulating tumor metabolism. Additionally, we employed Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry imaging (MALDI-FTICR-MSI) to evaluate tumor utilization of cellular metabolites.ResultsWe provide evidence that, in the absence of CCR5, the early events associated with rapid tumor growth in the MMTV-PyMT mouse model of spontaneous breast cancer development, are diminished, as demonstrated by a delay in tumor onset. In tumor transplant studies into immunocompromised mice we identify a direct correlation between reduced tumor proliferation and decreased metabolic activity, specifically associated with tumor expression of CCR5. The reduction in tumorigenesis is accompanied by decreases in glucose uptake, glucose transporter-1 (GLUT-1) cell surface expression, intracellular ATP and lactate levels, as well as reduced CCL5 production. Using MALDI-FTICR-MS, we show that the rapid early tumor growth of CCR5+/+ triple negative breast cancer cells in vivo is attributable to increased levels of glycolytic intermediates required for anabolic processes, in contrast to the slower growth rate of their corresponding CCR5−/− cells, that exhibit reduced glycolytic metabolism.ConclusionsThese findings suggest that CCL5-CCR5 interactions in the tumor microenvironment modulate metabolic events during tumor onset to promote tumorigenesis.

Metabolomic profiling of prostate cancer by matrix assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry imaging using Matrix Coating Assisted by an Electric Field (MCAEF)

In this work, we combined the use of two MALDI matrices (quercetin and 9-aminoacridine), a recently developed new matrix coating technique – matrix coating assisted by an electric field (MCAEF), and matrix-assisted laser desorption/ionization – Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICRMS) to detect and image endogenous compounds in the cancerous and non-cancerous regions of three human prostate cancer (stage II) tissue specimens. After three rounds of imaging data acquisitions (i.e., quercetin for positive and negative ion detection and 9-aminoacridine for negative ion detection), and metabolite identification, a total of 1091 metabolites including 1032 lipids and 59 other metabolites were routinely detected and successfully localized. Of these compounds, 250 and 217 were only detected in either the cancerous or the non-cancerous regions respectively, although we cannot rule out the presence of these metabolites at concentrations below the detection limit. In addition, 152 of the other 624 metabolites showed differential distributions (p<0.05, t-test) between the two regions of the tissues. Further studies on a larger number of clinical specimens will need to be carried out to confirm this large number of apparently cancer-related metabolites. The successful determination of the spatial locations and abundances of these endogenous biomolecules indicated significant metabolism abnormalities – e.g., increased energy charge and under-expression of neutral acyl glycerides, in the prostate cancer samples. To our knowledge, this work has resulted in MALDI-MS imaging of the largest group of metabolites in prostate cancer thus far and demonstrated the importance of using complementary matrices for comprehensive metabolomic imaging by MALDI-MS. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

High-mass-resolution MALDI mass spectrometry imaging of metabolites from formalin-fixed paraffin-embedded tissue.

Formalin-fixed and paraffin-embedded (FFPE) tissue specimens are the gold standard for histological examination, and they provide valuable molecular information in tissue-based research. Metabolite assessment from archived tissue samples has not been extensively conducted because of a lack of appropriate protocols and concerns about changes in metabolite content or chemical state due to tissue processing. We present a protocol for the in situ analysis of metabolite content from FFPE samples using a high-mass-resolution matrix-assisted laser desorption/ionization fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR-MSI) platform. The method involves FFPE tissue sections that undergo deparaffinization and matrix coating by 9-aminoacridine before MALDI-MSI. Using this platform, we previously detected ∼1,500 m/z species in the mass range m/z 50-1,000 in FFPE samples; the overlap compared with fresh frozen samples is 72% of m/z species, indicating that metabolites are largely conserved in FFPE tissue samples. This protocol can be reproducibly performed on FFPE tissues, including small samples such as tissue microarrays and biopsies. The procedure can be completed in a day, depending on the size of the sample measured and raster size used. Advantages of this approach include easy sample handling, reproducibility, high throughput and the ability to demonstrate molecular spatial distributions in situ. The data acquired with this protocol can be used in research and clinical practice.

Absorption Mode FTICR Mass Spectrometry Imaging

Fourier transform ion cyclotron resonance mass spectrometry offers the highest mass resolving power for molecular imaging experiments. This high mass resolving power ensures that closely spaced peaks at the same nominal mass are resolved for proper image generation. Typically higher magnetic fields are used to increase mass resolving power. However, a gain in mass resolving power can also be realized by phase correction of the data for absorption mode display. In addition to mass resolving power, absorption mode offers higher mass accuracy and signal-to-noise ratio over the conventional magnitude mode. Here, we present the first use of absorption mode for Fourier transform ion cyclotron resonance mass spectrometry imaging. The Autophaser algorithm is used to phase correct each spectrum (pixel) in the image, and then, these parameters are used by the Chameleon work-flow based data processing software to generate absorption mode "Datacubes" for image and spectral viewing. Absorption mode reveals new mass and spatial features that are not resolved in magnitude mode and results in improved selected ion image contrast.

Advanced Mass Calibration and Visualization for FT-ICR Mass Spectrometry Imaging

Mass spectrometry imaging by Fourier transform ion cyclotron resonance (FT-ICR) yields hundreds of unique peaks, many of which cannot be resolved by lower performance mass spectrometers. The high mass accuracy and high mass resolving power allow confident identification of small molecules and lipids directly from biological tissue sections. Here, calibration strategies for FT-ICR MS imaging were investigated. Sub-parts-per-million mass accuracy is demonstrated over an entire tissue section. Ion abundance fluctuations are corrected by addition of total and relative ion abundances for a root-mean-square error of 0.158 ppm on 16,764 peaks. A new approach for visualization of FT-ICR MS imaging data at high resolution is presented. The "Mosaic Datacube" provides a flexible means to visualize the entire mass range at a mass spectral bin width of 0.001 Da. The high resolution Mosaic Datacube resolves spectral features not visible at lower bin widths, while retaining the high mass accuracy from the calibration methods discussed.

Imaging and Identification of Phospholipids in Mouse Liver Tissue by Matrix Assisted Laser Desorption Ionization-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Imaging

Imaging and Identification of Phospholipids in Mouse Liver Tissue by Matrix Assisted Laser Desorption Ionization-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Imaging

Vacuum compatible sample positioning device for matrix assisted laser desorption∕ionization Fourier transform ion cyclotron resonance mass spectrometry imaging.

The high mass accuracy and resolving power of Fourier transform ion cyclotron resonance mass spectrometers (FT-ICR MS) make them ideal mass detectors for mass spectrometry imaging (MSI), promising to provide unmatched molecular resolution capabilities. The intrinsic low tolerance of FT-ICR MS to RF interference, however, along with typically vertical positioning of the sample, and MSI acquisition speed requirements present numerous engineering challenges in creating robotics capable of achieving the spatial resolution to match. This work discusses a two-dimensional positioning stage designed to address these issues. The stage is capable of operating in ∼1 × 10(-8) mbar vacuum. The range of motion is set to 100 mm × 100 mm to accommodate large samples, while the positioning accuracy is demonstrated to be less than 0.4 micron in both directions under vertical load over the entire range. This device was integrated into three different matrix assisted laser desorption∕ionization (MALDI) FT-ICR instruments and showed no detectable RF noise. The "oversampling" MALDI-MSI experiments, under which the sample is completely ablated at each position, followed by the target movement of the distance smaller than the laser beam, conducted on the custom-built 7T FT-ICR MS demonstrate the stability and positional accuracy of the stage robotics which delivers high spatial resolution mass spectral images at a fraction of the laser spot diameter.