High-resolution dual-polarity molecular imaging platform unraveling spatial metabolic heterogeneity in multiple plant tissues.

Development of high-spatial and high-mass resolution mass spectrometric imaging (MSI) and its application to the study of small metabolites and endogenous molecules of plants

Microscopy-Directed Imaging Mass Spectrometry for Rapid High Spatial Resolution Molecular Imaging of Glomeruli.

Examination of lipid distributions in hydrogel-expanded mouse brain tissue using imaging mass spectrometry.

Drug toxicity during the development of candidate pharmaceuticals is the leading cause of discontinuation in preclinical drug discovery and development. Traditionally, the cause of the toxicity is often determined by histological examination, clinical pathology, and the detection of drugs and/or metabolites by liquid chromatography-mass spectrometry (LC-MS). While these techniques individually provide information on the pathological effects of the drug and the detection of metabolites, they cannot provide specific molecular spatial information without additional experiments. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful, label-free technique for the simultaneous detection of pharmaceuticals, metabolites, and endogenous chemical species in tissue sections, which makes it suitable for mechanistic toxicological studies to directly correlate the distribution of the drug and its metabolites with histological findings. This capability was demonstrated by the analysis of the liver from dogs dosed with discontinued drug compound B and its N-desmethyl metabolite, compound A. Histological examination showed multifocal hepatocellular necrosis, bile duct hyperplasia, periportal fibrosis, and chronic inflammation. MALDI-MSI analysis of liver tissue dosed with only compound A indicated that liver lesions were associated exclusively with the parent compound, whereas liver lesions with compound B showed the presence of the parent compound and its two metabolites (compound A and an N-oxide metabolite). Using both positive and negative ion modes, simultaneous detection and identification of endogenous molecular markers of the connective tissue, blood vessels, liver parenchyma, and bile duct epithelium was achieved, allowing optimal visualization of histological lesions by mass spectrometry imaging.

A method is described for high-resolution label-free molecular imaging of human bone tissue. To preserve the lipid content and the heterogeneous structure of osseous tissue, 4 μm thick human bone sections were prepared via cryoembedding and tape-assisted cryosectioning, circumventing the application of organic solvents and a decalcification step. A protocol for comparative mass spectrometry imaging (MSI) on the same section was established for initial analysis with time-of-flight secondary ion mass spectrometry (TOF-SIMS) at a lateral resolution of 10 μm to <500 nm, followed by atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) Orbitrap MSI at a lateral resolution of 10 μm. This procedure ultimately enabled MSI of lipids, providing the lateral localization of major lipid classes such as glycero-, glycerophospho-, and sphingolipids. Additionally, the applicability of the recently emerged Orbitrap-TOF-SIMS hybrid system was exemplarily examined and compared to the before-mentioned MSI methods.

Integrating Ion Mobility Spectrometry and Mass Spectrometry Imaging for Characterizing the Distribution of Biologically Active Disaccharide Isomers.

Imaging Mass Spectrometry–Based Lipidomic Approach to Classification of Architectural Features in Nevi

Human striated muscle samples, from male control and Duchenne muscular dystrophy-affected children, were subjected to cluster-time-of-flight secondary ion mass spectrometry (cluster-ToF-SIMS) imaging using a 25 keV Bi(3)(+) liquid metal ion gun under static SIMS conditions. Spectra and ion density maps, or secondary ion images, were acquired in both positive and negative ion mode over several areas of 500 x 500 microm(2) (image resolution, 256 x 256 pixels). Characteristic distributions of various lipids were observed. Vitamin E and phosphatidylinositols were found to concentrate within the cells, whereas intact phosphocholines accumulated over the most damaged areas of the dystrophic muscles, together with cholesterol and sphingomyelin species. Fatty acyl chain composition varied depending on the region, allowing estimation of the local damage extent.

Metabolomics: Where seeing is believing

The accumulation of organic pollutants in vegetables is a major global food safety issue. The concentrations of pollutants in vegetables usually differ across different tissues because of different transport and accumulation pathways. However, owing to the limitations of conventional methods, in situ localization of typical organic pollutants such as phthalate esters (PAEs) in plant tissues has not yet been studied. Here, we developed a quick and efficient method for in situ detection and imaging of the spatial distribution of PAEs in a typical root vegetable, carrot, using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). The use of a 2,5-dihydroxybenzoic acid matrix with a spray-sublimation coating method led to the successful identification of PAEs ion signals. The IMS results showed that a typical PAE-di-(2-ethylhexyl)phthalate (DEHP) was broadly distributed in the cortex, phloem, and metaxylem, but was barely detectable in the cambium and protoxylem. Interestingly, MALDI-IMS data also revealed for the first time the spatial distribution of sugars and β-carotene in carrots. In summary, the developed method offers a new and practical methodology for the in situ analysis of PAEs and plant metabolites in plant tissues. As a result, it could provide a more intuitive understanding of the movement and transformation of organic pollutants in soil-plant systems.

Cardiovascular diseases are the world's number one cause of death, accounting for 17.1 million deaths a year. New high-resolution molecular and structural imaging strategies are needed to understand underlying pathophysiological mechanism. The aim of our study is (1) to provide a molecular basis of the heart animal model through the local identification of biomolecules by mass spectrometry imaging (MSI) (three-dimensional (3D) molecular reconstruction), (2) to perform a cross-species validation of secondary ion mass spectrometry (SIMS)-based cardiovascular molecular imaging, and (3) to demonstrate potential clinical relevance by the application of this innovative methodology to human heart specimens. We investigated a MSI approach using SIMS on the major areas of a rat and mouse heart: the pericardium, the myocardium, the endocardium, valves, and the great vessels. While several structures of the heart can be observed in individual two-dimensional sections analyzed by metal-assisted SIMS imaging, a full view of these structures in the total heart volume can be achieved only through the construction of the 3D heart model. The images of 3D reconstruction of the rat heart show a highly complementary localization between Na(+), K(+), and two ions at m/z 145 and 667. Principal component analysis of the MSI data clearly identified different morphology of the heart by their distinct correlated molecular signatures. The results reported here represent the first 3D molecular reconstruction of rat heart by SIMS imaging.

Space and time coherent mapping (STCM) is a technology developed in our laboratory for improved matrix-assisted laser desorption ionization (MALDI) time of flight (TOF) imaging mass spectrometry (IMS). STCM excels in high spatial resolutions, which probe-based scanning methods cannot attain in conventional MALDI IMS. By replacing a scanning probe with a large field laser beam, focusing ion optics, and position-sensitive detectors, STCM tracks the entire flight trajectories of individual ions throughout the ionization process and visualizes the ionization site on the sample surface with a subcellular scale of precision and a substantially short acquisition time. Results obtained in thinly sectioned leech segmental ganglia and epididymis demonstrate that STCM IMS is highly suited for (1) imaging bioactive lipid messengers such as endocannabinoids and the mediators of neuronal activities in situ with spatial resolution sufficient to detail subcellular localization, (2) integrating resultant images in mass spectrometry to optically defined cell anatomy, and (3) assembling a stack of ion maps derived from mass spectra for cluster analysis. We propose that STCM IMS is the choice over a probe-based scanning mass spectrometer for high-resolution single-cell molecular imaging.

Molecular events involved in successful embryo implantation are not well understood. In this study, we used MALDI imaging mass spectrometry (IMS) technologies to characterize the spatial and temporal distribution of phospholipid species associated with mouse embryo implantation. Molecular images showing phospholipid distribution within implantation sites changed markedly between distinct cellular areas during days 4-8 of pregnancy. For example, by day 8, linoleate- and docosahexaenoate-containing phospholipids localized to regions destined to undergo cell death, whereas oleate-containing phospholipids localized to angiogenic regions. Arachidonate-containing phospholipids showed different segregation patterns depending on the lipid class, revealing a strong correlation of phosphatidylethanolamines and phosphatidylinositols with cytosolic phospholipase A(2alpha) and cyclooxygenase-2 during embryo implantation. LC-ESI-MS/MS was used to validate MALDI IMS phospholipid distribution patterns. Overall, molecular images revealed the dynamic complexity of lipid distributions in early pregnancy, signifying the importance of complex interplay of lipid molecules in uterine biology and implantation.

The cftr knockout mouse model of cystic fibrosis (CF) shows intestinal obstruction; malabsorption and inflammation; and a fatty acid imbalance in intestinal mucosa. We performed a lipid mapping of colon sections from CF and control (WT) mice by cluster time of flight secondary-ion mass spectrometry (TOF-SIMS) imaging to localize lipid alterations. Data were processed either manually or by multivariate statistical methods. TOF-SIMS analysis showed a particular localization for cholesteryl sulfate at the epithelial border, C16:1 fatty acid in Lieberkühn glands, and C18:0 fatty acid in lamina propria and submucosa. Significant increases in vitamin E (vE) and C16:0 fatty acid in the epithelial border of CF colon were detected. Principal component analysis (PCA) and partitioning clustering allowed us to characterize different structural regions of colonic mucosa according to variations in C14:0, C16:0, C16:1, C18:0, C18:1, C18:2, C20:3, C20:4, and C22:6 fatty acids; phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol glycerolipids; cholesterol; vitamin E; and cholesteryl sulfate. PCA on spectra from Lieberkühn glands led to separation of CF and WT individuals. This study shows for the first time the spatial distribution of lipids in colonic mucosa and suggests TOF-SIMS plus multivariate analyses as a powerful tool to investigate disease-related tissue spatial lipid signatures.

Imaging with time-of-flight secondary ion mass spectrometry (TOF-SIMS) has expanded very rapidly with the development of gold cluster ion sources (Au(3+)). It is now possible to acquire ion density maps (ion images) on a tissue section without any treatment and with a lateral resolution of few micrometers. In this article, we have taken advantage of this technique to study the degeneration/regeneration process in muscles of a Duchenne muscular dystrophy model mouse. Specific distribution of different lipid classes (fatty acids, triglycerides, phospholipids, tocopherol, coenzyme Q9, and cholesterol) allows us to distinguish three different regions on a mouse leg section: one is destroyed, another is degenerating (oxidative stress and deregulation of the phosphoinositol cycle), and the last one is stable. TOF-SIMS imaging shows the ability to localize directly on a tissue section a great number of lipid compounds that reflect the state of the cellular metabolism.