Mass Spectrometry Imaging Techniques Research Articles

Unsupervised machine learning for exploratory data analysis in imaging mass spectrometry.

Imaging mass spectrometry (IMS) is a rapidly advancing molecular imaging modality that can map the spatial distribution of molecules with high chemical specificity. IMS does not require prior tagging of molecular targets and is able to measure a large number of ions concurrently in a single experiment. While this makes it particularly suited for exploratory analysis, the large amount and high‐dimensional nature of data generated by IMS techniques make automated computational analysis indispensable. Research into computational methods for IMS data has touched upon different aspects, including spectral preprocessing, data formats, dimensionality reduction, spatial registration, sample classification, differential analysis between IMS experiments, and data‐driven fusion methods to extract patterns corroborated by both IMS and other imaging modalities. In this work, we review unsupervised machine learning methods for exploratory analysis of IMS data, with particular focus on (a) factorization, (b) clustering, and (c) manifold learning. To provide a view across the various IMS modalities, we have attempted to include examples from a range of approaches including matrix assisted laser desorption/ionization, desorption electrospray ionization, and secondary ion mass spectrometry‐based IMS. This review aims to be an entry point for both (i) analytical chemists and mass spectrometry experts who want to explore computational techniques; and (ii) computer scientists and data mining specialists who want to enter the IMS field. © 2019 The Authors. Mass Spectrometry Reviews published by Wiley Periodicals, Inc. Mass SpecRev 00:1–47, 2019.

Spatial Mapping of Cellular Metabolites Using DESI Ion Mobility Mass Spectrometry.

Spatial mapping of cellular metabolites, such as neurotransmitters and lipids, on the tissue, can increase our understanding of the biological functions of those molecules. Mass spectrometry imaging (MSI) techniques, such as desorption electrospray ionization (DESI), have not demonstrated the ability to perform metabolite analysis at mammalian single cell level yet. However, they can be a valuable tool to provide insight into cellular metabolism in a very small population (tens) of cells. DESI MSI, coupled with ion mobility separation, improves the peak capacity and signal-to-noise ratio of detected analytes by separating a molecule of interest from interfering isobaric species found in a complex biological matrix. Here we present a protocol for mapping cellular metabolites neurotransmitters, such as serotonin, adenosine, and glutamine directly in brain tissue samples using DESI MSI.

Toward Single Cell Molecular Imaging by Matrix-Free Nanophotonic Laser Desorption Ionization Mass Spectrometry.

In recent years, innovations in mass spectrometry imaging (MSI) have enabled simultaneous detection and mapping of biomolecules and xenobiotics directly from biological tissues and single cells. Matrix-assisted laser desorption ionization (MALDI) has been the most widely embraced MSI technique. However, this technique can exhibit ion suppression effects hindering metabolite coverage and possesses a narrow dynamic range. Nanophotonic platforms, e.g., silicon nanopost array (NAPA) structures, can be used as an alternative for matrix-free imaging of biological tissues. Here, we present a protocol for MSI of large and small adherent cell clusters by laser desorption ionization from NAPA with minimal sample preparation.

Detection of a High-Turnover Serotonin Circuit in the Mouse Brain Using Mass Spectrometry Imaging.

SummaryMonoamine neurotransmitters are released by specialized neurons regulating behavioral, motor, and cognitive functions. Although the localization of monoaminergic neurons in the brain is well known, the distribution and kinetics of monoamines remain unclear. Here, we generated a murine brain atlas of serotonin (5-HT), dopamine (DA), and norepinephrine (NE) levels using mass spectrometry imaging (MSI). We found several nuclei rich in both 5-HT and a catecholamine (DA or NE) and identified the paraventricular nucleus of the thalamus (PVT), where 5-HT and NE are co-localized. The analysis of 5-HT fluctuations in response to acute tryptophan depletion and infusion of isotope-labeled tryptophan in vivo revealed a close kinetic association between the raphe nuclei, PVT, and amygdala but not the other nuclei. Our findings imply the existence of a highly dynamic 5-HT-mediated raphe to PVT pathway that likely plays a role in the brain monoamine system.

CB1 and LPA1 Receptors Relationship in the Mouse Central Nervous System

Neurolipids are a class of bioactive lipids that are produced locally through specific biosynthetic pathways in response to extracellular stimuli. Neurolipids are important endogenous regulators of neural cell proliferation, differentiation, oxidative stress, inflammation and apoptosis. Endocannabinoids (eCBs) and lysophosphatidic acid (LPA) are examples of this type of molecule and are involved in neuroprotection. The present study analyzes a possible relationship of the main receptor subtypes for both neurolipid systems that are present in the central nervous system, the CB1 and LPA1 receptors, by using brain slices from CB1 KO mice and LPA1-null mice. Receptor-mediated G protein activation and glycerophospholipid regulation of potential precursors of their endogenous neurotransmitters were measured by two different in vitro imaging techniques, functional autoradiography and imaging mass spectrometry (IMS), respectively. Possible crosstalk between CB1 and LPA1 receptors was identified in specific areas of the brain, such as the amygdala, where LPA1 receptor activity is upregulated in CB1 KO mice. More evidence of an interaction between both systems was that the CB1-mediated activity was clearly increased in the prefrontal cortex and cerebellum of LPA1-null mice. The eCB system was specifically over-activated in regions where LPA1 has an important signaling role during embryonic development. The modifications on phospholipids (PLs) observed in these genetically modified mice by using the IMS technique indicated the regulation of some of the PL precursors of both LPA and eCBs in specific brain areas. For example, phosphatidylcholine (PC) (36:1) was detected as a potential LPA precursor, and phosphatidylethanolamine (PE) (40:6) and PE (p18:0/22:6) as potential eCB precursors. The absence of the main cerebral receptors for LPA or eCB systems is able to induce modulation on the other at the levels of both signaling and synthesis of endogenous neurotransmitters, indicating adaptive responses between both systems during prenatal and/or postnatal development.

Genomics of Aerobic Photoheterotrophs in Wheat Phyllosphere Reveals Divergent Evolutionary Patterns of Photosynthetic Genes in Methylobacterium spp.

Phyllosphere is a habitat to a variety of viruses, bacteria, fungi, and other microorganisms, which play a fundamental role in maintaining the health of plants and mediating the interaction between plants and ambient environments. A recent addition to this catalogue of microbial diversity was the aerobic anoxygenic phototrophs (AAPs), a group of widespread bacteria that absorb light through bacteriochlorophyll α (BChl a) to produce energy without fixing carbon or producing molecular oxygen. However, culture representatives of AAPs from phyllosphere and their genome information are lacking, limiting our capability to assess their potential ecological roles in this unique niche. In this study, we investigated the presence of AAPs in the phyllosphere of a winter wheat (Triticum aestivum L.) in Denmark by employing bacterial colony based infrared imaging and MALDI-TOF mass spectrometry (MS) techniques. A total of ∼4,480 colonies were screened for the presence of cellular BChl a, resulting in 129 AAP isolates that were further clustered into 21 groups based on MALDI-TOF MS profiling, representatives of which were sequenced using the Illumina NextSeq and Oxford Nanopore MinION platforms. Seventeen draft and four complete genomes of AAPs were assembled belonging in Methylobacterium, Rhizobium, Roseomonas, and a novel Alsobacter. We observed a diverging pattern in the evolutionary rates of photosynthesis genes among the highly homogenous AAP strains of Methylobacterium (Alphaproteobacteria), highlighting an ongoing genomic innovation at the gene cluster level.

Mapping lipid C=C location isomers in organ tissues by coupling photochemical derivatization and rapid extractive mass spectrometry

Lipid desaturation plays important roles in biological processes and the disease states. Here, we report a simple but efficient method for mapping unsaturated phospholipids including the spatial distribution of lipid C=C location isomers in animal organs by coupling the C=C specific derivatization with direct analysis mass spectrometry (MS). Lipids are sampled directly by a stainless-steel wire from rat brain or kidney, extracted, and derivatized via the Paternò–Büchi reaction in a glass emitter of the nanoelectrospray ionization (nanoESI) source. Subsequent analysis by nanoESI-tandem mass spectrometry reveals C=C locations and relative quantities of lipid C=C location isomers. Unsaturated lipids, such as phospholipids and free fatty acids, have been identified with ion intensities spanning two orders of magnitude in rat brain. Typical sample consumption is less than 10 μg/measurement and the time for each analysis is about 3 min. This method should serve as a complementary method to high spatial resolution mass spectrometry imaging techniques, because it offers a streamlined experimental workflow for rapid profiling of lipids with C=C specificity to enable such applications as point-of-care disease diagnostics.

Absolute quantitative imaging of sphingolipids in brain tissue by exhaustive liquid microjunction surface sampling–liquid chromatography–mass spectrometry

It is challenging to achieve absolute quantitation and accurate identification with mass spectrometry imaging (MSI) techniques. The issues facing these techniques include the uncertainty of sampling and ionization efficiencies in a localized environment, the difficulty in defining the concentration of spiked standard on tissue, and the low identification capability of MS in distinguishing isobaric compounds. In this study, we coupled continuous flow liquid microjunction surface sampling (LMJSS) with ultrahigh-performance liquid chromatography (UPLC)–MS for quantitative MSI of brain tissue. LC separation could increase the dimensions of identification and reduce the matrix effects in the tissue. A new LMJSS extraction solvent was developed to achieve exhaustive surface sampling; therefore, direct internal standard addition in the extraction solvent could be used for spot-to-spot absolute quantitation. The results showed that sphingolipids were successfully separated from their isobaric counterparts with LC–MS, and 10 sphingolipids were identified and imaged in brain tissue. The matrix effects in different locations of brain tissue with the new solvent were all in the range of 80%–150%. Compared with the traditional LMJSS solvent (90% methanol-water), the new solvent (10% 1,1,1,3,3,3-Hexafluoro-2-propanol-10% isopropanol-80% methanol) led to higher detection coverage (more lipid features) and 6-10-fold higher sensitivity for 6 identified metabolites in brain tissue. Moreover, extraction efficiencies of 80–98% for targeted sphingolipids in brain tissue were obtained with the new solvent, which allowed direct standard addition in extracts for absolute quantitation. Finally, the absolute quantitation results with LMJSS–LC–MSI were compared with those using traditional bulk tissue extraction–LC–MS, and similar quantitation results with these two methods (relative recoveries=64–119%) were obtained for sphingolipids. The absolute quantitative spatial distributions of targeted metabolites largely matched previously reported results. The method was applied to a study on the quantitative spatial changes of sphingolipids and creatine in brain after traumatic brain injury (TBI). The biological replicate results showed that 2 metabolites had significant changes after TBI in several specific regions of brain tissue.

Distribution of Small Molecular Weight Drugs into the Porcine Lens: Studies on Imaging Mass Spectrometry, Partition Coefficients, and Implications in Ocular Pharmacokinetics.

Lens is the avascular tissue in the eye between the aqueous humor and vitreous. Drug binding to the lens might affect ocular pharmacokinetics, and the binding may also have a pharmacological role in drug-induced cataract and cataract treatment. Drug distribution in the lens has been studied in vitro with many compounds; however, the experimental methods vary, no detailed information on distribution between the lens sublayers exist, and the partition coefficients are reported rarely. Therefore, our objectives were to clarify drug localization in the lens layers and establish partition coefficients for a wide range of molecules. Furthermore, we aimed to illustrate the effect of lenticular drug binding on overall ocular drug pharmacokinetics. We studied the distribution of 16 drugs and three fluorescent dyes in whole porcine lenses in vitro with imaging mass spectrometry and fluorescence microscopy techniques. Furthermore, we determined lens/buffer partition coefficients with the same experimental setup for 28 drugs with mass spectrometry. Finally, the effect of lenticular binding of drugs on aqueous humor drug exposure was explored with pharmacokinetic simulations. After 4 h, the drugs and the dyes distributed only to the outermost lens layers (capsule and cortex). The lens/buffer partition coefficients for the drugs were low, ranging from 0.05 to 0.8. On the basis of the pharmacokinetic simulations, a high lens-aqueous humor partition coefficient increases drug exposure in the lens but does not significantly alter the pharmacokinetics in the aqueous humor. To conclude, the lens seems to act mainly as a physical barrier for drug distribution in the eye, and drug binding to the lens affects mainly the drug pharmacokinetics in the lens.

Conductive Adhesive Film Expands the Utility of Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

The matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technique is a promising approach for detecting the distribution of small molecules in a section of biological tissue. However, when a cryosection is created from fragile, hard, or whole-body samples, obtaining a high-quality section that maintains the distribution of the various components has been difficult. Since adhesive films have the potential to obtain high-quality cryosections, we attempted to utilize a conductive adhesive film for MALDI-MSI. To this end, cryosections of the whole body of a 9-day-old mouse were directly prepared on indium tin oxide (ITO) glass slides, nonconductive adhesive films, or conductive adhesive films, and the signal intensities from each section were measured by MALDI-MSI. We measured the differences in the ion intensity among these three slides/films by means of multivariate analyses and found that both the nonconductive and conductive adhesive films gave rise to high-quality sections in comparison with the ITO glass slide. The conductive adhesive film gave higher signals that were comparable to those of the ITO glass slide in comparison with the nonconductive adhesive film. We divided the frozen sections into two groups, a freeze-dried group and a thawed group, to examine the freeze-thaw effect on the signals of representative compounds of amino acids, cholesterol, and phosphatidylcholines. The freeze-dried samples were found to be useful for the analysis. These results indicate that the sections made with the conductive adhesive film under a freeze-dried condition can expand the utility of the MALDI-MSI analysis.

Dexmedetomidine enhances glymphatic brain delivery of intrathecally administered drugs

Drug delivery to the central nervous system remains a major problem due to biological barriers. The blood-brain-barrier can be bypassed by administering drugs intrathecally directly to the cerebrospinal fluid (CSF). The glymphatic system, a network of perivascular spaces promoting fluid exchange between CSF and interstitial space, could be utilized to enhance convective drug delivery from the CSF to the parenchyma. Glymphatic flow is highest during sleep and anesthesia regimens that induce a slow-wave sleep-like state. Here, using mass spectrometry and fluorescent imaging techniques, we show that the clinically used α2-adrenergic agonist dexmedetomidine that enhances EEG slow-wave activity, increases brain and spinal cord drug exposure of intrathecally administered drugs in mice and rats. Using oxycodone, naloxone, and an IgG-sized antibody as relevant model drugs we demonstrate that modulation of glymphatic flow has a distinct impact on the distribution of intrathecally administered therapeutics. These findings can be exploited in the clinic to improve the efficacy and safety of intrathecally administered therapeutics.

Spatiotemporal Transformation in the Alkaloid Profile of Pinus Roots in Response to Mycorrhization.

Root alkaloids remain highly unexplored in ectomycorrhizae development studies. By employing ultrahigh mass resolution mass spectrometry imaging techniques, we showed substantial relocation and transformation of piperidine alkaloids in pine root tips in response to Suillus mycorrhization. We imaged, in the time frame of ectomycorrhizae formation, a completely different alkaloid profile in Pinus strobus, where basidiospores of Suillus spraguei induce morphogenesis of symbiotic tissues, than in Pinus taeda, where such interaction fails to induce morphogenesis. On the basis of spatial colocalization studies, we proposed some alternative routes for biosynthesis of these alkaloids that supplement existing literature data.

Anticancer Drug Affects Metabolomic Profiles in Multicellular Spheroids: Studies Using Mass Spectrometry Imaging Combined with Machine Learning.

Multicellular spheroids (hereinafter referred to as spheroids) are 3D biological models. The metabolomic profiles inside spheroids provide crucial information reflecting the molecular phenotypes and microenvironment of cells. To study the influence of an anticancer drug on the spatially resolved metabolites, spheroids were cultured using HCT-116 colorectal cancer cells, treated with the anticancer drug Irinotecan under a series of time- and concentration-dependent conditions. The Single-probe mass spectrometry imaging (MSI) technique was utilized to conduct the experiments. The MSI data were analyzed using advanced data analysis methods to efficiently extract metabolomic information. Multivariate curve resolution alternating least square (MCR-ALS) was used to decompose each MS image into different components with grouped species. To improve the efficiency of data analysis, both supervised (Random Forest) and unsupervised (cluster large applications (CLARA)) machine learning (ML) methods were employed to cluster MS images according to their metabolomic features. Our results indicate that anticancer drug significantly affected the abundances of a variety of metabolites in different regions of spheroids. This integrated experiment and data analysis approach can facilitate the studies of metabolites in different types of 3D tumor models and tissues and potentially benefit the drug discovery, therapeutic resistance, and other biological research fields.

Monitoring indole alkaloid production by Penicillium digitatum during infection process in citrus by Mass Spectrometry Imaging and molecular networking

Green mold, caused by Penicillium digitatum, is the most destructive post-harvest disease in citrus. Secondary metabolites produced by fungal phytopathogens have been associated with toxicity to their respective host through the interaction with a wide range of cell targets. Natural products have also been described as important molecules for biocontrol and competition in their respective environment. For P. digitatum, the production of indole alkaloids, tryptoquialanines A and B, have been reported. However, their biological role remains unknown. Mass Spectrometry Imaging (MSI) technique was applied here for the first time to monitor the secondary metabolites produced on the orange surface during infection in order to gain insights about the P. digitatum-citrus interaction mechanisms. Through the combination of MSI and molecular networking it was possible to report, for the first time, the production of tryptoquivalines and fumiquinazolines by P. digitatum and also the accumulation of tryptoquialanines on the fruit surface from 4 to 7 d post inoculation. P. digitatum was also evaluated concerning the ability to sinthesize indole alkaloids in vivo in the different citrus hosts. The biological role of tryptoquialanines was investigated and tryptoquialanine A was submitted to insecticidal bioassays that revealed its high toxicity against Aedes Aegypti, suggesting an important insecticidal action during orange decay.

Application of hyperspectral imaging and mass spectrometry imaging technique to fingerprint visualization and trace analysis

Developing on advanced light sources, especially those applied in the areas of spectral imaging and mass spectrometry imaging, has made the trace analysis feasible and more reliable. These techniques show great potentials in various fields including forensic science, environment, food, pharmaceuticals, archaeology, etc. In many cases of trace analysis, it is expected to obtain both the spatial distributions and chemical compositions of the target objects. Through the combination of imaging technology with optical spectroscopy and mass spectrometry, it is possible to detect the trace chemicals on the surface of various materials as well as their spatial distributions, thus improving the accuracy of detection and the range of application. Moreover, trace analysis based on such methods can reduce or even avoid the use of special chemical reagents, and is compatible with the traditional chemical detection methods. In the paper, we focus on fingerprint visualization and analysis, as a typical trace analysis issue, to discuss the recent progress of the applicable chemical imaging technologies based on the advanced light sources. The effect of latent fingerprint development depends on not only features of fingerprint carrying object, but also the characteristics of fingerprint residues. In this paper, we provide an overview of two technical approaches: specific component targeted chemical imaging and nondirective chemical imaging. We describe the major technologies involved in this field, including visible-near infrared chemical imaging, mid-infrared chemical imaging, Raman imaging, and mass spectrometry imaging.

Matrix-free mass spectrometry imaging of mouse brain tissue sections on silicon nanopost arrays.

Mass spectrometry imaging (MSI) is capable of detection and identification of diverse classes of compounds in brain tissue sections, whereas simultaneously mapping their spatial distributions. Given the vast array of chemical components present in neurological systems, as well as the innate diversity within molecular classes, MSI platforms capable of detecting a wide array of species are useful for achieving a more comprehensive understanding of their biological roles and significance. Currently, matrix-assisted laser desorption ionization (MALDI) is the method of choice for the molecular imaging of brain samples by mass spectrometry. However, nanostructured laser desorption ionization platforms, such as silicon nanopost arrays (NAPA), are emerging as alternative MSI techniques that can provide complementary insight into molecular distributions in the central nervous system. In this work, the molecular coverage of mouse brain lipids afforded by NAPA-MSI is compared to that of MALDI-MSI using two common MALDI matrices. In positive ion mode, MALDI spectra were dominated by phosphatidylcholines and phosphatidic acids. NAPA favored the ionization of phosphatidylethanolamines and glycosylated ceramides, which were poorly detected in MALDI-MSI. In negative ion mode, MALDI favored sulfatides and free fatty acids, whereas NAPA spectra were dominated by signal from phosphatidylethanolamines. The complementarity in lipid coverages between the NAPA- and MALDI-MSI platforms presents the possibility of selective lipid analysis and imaging dependent upon which platform is used. Nanofabrication of the NAPA platform offers better uniformity compared to MALDI, and the wider dynamic range offered by NAPA promises improved quantitation in imaging.

The influence of polyatomic primary ion chemistry on matrix effects in secondary ion mass spectrometry analysis

The application of mass spectrometry imaging techniques to determine two- (2D) and three- (3D) dimensional chemical distribution ideally provides uniform, high sensitivity to multiple components and reliable quantification. These criteria are typically not met due to variations in sensitivity due to the chemistry of the analyte and surrounding surface chemistry. Here we explore the influence of projectile beam chemistry and sample chemistry in time-of-flight secondary ion mass spectrometry (TOF-SIMS). To the authors' knowledge this is the first time the combined effects of projectile chemistry and sample environment on the quantitative determination of mixed samples have been systematically studied. Secondary ion yields of lipid and amino acid mixtures were measured under 20 keV C60 , Arn , and (H2 O)n cluster ion bombardment (n= 2000 or 4000) using TOF-SIMS. Ion suppression/enhancement effects were studied in dry sample films and in trehalose and water ice matrices. The extent of the matrix effects and the secondary ion yield were found to depend on the chemistry of the primary ion beam and (for C60 , Arn ) on the nature of the sample matrix. Under (H2 O)n bombardment the sample matrix had negligible effect on the analysis. Compared with C60 and Arn , water-containing cluster projectiles enhanced the sensitivity of TOF-SIMS determination of the chosen analytes and reduced the effect of signal suppression/enhancement in multicomponent samples and in different sample matrices. One possible explanation for this is that the (H2 O)4000 projectile initiates on impact a nanoscale matrix environment that is very similar to that in frozen-hydrated samples in terms of the resulting ionisation effects. The competition between analytes for protons and the effect of the sample matrix are reduced with water-containing cluster projectiles. These chemically reactive projectile beams have improved characteristics for quantitative chemical imaging by TOF-SIMS compared with their non-reactive counterparts.

Biomolecular Imaging of Regeneration of Zebrafish Caudal Fins Using High Spatial Resolution Ambient Mass Spectrometry.

We observed the molecular distribution changes that occurred during the regeneration of fresh zebrafish caudal fins using the recently developed ambient high-resolution mass spectrometry (MS) imaging technique of atmospheric pressure-nanoparticle and plasma-assisted laser desorption ionization (AP-nanoPALDI). AP-nanoPALDI analyses of fresh zebrafish caudal fins revealed that the small molecules, including neurotransmitters, amino acids, lipids, and metabolites of the regenerated area, were more evenly distributed throughout the bony rays and inter-ray mesenchymal tissues compared to the original area in the early stage. Zebrafish caudal fins of less than 200 μm thickness can be very useful for tissue regeneration studies using ambient MS imaging by providing sufficient biomolecular information at the molecular level for wound-healing studies. AP-nanoPALDI imaging was compared with a complementary MS imaging tool, surface sensitive time-of-flight secondary ion MS (ToF-SIMS).

Ambient ionization mass spectrometry imaging for disease diagnosis: Excitements and challenges

Tissue analysis in histology is extremely important and also considered to be a gold standard to diagnose and prognosticate several diseases including cancer. Intraoperative evaluation of surgical margin of tumor also relies on frozen section histopathology, which is time consuming, challenging and often subjective. Recent development in the ambient ionization mass spectrometry imaging (MSI) technique has enabled us to simultaneously visualize hundreds to thousands of molecules (ion images) in the biopsy specimen, which are strikingly different and more powerful than the single optical tissue image analysis in conventional histopathology. This paper will highlight the emergence of the desorption electrospray ionization MSI (DESI-MSI) technique, which is label-free, requires minimal or no sample preparation and operates under ambient conditions. DESI-MSI can record ion images of lipid/metabolite distributions on biopsy specimens, providing a wealth of diagnostic information based on differential distributions of these molecular species in healthy and unhealthy tissues. Remarkable success of this technology in rapidly evaluating the cancer margin intraoperatively with very high accuracy also promises to bring this imaging technique from bench to bedside.

Spatio-temporal visualization of the distribution of acetaminophen as well as its metabolites and adducts in mouse livers by MALDI MSI.

Acetaminophen (APAP) is one of the most intensively studied compounds that causes hepatotoxicity in the pericentral region of the liver lobules. However, spatio-temporal information on the distribution of APAP, its metabolites and GSH adducts in the liver tissue is not yet available. Here, we addressed the question, whether APAP-GSH adducts and GSH depletion show a zonated pattern and whether the distribution of APAP and its glucuronide as well as sulfate conjugates in liver lobules are zonated. For this purpose, a matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) technique was established, where the MSI images were superimposed onto CYP2E1 immunostained tissue. A time-dependent analysis (5, 15, 30, 60, 120, 240, 480min) after intraperitoneal administration of 300mg/kg APAP and a dose-dependent analysis (56 up to 500mg APAP/kg) at 30min were performed. The results demonstrate that the MALDI MSI technique allows the assignment of compounds and their metabolites to specific lobular zones. APAP-GSH adducts and GSH depletion occurred predominantly in the CYP2E1-positive zone of the liver, although GSH also decreased in the periportal region. In contrast, the parent compound, APAP sulfate and APAP glucuronide did not show a zonated pattern and tissue concentrations showed a similar time course as the corresponding analyses were performed with blood from the portal and liver veins. In conclusion, the present study is in agreement with the concept that pericentral CYPs form NAPQI that in the same cell binds to and depletes GSH but a lower level of GSH adducts is also observed in the periportal region. The results also provide further evidence of the recently published concept of 'aggravated loss of clearance capacity' according to which also liver tissue that survives intoxication may transiently show decreased metabolic capacity.