Validation of an Organ Mapping Antibody Panel for Cyclical Immunofluorescence Microscopy on Normal Human Kidneys.

The lack of standardization in antibody validation remains a major contributor to irreproducibility of human research. To address this, we have applied a standardized approach to validate a panel antibodies to identify 18 major cell types and 5 extracellular matrix compartments in the human kidney by immunofluorescence (IF) microscopy. We have used these to generate an organ mapping antibody panel for 2-D and 3-D Cyclical Immunofluorescence (CyCIF) to provide a more detailed method to evaluate of tissue segmentation and volumes using a larger panel of markers than would normally be possible using standard fluorescence microscopy. CyCIF also makes it possible to perform multiplexed IF microscopy of whole slide images, which is a distinct advantage over other multiplexed imaging technologies that are applicable to limited fields of view. This enables a broader view of cell distributions across larger anatomical regions, allowing a better chance to capture localized regions of dysfunction in diseased tissues. These methods are broadly accessible to any laboratory with a fluorescence microscope, enabling spatial cellular phenotyping in normal and disease states. We also provide a detailed solution for image alignment between CyCIF cycles that can be used by investigators to perform these studies without programming experience using open-sourced software. This ability to perform multiplexed imaging without specialized instrumentation or computational skills, opens the door to integration with more highly dimensional molecular imaging modalities such as spatial transcriptomics and imaging mass spectrometry, enabling the discovery of molecular markers of specific cell types and how these are altered in disease.

Blind Polarization Demultiplexing of Shaped QAM Signals Assisted by Temporal Correlations.

While probabilistic constellation shaping (PCS) enables rate and reach adaption with finer granularity [1], it imposes signal processing challenges at the receiver. Since the distribution of PCS-quadrature amplitude modulation (QAM) signals tends to be Gaussian, conventional blind polarization demultiplexing algorithms are not suitable for them [2]. It is known that independently and identically distributed (iid) Gaussian signals, when mixed, cannot be recovered/separated from their mixture. For PCS-QAM signals, there are algorithms such as [3], [4] which are designed by extending conventional blind algorithms used for uniform QAM signals. In these algorithms, an initialization point is obtained by processing only a part of the mixed signal, which have non-Gaussian statistics. In this paper, we propose an alternative method wherein we add temporal correlations at the transmitter, which are subsequently exploited at the receiver in order to separate the polarizations. We will refer to the proposed method as frequency domain (FD) joint diagonalization (JD) probability aware-multi modulus algorithm (pr-MMA), and it is suited to channels with moderate polarization mode dispersion (PMD) effects. Furthermore, we extend our previously proposed JD-MMA [5] by replacing the standard MMA with a pr-MMA, improving its performance. Both FDJD-pr-MMA and JD-pr-MMA are evaluated for a diverse range of PCS (entropy 𝓗) over a first-order PMD channel that is simulated in a proof-of-concept setup. A MMA initialized with a memoryless constant modulus algorithm (CMA) is used as a benchmark. We show that at a differential group delay (DGD) of 10% of symbol period Tsymb and 18 dB SNR/pol., JD-pr-MMA successfully demultiplexes the PCS signals, while CMA-MMA fails drastically. Furthermore, we demonstrate that the newly proposed FDJD-pr-MMA is robust against moderate PMD effects by evaluating it over a DGD of up to 40% of Tsymb. Our results show that the proposed FDJD-pr-MMA successfully equalizes PMD channels with a DGD up to 20% of Tsymb.

Advances and prospects for the Human BioMolecular Atlas Program (HuBMAP).

The Human BioMolecular Atlas Program (HuBMAP) aims to create a multi-scale spatial atlas of the healthy human body at single-cell resolution by applying advanced technologies and disseminating resources to the community. As the HuBMAP moves past its first phase, creating ontologies, protocols and pipelines, this Perspective introduces the production phase: the generation of reference spatial maps of functional tissue units across many organs from diverse populations and the creation of mapping tools and infrastructure to advance biomedical research.

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

The glomerulus is a multicellular functional tissue unit (FTU) of the nephron that is responsible for blood filtration. Each glomerulus contains multiple substructures and cell types that are crucial for their function. To understand normal aging and disease in kidneys, methods for high spatial resolution molecular imaging within these FTUs across whole slide images is required. Here we demonstrate a workflow using microscopy-driven selected sampling to enable 5 µm pixel size matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) of all glomeruli within whole slide human kidney tissues. Such high spatial resolution imaging entails large numbers of pixels, increasing the data acquisition times. Automating FTU-specific tissue sampling enables high-resolution analysis of critical tissue structures, while concurrently maintaining throughput. Glomeruli were automatically segmented using coregistered autofluorescence microscopy data, and these segmentations were translated into MALDI IMS measurement regions. This allowed high-throughput acquisition of 268 glomeruli from a single whole slide human kidney tissue section. Unsupervised machine learning methods were used to discover molecular profiles of glomerular subregions and differentiate between healthy and diseased glomeruli. Average spectra for each glomerulus were analyzed using Uniform Manifold Approximation and Projection (UMAP) and k-means clustering, yielding 7 distinct groups of differentiated healthy and diseased glomeruli. Pixel-wise k-means clustering was applied to all glomeruli, showing unique molecular profiles localized to subregions within each glomerulus. Automated microscopy-driven, FTU-targeted acquisition for high spatial resolution molecular imaging maintains high-throughput and enables rapid assessment of whole slide images at cellular resolution and identification of tissue features associated with normal aging and disease.

MALDI IMS-Derived Molecular Contour Maps: Augmenting Histology Whole-Slide Images.

Imaging mass spectrometry (IMS) provides untargeted, highly multiplexed maps of molecular distributions in tissue. Ion images are routinely presented as heatmaps and can be overlaid onto complementary microscopy images that provide greater context. However, heatmaps use transparency blending to visualize both images, obscuring subtle quantitative differences and distribution gradients. Here, we developed a contour mapping approach that combines information from IMS ion intensity distributions with that of stained microscopy. As a case study, we applied this approach to imaging data from Staphylococcus aureus-infected murine kidney. In a univariate, or single molecular species, use-case of the contour map representation of IMS data, certain lipids colocalizing with regions of infection were selected using Pearson's correlation coefficient. Contour maps of these lipids overlaid with stained microscopy showed enhanced visualization of lipid distributions and spatial gradients in and around the bacterial abscess as compared to traditional heatmaps. The full IMS data set comprising hundreds of individual ion images was then grouped into a smaller subset of representative patterns using non-negative matrix factorization (NMF). Contour maps of these multivariate NMF images revealed distinct molecular profiles of the major abscesses and surrounding immune response. This contour mapping workflow also enabled a molecular visualization of the transition zone at the host-pathogen interface, providing potential clues about the spatial molecular dynamics beyond what histological staining alone provides. In summary, we developed a new IMS-based contour mapping approach to augment classical stained microscopy images, providing an enhanced and more interpretable visualization of IMS-microscopy multimodal molecular imaging data sets.

MALDI TIMS IMS of Disialoganglioside Isomers─GD1a and GD1b in Murine Brain Tissue.

Gangliosides are acidic glycosphingolipids, containing ceramide moieties and oligosaccharide chains with one or more sialic acid residue(s) and are highly diverse isomeric structures with distinct biological roles. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) enables the untargeted spatial analysis of gangliosides, among other biomolecules, directly from tissue sections. Integrating trapped ion mobility spectrometry with MALDI IMS allows for the analysis of isomeric lipid structures in situ. Here, we demonstrate the gas-phase separation and identification of disialoganglioside isomers GD1a and GD1b that differ in the position of a sialic acid residue, in multiple samples, including a standard mixture of both isomers, a biological extract, and directly from thin tissue sections. The unique spatial distributions of GD1a/b (d36:1) and GD1a/b (d38:1) isomers were determined in rat hippocampus and spinal cord tissue sections, demonstrating the ability to structurally characterize and spatially map gangliosides based on both the carbohydrate chain and ceramide moieties.

Rapid Multivariate Analysis Approach to Explore Differential Spatial Protein Profiles in Tissue.

Spatially targeted proteomics analyzes the proteome of specific cell types and functional regions within tissue. While spatial context is often essential to understanding biological processes, interpreting sub-region-specific protein profiles can pose a challenge due to the high-dimensional nature of the data. Here, we develop a multivariate approach for rapid exploration of differential protein profiles acquired from distinct tissue regions and apply it to analyze a published spatially targeted proteomics data set collected from Staphylococcus aureus-infected murine kidney, 4 and 10 days postinfection. The data analysis process rapidly filters high-dimensional proteomic data to reveal relevant differentiating species among hundreds to thousands of measured molecules. We employ principal component analysis (PCA) for dimensionality reduction of protein profiles measured by microliquid extraction surface analysis mass spectrometry. Subsequently, k-means clustering of the PCA-processed data groups samples by chemical similarity. Cluster center interpretation revealed a subset of proteins that differentiate between spatial regions of infection over two time points. These proteins appear involved in tricarboxylic acid metabolomic pathways, calcium-dependent processes, and cytoskeletal organization. Gene ontology analysis further uncovered relationships to tissue damage/repair and calcium-related defense mechanisms. Applying our analysis in infectious disease highlighted differential proteomic changes across abscess regions over time, reflecting the dynamic nature of host-pathogen interactions.

Lysolipids are prominent in subretinal drusenoid deposits, a high-risk phenotype in age-related macular degeneration.

Introduction: Age related macular degeneration (AMD) causes legal blindness worldwide, with few therapeutic targets in early disease and no treatments for 80% of cases. Extracellular deposits, including drusen and subretinal drusenoid deposits (SDD; also called reticular pseudodrusen), disrupt cone and rod photoreceptor functions and strongly confer risk for advanced disease. Due to the differential cholesterol composition of drusen and SDD, lipid transfer and cycling between photoreceptors and support cells are candidate dysregulated pathways leading to deposit formation. The current study explores this hypothesis through a comprehensive lipid compositional analysis of SDD. Methods: Histology and transmission electron microscopy were used to characterize the morphology of SDD. Highly sensitive tools of imaging mass spectrometry (IMS) and nano liquid chromatography tandem mass spectrometry (nLC-MS/MS) in positive and negative ion modes were used to spatially map and identify SDD lipids, respectively. An interpretable supervised machine learning approach was utilized to compare the lipid composition of SDD to regions of uninvolved retina across 1873 IMS features and to automatically discern candidate markers for SDD. Immunohistochemistry (IHC) was used to localize secretory phospholipase A2 group 5 (PLA2G5). Results: Among the 1873 detected features in IMS data, three lipid classes, including lysophosphatidylcholine (LysoPC), lysophosphatidylethanolamine (LysoPE) and lysophosphatidic acid (LysoPA) were observed nearly exclusively in SDD while presumed precursors, including phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidic acid (PA) lipids were detected in SDD and adjacent photoreceptor outer segments. Molecular signals specific to SDD were found in central retina and elsewhere. IHC results indicated abundant PLA2G5 in photoreceptors and retinal pigment epithelium (RPE). Discussion: The abundance of lysolipids in SDD implicates lipid remodeling or degradation in deposit formation, consistent with ultrastructural evidence of electron dense lipid-containing structures distinct from photoreceptor outer segment disks and immunolocalization of secretory PLA2G5 in photoreceptors and RPE. Further studies are required to understand the role of lipid signals observed in and around SDD.

Imaging mass spectrometry reveals complex lipid distributions across Staphylococcus aureus biofilm layers.

Introduction: Although Staphylococcus aureus is the leading cause of biofilm-related infections, the lipidomic distributions within these biofilms is poorly understood. Here, lipidomic mapping of S. aureus biofilm cross-sections was performed to investigate heterogeneity between horizontal biofilm layers. Methods: S. aureus biofilms were grown statically, embedded in a mixture of carboxymethylcellulose/gelatin, and prepared for downstream matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). Trapped ion mobility spectrometry (TIMS) was also applied prior to mass analysis. Results: Implementation of TIMS led to a âˆ¼ threefold increase in the number of lipid species detected. Washing biofilm samples with ammonium formate (150 mM) increased signal intensity for some bacterial lipids by as much as tenfold, with minimal disruption of the biofilm structure. MALDI TIMS IMS revealed that most lipids localize primarily to a single biofilm layer, and species from the same lipid class such as cardiolipins CL(57:0) - CL(66:0) display starkly different localizations, exhibiting between 1.5 and 6.3-fold intensity differences between layers (n = 3, p < 0.03). No horizontal layers were observed within biofilms grown anaerobically, and lipids were distributed homogenously. Conclusions: High spatial resolution analysis of S. aureus biofilm cross-sections by MALDI TIMS IMS revealed stark lipidomic heterogeneity between horizontal S. aureus biofilm layers demonstrating that each layer was molecularly distinct. Finally, this workflow uncovered an absence of layers in biofilms grown under anaerobic conditions, possibly indicating that oxygen contributes to the observed heterogeneity under aerobic conditions. Future applications of this workflow to study spatially localized molecular responses to antimicrobials could provide new therapeutic strategies.

Visualizing Staphylococcus aureus pathogenic membrane modification within the host infection environment by multimodal imaging mass spectrometry.

Bacterial pathogens have evolved virulence factors to colonize, replicate, and disseminate within the vertebrate host. Although there is an expanding body of literature describing how bacterial pathogens regulate their virulence repertoire in response to environmental signals, it is challenging to directly visualize virulence response within the host tissue microenvironment. Multimodal imaging approaches enable visualization of host-pathogen molecular interactions. Here we demonstrate multimodal integration of high spatial resolution imaging mass spectrometry and microscopy to visualize Staphylococcus aureus envelope modifications within infected murine and human tissues. Data-driven image fusion of fluorescent bacterial reporters and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance imaging mass spectrometry uncovered S. aureus lysyl-phosphatidylglycerol lipids, localizing to select bacterial communities within infected tissue. Absence of lysyl-phosphatidylglycerols is associated with decreased pathogenicity during vertebrate colonization as these lipids provide protection against the innate immune system. The presence of distinct staphylococcal lysyl-phosphatidylglycerol distributions within murine and human infections suggests a heterogeneous, spatially oriented microbial response to host defenses.

Automated biomarker candidate discovery in imaging mass spectrometry data through spatially localized Shapley additive explanations.

The search for molecular species that are differentially expressed between biological states is an important step towards discovering promising biomarker candidates. In imaging mass spectrometry (IMS), performing this search manually is often impractical due to the large size and high-dimensionality of IMS datasets. Instead, we propose an interpretable machine learning workflow that automatically identifies biomarker candidates by their mass-to-charge ratios, and that quantitatively estimates their relevance to recognizing a given biological class using Shapley additive explanations (SHAP). The task of biomarker candidate discovery is translated into a feature ranking problem: given a classification model that assigns pixels to different biological classes on the basis of their mass spectra, the molecular species that the model uses as features are ranked in descending order of relative predictive importance such that the top-ranking features have a higher likelihood of being useful biomarkers. Besides providing the user with an experiment-wide measure of a molecular species' biomarker potential, our workflow delivers spatially localized explanations of the classification model's decision-making process in the form of a novel representation called SHAP maps. SHAP maps deliver insight into the spatial specificity of biomarker candidates by highlighting in which regions of the tissue sample each feature provides discriminative information and in which regions it does not. SHAP maps also enable one to determine whether the relationship between a biomarker candidate and a biological state of interest is correlative or anticorrelative. Our automated approach to estimating a molecular species' potential for characterizing a user-provided biological class, combined with the untargeted and multiplexed nature of IMS, allows for the rapid screening of thousands of molecular species and the obtention of a broader biomarker candidate shortlist than would be possible through targeted manual assessment. Our biomarker candidate discovery workflow is demonstrated on mouse-pup and rat kidney case studies.

Molecular Mapping of Neutral Lipids Using Silicon Nanopost Arrays and TIMS Imaging Mass Spectrometry.

We demonstrate the utility of combining silicon nanopost arrays (NAPA) and trapped ion mobility imaging mass spectrometry (TIMS IMS) for high spatial resolution and specificity mapping of neutral lipid classes in tissue. Ionization of neutral lipid species such as triglycerides (TGs), cholestryl esters (CEs), and hexosylceramides (HexCers) from biological tissues has remained a challenge for imaging applications. NAPA, a matrix-free laser desorption ionization substrate, provides enhanced ionization efficiency for the above-mentioned neutral lipid species, providing complementary lipid coverage to matrix-assisted laser desorption ionization (MALDI). The combination of NAPA and TIMS IMS enables imaging of neutral lipid species at 20 µm spatial resolution while also increasing molecular coverage greater than 2-fold using gas-phase ion mobility separations. This is a significant improvement with respect to sensitivity, specificity, and spatial resolution compared to previously reported imaging studies using NAPA alone. Improved specificity for neutral lipid analysis using TIMS IMS was shown using rat kidney tissue to separate TGs, CEs, HexCers, and phospholipids into distinct ion mobility trendlines. Further, this technology allowed for the separation of isomeric species, including mobility resolved isomers of Cer(d42:2) (m/z 686.585) with distinct spatial localizations measured in rat kidney tissue section.

Protocol for multimodal analysis of human kidney tissue by imaging mass spectrometry and CODEX multiplexed immunofluorescence.

Here, we describe the preservation and preparation of human kidney tissue for interrogation by histopathology, imaging mass spectrometry, and multiplexed immunofluorescence. Custom image registration and integration techniques are used to create cellular and molecular atlases of this organ system. Through careful optimization, we ensure high-quality and reproducible datasets suitable for cross-patient comparisons that are essential to understanding human health and disease. Moreover, each of these steps can be adapted to other organ systems or diseases, enabling additional atlas efforts.

Resolving the Complexity of Spatial Lipidomics Using MALDI TIMS Imaging Mass Spectrometry.

Lipids are a structurally diverse class of molecules with important biological functions including cellular signaling and energy storage. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows for direct mapping of biomolecules in tissues. Fully characterizing the structural diversity of lipids remains a challenge due to the presence of isobaric and isomeric species, which greatly complicates data interpretation when only m/z information is available. Integrating ion mobility separations aids in deconvoluting these complex mixtures and addressing the challenges of lipid IMS. Here, we demonstrate that a MALDI quadrupole time-of-flight (Q-TOF) mass spectrometer with trapped ion mobility spectrometry (TIMS) enables a >250% increase in the peak capacity during IMS experiments. MALDI TIMS-MS separation of lipid isomer standards, including sn backbone isomers, acyl chain isomers, and double-bond position and stereoisomers, is demonstrated. As a proof of concept, in situ separation and imaging of lipid isomers with distinct spatial distributions were performed using tissue sections from a whole-body mouse pup.

Spatial Metabolomics of the Human Kidney using MALDI Trapped Ion Mobility Imaging Mass Spectrometry.

Low molecular weight metabolites are essential for defining the molecular phenotypes of cells. However, spatial metabolomics tools often lack the sensitivity, specify, and spatial resolution to provide comprehensive descriptions of these species in tissue. MALDI imaging mass spectrometry (IMS) of low molecular weight ions is particularly challenging as MALDI matrix clusters are often nominally isobaric with multiple metabolite ions, requiring high resolving power instrumentation or derivatization to circumvent this issue. An alternative to this is to perform ion mobility separation before ion detection, enabling the visualization of metabolites without the interference of matrix ions. Additional difficulties surrounding low weight metabolite visualization include high resolution imaging, while maintaining sufficient ion numbers for broad and representative analysis of the tissue chemical complement. Here, we use MALDI timsTOF IMS to image low molecular weight metabolites at higher spatial resolution than most metabolite MALDI IMS experiments (20 µm) while maintaining broad coverage within the human kidney. We demonstrate that trapped ion mobility spectrometry (TIMS) can resolve matrix peaks from metabolite signal and separate both isobaric and isomeric metabolites with different distributions within the kidney. The added ion mobility data dimension dramatically increased the peak capacity for spatial metabolomics experiments. Through this improved sensitivity, we have found >40 low molecular weight metabolites in human kidney tissue, such as argininic acid, acetylcarnitine, and choline that localize to the cortex, medulla, and renal pelvis, respectively. Future work will involve further exploring metabolomic profiles of human kidneys as a function of age, sex, and race.

Discovering New Lipidomic Features Using Cell Type Specific Fluorophore Expression to Provide Spatial and Biological Specificity in a Multimodal Workflow with MALDI Imaging Mass Spectrometry.

Identifying the spatial distributions of biomolecules in tissue is crucial for understanding integrated function. Imaging mass spectrometry (IMS) allows simultaneous mapping of thousands of biosynthetic products such as lipids but has needed a means of identifying specific cell-types or functional states to correlate with molecular localization. We report, here, advances starting from identity marking with a genetically encoded fluorophore. The fluorescence emission data were integrated with IMS data through multimodal image processing with advanced registration techniques and data-driven image fusion. In an unbiased analysis of spleens, this integrated technology enabled identification of ether lipid species preferentially enriched in germinal centers. We propose that this use of genetic marking for microanatomical regions of interest can be paired with molecular information from IMS for any tissue, cell-type, or activity state for which fluorescence is driven by a gene-tracking allele and ultimately with outputs of other means of spatial mapping.

Integrated molecular imaging technologies for investigation of metals in biological systems: A brief review.

Metals play an essential role in biological systems and are required as structural or catalytic co-factors in many proteins. Disruption of the homeostatic control and/or spatial distributions of metals can lead to disease. Imaging technologies have been developed to visualize elemental distributions across a biological sample. Measurement of elemental distributions by imaging mass spectrometry and imaging X-ray fluorescence are increasingly employed with technologies that can assess histological features and molecular compositions. Data from several modalities can be interrogated as multimodal images to correlate morphological, elemental, and molecular properties. Elemental and molecular distributions have also been axially resolved to achieve three-dimensional volumes, dramatically increasing the biological information. In this review, we provide an overview of recent developments in the field of metal imaging with an emphasis on multimodal studies in two and three dimensions. We specifically highlight studies that present technological advancements and biological applications of how metal homeostasis affects human health.

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.

High-Performance Molecular Imaging with MALDI Trapped Ion-Mobility Time-of-Flight (timsTOF) Mass Spectrometry.

Imaging mass spectrometry (IMS) enables the spatially targeted molecular assessment of biological tissues at cellular resolutions. New developments and technologies are essential for uncovering the molecular drivers of native physiological function and disease. Instrumentation must maximize spatial resolution, throughput, sensitivity, and specificity, because tissue imaging experiments consist of thousands to millions of pixels. Here, we report the development and application of a matrix-assisted laser desorption/ionization (MALDI) trapped ion-mobility spectrometry (TIMS) imaging platform. This prototype MALDI timsTOF instrument is capable of 10 µm spatial resolutions and 20 pixels/s throughput molecular imaging. The MALDI source utilizes a Bruker SmartBeam 3-D laser system that can generate a square burn pattern of <10 × 10 µm at the sample surface. General image performance was assessed using murine kidney and brain tissues and demonstrate that high-spatial-resolution imaging data can be generated rapidly with mass measurement errors <5 ppm and ∼40 000 resolving power. Initial TIMS-based imaging experiments were performed on whole-body mouse pup tissue demonstrating the separation of closely isobaric [PC(32:0) + Na]+ and [PC(34:3) + H]+ (3 mDa mass difference) in the gas phase. We have shown that the MALDI timsTOF platform can maintain reasonable data acquisition rates (>2 pixels/s) while providing the specificity necessary to differentiate components in complex mixtures of lipid adducts. The combination of high-spatial-resolution and throughput imaging capabilities with high-performance TIMS separations provides a uniquely tunable platform to address many challenges associated with advanced molecular imaging applications.