Organ Mapping Antibody Panels: a community resource for standardized multiplexed tissue imaging.

Multiplexed antibody-based imaging enables the detailed characterization of molecular and cellular organization in tissues. Advances in the field now allow high-parameter data collection (>60 targets); however, considerable expertise and capital are needed to construct the antibody panels employed by these methods. Organ mapping antibody panels are community-validated resources that save time and money, increase reproducibility, accelerate discovery and support the construction of a Human Reference Atlas.

Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging.

Tissues and organs are composed of distinct cell types that must operate in concert to perform physiological functions. Efforts to create high-dimensional biomarker catalogs of these cells have been largely based on single-cell sequencing approaches, which lack the spatial context required to understand critical cellular communication and correlated structural organization. To probe in situ biology with sufficient depth, several multiplexed protein imaging methods have been recently developed. Though these technologies differ in strategy and mode of immunolabeling and detection tags, they commonly utilize antibodies directed against protein biomarkers to provide detailed spatial and functional maps of complex tissues. As these promising antibody-based multiplexing approaches become more widely adopted, new frameworks and considerations are critical for training future users, generating molecular tools, validating antibody panels, and harmonizing datasets. In this Perspective, we provide essential resources, key considerations for obtaining robust and reproducible imaging data, and specialized knowledge from domain experts and technology developers.

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.

Sample Preparation Method for MALDI Mass Spectrometry Imaging of Fresh-Frozen Spines.

Technologies assessing the lipidomics, genomics, epigenomics, transcriptomics, and proteomics of tissue samples at single-cell resolution have deepened our understanding of physiology and pathophysiology at an unprecedented level of detail. However, the study of single-cell spatial metabolomics in undecalcified bones faces several significant challenges, such as the fragility of bone, which often requires decalcification or fixation leading to the degradation or removal of lipids and other molecules. As such, we describe a method for performing mass spectrometry imaging on undecalcified spine that is compatible with other spatial omics measurements. In brief, we use fresh-frozen rat spines and a system of carboxyl methylcellulose embedding, cryofilm, and polytetrafluoroethylene rollers to maintain tissue integrity while avoiding signal loss from variations in laser focus and artifacts from traditional tissue processing. This reveals various tissue types and lipidomic profiles of spinal regions at 10 µm spatial resolutions using matrix-assisted laser desorption/ionization mass spectrometry imaging. We expect this method to be adapted and applied to the analysis of the spinal cord, shedding light on the mechanistic aspects of cellular heterogeneity, development, and disease pathogenesis underlying different bone-related conditions and diseases. This study furthers the methodology for high spatial metabolomics of spines and adds to the collective efforts to achieve a holistic understanding of diseases via single-cell spatial multiomics.

Highly multiplexed immunofluorescence of the human kidney using co-detection by indexing.

The human kidney is composed of many cell types that vary in their abundance and distribution from normal to diseased organ. As these cell types perform unique and essential functions, it is important to confidently label each within a single tissue to accurately assess tissue architecture and microenvironments. Towards this goal, we demonstrate the use of co-detection by indexing (CODEX) multiplexed immunofluorescence for visualizing 23 antigens within the human kidney. Using CODEX, many of the major cell types and substructures, such as collecting ducts, glomeruli, and thick ascending limb, were visualized within a single tissue section. Of these antibodies, 19 were conjugated in-house, demonstrating the flexibility and utility of this approach for studying the human kidney using custom and commercially available antibodies. We performed a pilot study that compared both fresh frozen and formalin-fixed paraffin-embedded healthy non-neoplastic and diabetic nephropathy kidney tissues. The largest cellular differences between the two groups was observed in cells labeled with aquaporin 1, cytokeratin 7, and α-smooth muscle actin. Thus, our data show the power of CODEX multiplexed immunofluorescence for surveying the cellular diversity of the human kidney and the potential for applications within pathology, histology, and building anatomical atlases.

Referenced Kendrick Mass Defect Annotation and Class-Based Filtering of Imaging MS Lipidomics Experiments.

Because of their diverse functionalities in cells, lipids are of primary importance when characterizing molecular profiles of physiological and disease states. Imaging mass spectrometry (IMS) provides the spatial distributions of lipid populations in tissues. Referenced Kendrick mass defect (RKMD) analysis is an effective mass spectrometry (MS) data analysis tool for classification and annotation of lipids. Herein, we extend the capabilities of RKMD analysis and demonstrate an integrated method for lipid annotation and chemical structure-based filtering for IMS datasets. Annotation of lipid features with lipid molecular class, radyl carbon chain length, and degree of unsaturation allows image reconstruction and visualization based on each structural characteristic. We show a proof-of-concept application of the method to a computationally generated IMS dataset and validate that the RKMD method is highly specific for lipid components in the presence of confounding background ions. Moreover, we demonstrate an application of the RKMD-based annotation and filtering to matrix-assisted laser desorption/ionization (MALDI) IMS lipidomic data from human kidney tissue analysis.

High Spatial Resolution MALDI Imaging Mass Spectrometry of Fresh-Frozen Bone.

Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to molecular alterations giving rise to debilitating diseases like rheumatoid arthritis and osteomyelitis. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) facilitate the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of osseous tissue for MALDI IMS has been difficult due to its mineralized composition and heterogeneous morphology, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs, allowing the distribution of endogenous lipids to be linked to tissue structures and cell types. Adhesive-bound bone sections were mounted onto conductive glass slides with microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. High spatial resolution (10 µm) MALDI IMS was employed to characterize lipid distributions, and use of complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to the bone marrow, adipose tissue, marrow adipose tissue, and muscle. Further, sphingomyelin(42:1) was abundant in megakaryocytes, whereas sphingomyelin(42:2) was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage with spatial relevance in a preserved native bone microenvironment.

Strain-Release Pentafluorosulfanylation and Tetrafluoro(aryl)sulfanylation of [1.1.1]Propellane: Reactivity and Structural Insight.

We leveraged the recent increase in synthetic accessibility of SF5 Cl and Ar-SF4 Cl compounds to combine chemistry of the SF5 and SF4 Ar groups with strain-release functionalization. By effectively adding SF5 and SF4 Ar radicals across [1.1.1]propellane, we accessed structurally unique bicyclopentanes, bearing two distinct elements of bioisosterism. Upon evaluating these "hybrid isostere" motifs in the solid state, we measured exceptionally short transannular distances; in one case, the distance rivals the shortest nonbonding C⋅⋅⋅C contact reported to date. This prompted SC-XRD and DFT analyses that support the notion that a donor-acceptor interaction involving the "wing" C-C bonds is playing an important role in stabilization. Thus, these heretofore unknown structures expand the palette for highly coveted three-dimensional fluorinated building blocks and provide insight to a more general effect observed in bicyclopentanes.

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.

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.

Lipid Analysis of 30 000 Individual Rodent Cerebellar Cells Using High-Resolution Mass Spectrometry.

Single-cell measurements aid our understanding of chemically heterogeneous systems such as the brain. Lipids are one of the least studied chemical classes, and their cell-to-cell heterogeneity remains largely unexplored. We adapted microscopy-guided single-cell profiling using matrix-assisted laser desorption/ionization ion cyclotron resonance mass spectrometry to profile the lipid composition of over 30 000 individual rat cerebellar cells. We detected 520 lipid features, many of which were found in subsets of cells; Louvain clustering identified 101 distinct groups that can be correlated to neuronal and astrocytic classifications and lipid classes. Overall, the two most common lipids found were [PC(32:0)+H]+ and [PC(34:1)+H]+, which were present within 98.9 and 89.5% of cells, respectively; lipid signals present in <1% of cells were also detected, including [PC(34:1)+K]+ and [PG(40:2(OH))+Na]+. These results illustrate the vast lipid heterogeneity found within rodent cerebellar cells and hint at the distinct functional consequences of this heterogeneity.

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.

Exploring the Fundamental Structures of Life: Non-Targeted, Chemical Analysis of Single Cells and Subcellular Structures.

Cells are a basic functional and structural unit of living organisms. Both unicellular communities and multicellular species produce an astonishing chemical diversity, enabling a wide range of divergent functions, yet each cell shares numerous aspects that are common to all living organisms. While there are many approaches for studying this chemical diversity, only a few are non-targeted and capable of analyzing hundreds of different chemicals at cellular resolution. Here, we review the non-targeted approaches used to perform comprehensive chemical analyses, provide chemical imaging information, or obtain high-throughput single-cell profiling data. Single-cell measurement capabilities are rapidly increasing in terms of throughput, limits of detection, and completeness of the chemical analyses; these improvements enable their application to understand ever more complex physiological phenomena, such as learning, memory, and behavior.

Lipid Heterogeneity between Astrocytes and Neurons Revealed by Single-Cell MALDI-MS Combined with Immunocytochemical Classification.

Transcriptomics characterizes cells based on their potential molecular repertoire whereas direct mass spectrometry (MS) provides information on the actual compounds present within cells. Single-cell matrix-assisted laser desorption/ionization (MALDI) MS can measure the chemical contents of individual cells but spectra are difficult to correlate to conventional cell types, limiting the metabolic information obtained. We present a protocol that combines MALDI-MS with immunocytochemistry to assay over a thousand individual rat brain cells. The approach entwines the wealth of knowledge obtained by immunocytochemical profiling with mass spectral information on the predominant lipids within each cell. While many lipid species showed a high degree of similarity between neurons and astrocytes, seventeen significantly differed between the two cell types, including four phosphatidylethanolamines elevated in astrocytes and nine phosphatidylcholines in neurons.

Multimodal Chemical Analysis of the Brain by High Mass Resolution Mass Spectrometry and Infrared Spectroscopic Imaging.

The brain functions through chemical interactions between many different cell types, including neurons and glia. Acquiring comprehensive information on complex, heterogeneous systems requires multiple analytical tools, each of which have unique chemical specificity and spatial resolution. Multimodal imaging generates complementary chemical information via spatially localized molecular maps, ideally from the same sample, but requires method enhancements that span from data acquisition to interpretation. We devised a protocol for performing matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance-mass spectrometry imaging (MSI), followed by infrared (IR) spectroscopic imaging on the same specimen. Multimodal measurements from the same tissue provide precise spatial alignment between modalities, enabling more advanced image processing such as image fusion and sharpening. Performing MSI first produces higher quality data from each technique compared to performing IR imaging before MSI. The difference is likely due to fixing the tissue section during MALDI matrix removal, thereby preventing analyte degradation occurring during IR imaging from an unfixed specimen. Leveraging the unique capabilities of each modality, we utilized pan sharpening of MS (mass spectrometry) ion images with selected bands from IR spectroscopy and midlevel data fusion. In comparison to sharpening with histological images, pan sharpening can employ a plethora of IR bands, producing sharpened MS images while retaining the fidelity of the initial ion images. Using Laplacian pyramid sharpening, we determine the localization of several lipids present within the hippocampus with high mass accuracy at 5 µm pixel widths. Further, through midlevel data fusion of the imaging data sets combined with k-means clustering, the combined data set discriminates between additional anatomical structures unrecognized by the individual imaging approaches. Significant differences between molecular ion abundances are detected between relevant structures within the hippocampus, such as the CA1 and CA3 regions. Our methodology provides high quality multiplex and multimodal chemical imaging of the same tissue sample, enabling more advanced data processing and analysis routines.

Interrogation of spatial metabolome of Ginkgo biloba with high-resolution matrix-assisted laser desorption/ionization and laser desorption/ionization mass spectrometry imaging.

Ginkgo biloba is one of the oldest extant seed plants and has a number of unique properties and uses. Numerous efforts have characterized metabolites within the ginkgo plant and their corresponding biosynthesis pathways, but spatio-chemical information on ginkgo metabolites is lacking. Mass spectrometry (MS) imaging was used to interrogate the spatio-chemical localization of metabolites with matrix-assisted laser desorption/ionization and laser desorption/ionization Fourier-transform ion cyclotron resonance MS across the ginkgo leaf. Flavonoids, particularly unexpected and rare flavonoid cyclodimers, were detected predominately from leaf epidermis; ginkgolic acids and cardanols were observed exclusively in the secretory cavities. A non-uniform distribution of flavonoids observed between the upper and lower leaf epidermis was verified by liquid chromatography-MS analyses. Other metabolites, such as saccharides, phospholipids, and chlorophylls, occurred mainly in mesophyll cells. Furthermore, organ- and tissue-specific distributions of ginkgolides were revealed in the ginkgo root, young stem, and leaf. The acquired ion images provide important information regarding biosynthesis, transportation, and accumulation of metabolites throughout the ginkgo plant and should help us to understand the physiological roles of several plant secondary metabolites.

Optically Guided Single Cell Mass Spectrometry of Rat Dorsal Root Ganglia to Profile Lipids, Peptides and Proteins.

The mammalian dorsal root ganglia (DRG) are located on the dorsal roots of the spinal nerves and contain cell bodies of primary sensory neurons. DRG cells have been classified into subpopulations based on their size, morphology, intracellular markers, response to stimuli, and neuropeptides. To understand the connections between DRG chemical heterogeneity and cellular function, we performed optically guided, high-throughput single cell profiling using sequential matrix-assisted laser desorption/ionization mass spectrometry (MS) to detect lipids, peptides, and several proteins in individual DRG cells. Statistical analysis of the resulting mass spectra allows stratification of the DRG population according to cellular morphology and, presumably, major cell types. A subpopulation of small cells contained myelin proteins, which are abundant in Schwann cells, and mass spectra of several larger cells contained peaks matching neurofilament, vimentin, myelin basic protein S, and thymosin beta proteins. Of the over 1000 cells analyzed, approximately 78 % produced putative peptide-rich spectra, allowing the population to be classified into three distinct cell types. Two signals with m/z 4404 and 5487 were exclusively observed in a cell type, but could not be matched to results of our previous liquid chromatography-MS analyses.

Optimized combination of MALDI MSI and immunofluorescence for neuroimaging of lipids within cellular microenvironments.

Proper neurological function relies on the cellular and molecular microenvironment of the brain, with perturbations of this environment leading to neurological disorders. However, studying the microenvironments of neurological tissue has proven difficult because of its inherent complexity. Both the cell type and metabolomic underpinnings of the cell have crucial functional roles, thus making multimodal characterization methods key to acquiring a holistic view of the brain's microenvironment. This study investigates methods for combining matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and immunofluorescence (IF) microscopy to enable the concurrent investigation of cell types and lipid profiles on the same sample. In brief, 1,5-diaminonaphthalene (DAN), α-cyano-4-hydroxy-cinnamic acid (CHCA), and 2,5-dihydroxybenzoic acid (DHB) were tested in addition to instrument-specific parameters for compatibility with IF. Alternatively, the effects of IF protocols on MALDI MSI were also tested, showing significant signal loss with all tested permutations. Ultimately, the use of CHCA for MALDI MSI resulted in the best IF images, while the use of DAN gave the lowest quality IF images. Overall, increasing the laser power and number of shots per laser burst resulted in the most tissue ablation. However, optimized parameter settings allowed for minimal tissue ablation while maintaining sufficient MALDI MSI signal.

Effects of Sex and Western Diet on Spatial Lipidomic Profiles for the Hippocampus, Cortex, and Corpus Callosum in Mice Using MALDI MSI.

Diet is inextricably linked to human health and biological functionality. Reduced cognitive function among other health issues has been correlated with a western diet (WD) in mouse models, indicating that increases in neurodegeneration could be fueled in part by a poor diet. In this study, we use matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) to spatially map the lipidomic profiles of male and female mice that were fed a high-fat, high-sucrose WD for a period of 7 weeks. Our findings concluded that the cortex and corpus callosum showed significant lipid variation by WD in female mice, while there was little to no variation in the hippocampus, regardless of sex. On the other hand, lipid profiles were significantly affected by sex in all regions. Overall, 83 lipids were putatively identified in the mouse brain; among them, HexCer(40:1;O3) and PE(34:0) were found to have the largest statistical difference based on diet for female mice in the cortex and corpus callosum, respectively. Additional lipid changes are noted and can serve as a metric for understanding the brain's metabolomic response to changes in diet, particularly as it relates to disease.

Spatial lipidomics of fresh-frozen spines.

Technologies assessing the lipidomics, genomics, epigenomics, transcriptomics, and proteomics of tissue samples at single-cell resolution have deepened our understanding of physiology and pathophysiology at an unprecedented level of detail. However, the study of single-cell spatial metabolomics in undecalcified bones faces several significant challenges, such as the fragility of bone which often requires decalcification or fixation leading to the degradation or removal of lipids and other molecules and. As such, we describe a method for performing mass spectrometry imaging on undecalcified spine that is compatible with other spatial omics measurements. In brief, we use fresh-freeze rat spines and a system of carboxyl methylcellulose embedding, cryofilm, and polytetrafluoroethylene rollers to maintain tissue integrity, while avoiding signal loss from variations in laser focus and artifacts from traditional tissue processing. This reveals various tissue types and lipidomic profiles of spinal regions at 10 µm spatial resolutions using matrix-assisted laser desorption/ionization mass spectrometry imaging. We expect this method to be adapted and applied to the analysis of spinal cord, shedding light on the mechanistic aspects of cellular heterogeneity, development, and disease pathogenesis underlying different bone-related conditions and diseases. This study furthers the methodology for high spatial metabolomics of spines, as well as adds to the collective efforts to achieve a holistic understanding of diseases via single-cell spatial multi-omics.