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 of 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 two-dimensional (2-D) and three-dimensional (3-D) cyclical IF (CyCIF) to provide a more detailed method for evaluating 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.NEW & NOTEWORTHY We describe here validation criteria used to define on organ mapping panel of antibodies that can be used to define 18 cell types and five extracellular matrix compartments using cyclical immunofluorescence (CyCIF) microscopy. As CyCIF does not require specialized instrumentation, and image registration required to assemble CyCIF images can be performed by any laboratory without specialized computational skills, this technology is accessible to any laboratory with access to a fluorescence microscope and digital scanner.

High-Specificity Imaging Mass Spectrometry.

Imaging mass spectrometry (IMS) enables highly multiplexed, untargeted tissue mapping for a broad range of molecular classes, facilitating in situ biological discovery. Yet, challenges persist in molecular specificity, which is the ability to discern one molecule from another, and spatial specificity, which is the ability to link untargeted imaging data to specific tissue features. Instrumental developments have dramatically improved IMS spatial resolution, allowing molecular observations to be more readily associated with distinct tissue features across spatial scales, ranging from larger anatomical regions to single cells. High-performance mass analyzers and systems integrating ion mobility technologies are also becoming more prevalent, further improving molecular coverage and the ability to discern chemical identity. This review provides an overview of recent advancements in high-specificity IMS that are providing critical biological context to untargeted molecular imaging, enabling integrated analyses, and addressing advanced biomedical research applications.

Imaging Mass Spectrometry of Isotopically Resolved Intact Proteins on a Trapped Ion-Mobility Quadrupole Time-of-Flight Mass Spectrometer.

In this work, we demonstrate rapid, high spatial, and high spectral resolution imaging of intact proteins by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on a hybrid quadrupole-reflectron time-of-flight (qTOF) mass spectrometer equipped with trapped ion mobility spectrometry (TIMS). Historically, untargeted MALDI IMS of proteins has been performed on TOF mass spectrometers. While advances in TOF instrumentation have enabled rapid, high spatial resolution IMS of intact proteins, TOF mass spectrometers generate relatively low-resolution mass spectra with limited mass accuracy. Conversely, the implementation of MALDI sources on high-resolving power Fourier transform (FT) mass spectrometers has allowed IMS experiments to be conducted with high spectral resolution with the caveat of increasingly long data acquisition times. As illustrated here, qTOF mass spectrometers enable protein imaging with the combined advantages of TOF and FT mass spectrometers. Protein isotope distributions were resolved for both a protein standard mixture and proteins detected from a whole-body mouse pup tissue section. Rapid (∼10 pixels/s) 10 µm lateral spatial resolution IMS was performed on a rat brain tissue section while maintaining isotopic spectral resolution. Lastly, proof-of-concept MALDI-TIMS data was acquired from a protein mixture to demonstrate the ability to differentiate charge states by ion mobility. These experiments highlight the advantages of qTOF and timsTOF platforms for resolving and interpreting complex protein spectra generated from tissue by IMS.

Harmonizing the Generation and Pre-publication Stewardship of FAIR Image data.

Together with the molecular knowledge of genes and proteins, biological images promise to significantly enhance the scientific understanding of complex cellular systems and to advance predictive and personalized therapeutic products for human health. For this potential to be realized, quality-assured image data must be shared among labs at a global scale to be compared, pooled, and reanalyzed, thus unleashing untold potential beyond the original purpose for which the data was generated. There are two broad sets of requirements to enable image data sharing in the life sciences. One set of requirements is articulated in the companion White Paper entitled "Enabling Global Image Data Sharing in the Life Sciences," which is published in parallel and addresses the need to build the cyberinfrastructure for sharing the digital array data (arXiv:2401.13023 [q-bio.OT], https://doi.org/10.48550/arXiv.2401.13023). In this White Paper, we detail a broad set of requirements, which involves collecting, managing, presenting, and propagating contextual information essential to assess the quality, understand the content, interpret the scientific implications, and reuse image data in the context of the experimental details. We start by providing an overview of the main lessons learned to date through international community activities, which have recently made considerable progress toward generating community standard practices for imaging Quality Control (QC) and metadata. We then provide a clear set of recommendations for amplifying this work. The driving goal is to address remaining challenges, and democratize access to common practices and tools for a spectrum of biomedical researchers, regardless of their expertise, access to resources, and geographical location.

Advances in Imaging Mass Spectrometry for Biomedical and Clinical Research.

Imaging mass spectrometry (IMS) allows for the untargeted mapping of biomolecules directly from tissue sections. This technology is increasingly integrated into biomedical and clinical research environments to supplement traditional microscopy and provide molecular context for tissue imaging. IMS has widespread clinical applicability in the fields of oncology, dermatology, microbiology, and others. This review summarizes the two most widely employed IMS technologies, matrix-assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI), and covers technological advancements, including efforts to increase spatial resolution, specificity, and throughput. We also highlight recent biomedical applications of IMS, primarily focusing on disease diagnosis, classification, and subtyping.

In situ lipidomics of Staphylococcus aureus osteomyelitis using imaging mass spectrometry.

Osteomyelitis occurs when Staphylococcus aureus invades the bone microenvironment, resulting in a bone marrow abscess with a spatially defined architecture of cells and biomolecules. Imaging mass spectrometry and microscopy are invaluable tools that can be employed to interrogate the lipidome of S. aureus-infected murine femurs to reveal metabolic and signaling consequences of infection. Here, nearly 250 lipids were spatially mapped to healthy and infection-associated morphological features throughout the femur, establishing composition profiles for tissue types. Ether lipids and arachidonoyl lipids were significantly altered between cells and tissue structures in abscesses, suggesting their roles in abscess formation and inflammatory signaling. Sterols, triglycerides, bis(monoacylglycero)phosphates, and gangliosides possessed ring-like distributions throughout the abscess, indicating dysregulated lipid metabolism in a subpopulation of leukocytes that cannot be discerned with traditional microscopy. These data provide chemical insight into the signaling function and metabolism of cells in the fibrotic border of abscesses, likely characteristic of lipid-laden macrophages.

Clostridioides difficile ferrosome organelles combat nutritional immunity.

Iron is indispensable for almost all forms of life but toxic at elevated levels1-4. To survive within their hosts, bacterial pathogens have evolved iron uptake, storage and detoxification strategies to maintain iron homeostasis1,5,6. Recent studies showed that three Gram-negative environmental anaerobes produce iron-containing ferrosome granules7,8. However, it remains unclear whether ferrosomes are generated exclusively by Gram-negative bacteria. The Gram-positive bacterium Clostridioides difficile is the leading cause of nosocomial and antibiotic-associated infections in the USA9. Here we report that C. difficile undergoes an intracellular iron biomineralization process and stores iron in membrane-bound ferrosome organelles containing non-crystalline iron phosphate biominerals. We found that a membrane protein (FezA) and a P1B6-ATPase transporter (FezB), repressed by both iron and the ferric uptake regulator Fur, are required for ferrosome formation and play an important role in iron homeostasis during transition from iron deficiency to excess. Additionally, ferrosomes are often localized adjacent to cellular membranes as shown by cryo-electron tomography. Furthermore, using two mouse models of C. difficile infection, we demonstrated that the ferrosome system is activated in the inflamed gut to combat calprotectin-mediated iron sequestration and is important for bacterial colonization and survival during C. difficile infection.

Untangling Alzheimer's disease with spatial multi-omics: a brief review.

Alzheimer's disease (AD) is the most common form of neurological dementia, specified by extracellular ß-amyloid plaque deposition, neurofibrillary tangles, and cognitive impairment. AD-associated pathologies like cerebral amyloid angiopathy (CAA) are also affiliated with cognitive impairment and have overlapping molecular drivers, including amyloid buildup. Discerning the complexity of these neurological disorders remains a significant challenge, and the spatiomolecular relationships between pathogenic features of AD and AD-associated pathologies remain poorly understood. This review highlights recent developments in spatial omics, including profiling and molecular imaging methods, and how they are applied to AD. These emerging technologies aim to characterize the relationship between how specific cell types and tissue features are organized in combination with mapping molecular distributions to provide a systems biology view of the tissue microenvironment around these neuropathologies. As spatial omics methods achieve greater resolution and improved molecular coverage, they are enabling deeper characterization of the molecular drivers of AD, leading to new possibilities for the prediction, diagnosis, and mitigation of this debilitating disease.

Segmentation of human functional tissue units in support of a Human Reference Atlas.

The Human BioMolecular Atlas Program (HuBMAP) aims to compile a Human Reference Atlas (HRA) for the healthy adult body at the cellular level. Functional tissue units (FTUs), relevant for HRA construction, are of pathobiological significance. Manual segmentation of FTUs does not scale; highly accurate and performant, open-source machine-learning algorithms are needed. We designed and hosted a Kaggle competition that focused on development of such algorithms and 1200 teams from 60 countries participated. We present the competition outcomes and an expanded analysis of the winning algorithms on additional kidney and colon tissue data, and conduct a pilot study to understand spatial location and density of FTUs across the kidney. The top algorithm from the competition, Tom, outperforms other algorithms in the expanded study, while using fewer computational resources. Tom was added to the HuBMAP infrastructure to run kidney FTU segmentation at scale-showcasing the value of Kaggle competitions for advancing research.

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.

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.

High-Resolution Imaging Mass Spectrometry of Human Donor Eye: Photoreceptors Cells and Basal Laminar Deposit of Age-Related Macular Degeneration.

Pathologies of the retina are clinically visualized in vivo with OCT and ex vivo with immunohistochemistry. Although both techniques provide valuable information on prognosis and disease state, a comprehensive method for fully elucidating molecular constituents present in locations of interest is desirable. The purpose of this work was to use multimodal imaging technologies to localize the vast number of molecular species observed with matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) in aged and diseased retinal tissues. Herein, MALDI IMS was utilized to observe molecular species that reside in photoreceptor cells and also a basal laminar deposit from two human donor eyes. The molecular species observed to accumulate in these discrete regions can be further identified and studied to attempt to gain a greater understanding of biological processes occurring in debilitating eye diseases such as age-related macular degeneration (AMD).

Unsupervised Registration Refinement for Generating Unbiased Eye Atlas.

With the confounding effects of demographics across large-scale imaging surveys, substantial variation is demonstrated with the volumetric structure of orbit and eye anthropometry. Such variability increases the level of difficulty to localize the anatomical features of the eye organs for populational analysis. To adapt the variability of eye organs with stable registration transfer, we propose an unbiased eye atlas template followed by a hierarchical coarse-to-fine approach to provide generalized eye organ context across populations. Furthermore, we retrieved volumetric scans from 1842 healthy patients for generating an eye atlas template with minimal biases. Briefly, we select 20 subject scans and use an iterative approach to generate an initial unbiased template. We then perform metric-based registration to the remaining samples with the unbiased template and generate coarse registered outputs. The coarse registered outputs are further leveraged to train a deep probabilistic network, which aims to refine the organ deformation in unsupervised setting. Computed tomography (CT) scans of 100 de-identified subjects are used to generate and evaluate the unbiased atlas template with the hierarchical pipeline. The refined registration shows the stable transfer of the eye organs, which were well-localized in the high-resolution (0.5 mm3) atlas space and demonstrated a significant improvement of 2.37% Dice for inverse label transfer performance. The subject-wise qualitative representations with surface rendering successfully demonstrate the transfer details of the organ context and showed the applicability of generalizing the morphological variation across patients.

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.

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.

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.

Tissue registration and exploration user interfaces in support of a human reference atlas.

Seventeen international consortia are collaborating on a human reference atlas (HRA), a comprehensive, high-resolution, three-dimensional atlas of all the cells in the healthy human body. Laboratories around the world are collecting tissue specimens from donors varying in sex, age, ethnicity, and body mass index. However, harmonizing tissue data across 25 organs and more than 15 bulk and spatial single-cell assay types poses challenges. Here, we present software tools and user interfaces developed to spatially and semantically annotate ("register") and explore the tissue data and the evolving HRA. A key part of these tools is a common coordinate framework, providing standard terminologies and data structures for describing specimen, biological structure, and spatial data linked to existing ontologies. As of April 22, 2022, the "registration" user interface has been used to harmonize and publish data on 5,909 tissue blocks collected by the Human Biomolecular Atlas Program (HuBMAP), the Stimulating Peripheral Activity to Relieve Conditions program (SPARC), the Human Cell Atlas (HCA), the Kidney Precision Medicine Project (KPMP), and the Genotype Tissue Expression project (GTEx). Further, 5,856 tissue sections were derived from 506 HuBMAP tissue blocks. The second "exploration" user interface enables consortia to evaluate data quality, explore tissue data spatially within the context of the HRA, and guide data acquisition. A companion website is at https://cns-iu.github.io/HRA-supporting-information/ .