Spatially Resolved CRISPR Screen Sequencing via Perturb-DBiT.

Perturb-seq enabled the profiling of transcriptional effects of genetic perturbations in single cells but lacks the ability to examine the impact on tissue environments. We present Perturb-DBiT for simultaneous co-sequencing of spatial transcriptome and guide RNAs (gRNAs) on the same tissue section for in vivo CRISPR screen with genome-scale gRNA libraries, offering a comprehensive understanding of how genetic modifications affect cellular behavior and tissue architecture. This platform supports a variety of delivery vectors, gRNA library sizes, and tissue preparations, along with two distinct gRNA capture methods, making it adaptable to a wide range of experimental setups.

In situ molecular profiles of glomerular cells by integrated imaging mass spectrometry and multiplexed immunofluorescence microscopy.

Glomeruli filter blood through the coordination of podocytes, mesangial cells, fenestrated endothelial cells, and the glomerular basement membrane. Cellular changes, such as podocyte loss, are associated with pathologies like diabetic kidney disease. However, little is known regarding the in situ molecular profiles of specific cell types and how these profiles change with disease.

Alleviating tiling effect by random walk sliding window in high-resolution histological whole slide image synthesis.

Multiplex immunofluorescence (MxIF) is an advanced molecular imaging technique that can simultaneously provide biologists with multiple (i.e., more than 20) molecular markers on a single histological tissue section.

Multimodal MALDI imaging mass spectrometry for improved diagnosis of melanoma.

Imaging mass spectrometry (IMS) provides promising avenues to augment histopathological investigation with rich spatio-molecular information. We have previously developed a classification model to differentiate melanoma from nevi lesions based on IMS protein data, a task that is challenging solely by histopathologic evaluation. Most IMS-focused studies collect microscopy in tandem with IMS data, but this microscopy data is generally omitted in downstream data analysis.

Multidimensional mass profiles increase confidence in bacterial identification when using low-resolution mass spectrometers.

Field-forward analytical technologies, such as portable mass spectrometry (MS), enable essential capabilities for real-time monitoring and point-of-care diagnostic applications. Significant and recent investments improving the features of miniaturized mass spectrometers enable various new applications outside of small molecule detection. Most notably, the addition of tandem mass spectrometry scans (MS/MS) allows the instrument to isolate and fragment ions and increase the analytical specificity by measuring unique chemical signatures for ions of interest.

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.

Toward Omics-Scale Quantitative Mass Spectrometry Imaging of Lipids in Brain Tissue Using a Multiclass Internal Standard Mixture.

Mass spectrometry imaging (MSI) has accelerated our understanding of lipid metabolism and spatial distribution in tissues and cells. However, few MSI studies have approached lipid imaging quantitatively and those that have focused on a single lipid class. We overcome this limitation by using a multiclass internal standard (IS) mixture sprayed homogeneously over the tissue surface with concentrations that reflect those of endogenous lipids.

Topological-Preserving Membrane Skeleton Segmentation in Multiplex Immunofluorescence Imaging.

Multiplex immunofluorescence (MxIF) is an emerging imaging technology whose downstream molecular analytics highly rely upon the effectiveness of cell segmentation. In practice, multiple membrane markers (e.g.

Addressing persistent challenges in digital image analysis of cancerous tissues.

The National Cancer Institute (NCI) supports many research programs and consortia, many of which use imaging as a major modality for characterizing cancerous tissue. A trans-consortia Image Analysis Working Group (IAWG) was established in 2019 with a mission to disseminate imaging-related work and foster collaborations. In 2022, the IAWG held a virtual hackathon focused on addressing challenges of analyzing high dimensional datasets from fixed cancerous tissues.

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.

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.

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.

Prospective on Imaging Mass Spectrometry in Clinical Diagnostics.

Imaging mass spectrometry (IMS) is a molecular technology utilized for spatially driven research, providing molecular maps from tissue sections. This article reviews matrix-assisted laser desorption ionization (MALDI) IMS and its progress as a primary tool in the clinical laboratory. MALDI mass spectrometry has been used to classify bacteria and perform other bulk analyses for plate-based assays for many years.

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.

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 -infected murine kidney, 4 and 10 days postinfection.

Tissue fixation effects on human retinal lipid analysis by MALDI imaging and LC-MS/MS technologies.

Imaging mass spectrometry (IMS) allows the location and abundance of lipids to be mapped across tissue sections of human retina. For reproducible and accurate information, sample preparation methods need to be optimized. Paraformaldehyde fixation of a delicate multilayer structure like human retina facilitates the preservation of tissue morphology by forming methylene bridge crosslinks between formaldehyde and amine/thiols in biomolecules; however, retina sections analyzed by IMS are typically fresh-frozen.

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.

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).

α-Cyano-4-hydroxycinnamic Acid and Tri-Potassium Citrate Salt Pre-Coated Silicon Nanopost Array Provides Enhanced Lipid Detection for High Spatial Resolution MALDI Imaging Mass Spectrometry.

We have developed a pre-coated substrate for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) that enables high spatial resolution mapping of both phospholipids and neutral lipid classes in positive ion mode as metal cation adducts. The MALDI substrates are constructed by depositing a layer of α-cyano-4-hydroxycinnamic acid (CHCA) and potassium salts onto silicon nanopost arrays (NAPA) prior to tissue mounting. The matrix/salt pre-coated NAPA substrate significantly enhances all detected lipid signals allowing lipids to be detected at lower laser energies than bare NAPA.

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.

Diagnosis of melanoma by imaging mass spectrometry: Development and validation of a melanoma prediction model.

Background: The definitive diagnosis of melanocytic neoplasia using solely histopathologic evaluation can be challenging. Novel techniques that objectively confirm diagnoses are needed. This study details the development and validation of a melanoma prediction model from spatially resolved multivariate protein expression profiles generated by imaging mass spectrometry (IMS).

An orthogonal methods assessment of topical drug concentrations in skin and the impact for risk assessment in the viable epidermis.

Systemic toxicity assessments for oral or parenteral drugs often utilize the concentration of drug in plasma to enable safety margin calculations for human risk assessment. For topical drugs, there is no standard method for measuring drug concentrations in the stratum basale of the viable epidermis. This is particularly important since the superficial part of the epidermis, the stratum corneum (SC), is nonviable and where most of a topically applied drug remains, never penetrating deeper into the skin.

Lipid Landscape of the Human Retina and Supporting Tissues Revealed by High-Resolution Imaging Mass Spectrometry.

The human retina provides vision at light levels ranging from starlight to sunlight. Its supporting tissues regulate plasma-delivered lipophilic essentials for vision, including retinoids. The macula is an anatomic specialization for high-acuity and color vision that is also vulnerable to prevalent blinding diseases.