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New tools for pathology: a user's review of a highly multiplexed method for <i>in situ</i> analysis of protein and RNA expression in tissue
摘要： Tumor cell heterogeneity and tumor cell - stromal interactions are being explored as determinants of disease progression and treatment resistance in solid tumor and hematologic malignancies. As such, tools simultaneously capable of highly-multiplexed profiling of tissues’ protein and RNA content, as well as interrogation of rare or single cells, are required to precisely characterize constituent tumor cell populations, infiltrating lymphocytes and stromal elements. Access to spatial relationships will enable more precise characterization of tumors, support patient stratification and may help to identify novel drug targets. Multiple platforms are being developed to address these critical unmet needs. The NanoString Digital Spatial Profiling (DSP) platform enables highly multiplexed, spatial assessment of protein and/or RNA targets in tissues by detecting oligonucleotide barcodes conjugated via a photocleavable linker to primary antibodies or nucleic acid probes. While this platform enables high-dimensional spatial interrogation of tissue protein and RNA expression, a detailed understanding of its composition, function and chemistry is advisable to guide experimental design and data interpretation. The purpose of this review is to provide an independent, comprehensive description of the DSP technology including an overview of NanoString’s capture and antibody barcode conjugation chemistries, experimental workflow, data output and analysis methods. The DSP technology will be discussed in the context of other highly multiplexed immunohistochemistry methods, including imaging mass cytometry (IMC) and multiplexed ion beam imaging (MIBI), to inform potential users of the advantages and limitations of each. Additional issues such as pre-analytical variability, sampling and specimen adequacy will be considered with respect to the platforms to inform potential experimental design.
关键词： tissue biomarkers,in situ hybridization,multiplexed immunohistochemistry,NanoString,tumor microenvironment
更新于2025-09-23 15:19:57
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Beyond the H&E: Advanced Technologies for in situ Tissue Biomarker Imaging
摘要： For decades, histopathology with routine hematoxylin and eosin staining has been and remains the gold standard for reaching a morphologic diagnosis in tissue samples from humans and veterinary species. However, within the past decade, there has been exponential growth in advanced techniques for in situ tissue biomarker imaging that bridge the divide between anatomic and molecular pathology. It is now possible to simultaneously observe localization and expression magnitude of multiple protein, nucleic acid, and molecular targets in tissue sections and apply machine learning to synthesize vast, image-derived datasets. As these technologies become more sophisticated and widely available, a team-science approach involving subspecialists with medical, engineering, and physics backgrounds is critical to upholding quality and validity in studies generating these data. The purpose of this manuscript is to detail the scientific premise, tools and training, quality control, and data collection and analysis considerations needed for the most prominent advanced imaging technologies currently applied in tissue sections: immunofluorescence, in situ hybridization, laser capture microdissection, matrix-assisted laser desorption ionization imaging mass spectrometry, and spectroscopic/optical methods. We conclude with a brief overview of future directions for ex vivo and in vivo imaging techniques.
关键词： laser capture microdissection,immunohistochemistry,quality control,in situ hybridization,biomarkers,molecular pathology,fluorescence microscopy,MALDI
更新于2025-09-10 09:29:36
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Clarification and confocal imaging of the nonhuman primate placental micro-anatomy
摘要： Geometry of the placental villous vasculature is a key determinant of maternal–fetal nutrient exchange for optimal fetal growth. Recent advances in tissue clarification techniques allow for deep high-resolution imaging with confocal microscopy; however, the methodology lacks a signal:noise ratio of sufficient magnitude to allow for quantitative analysis. Thus, we sought to develop a reproducible method to investigate the 3D vasculature of the nonhuman primate placenta for subsequent data analysis. Fresh placental tissue was dissected, formalin fixed, clarified using a modified Visikol? protocol and immunolabeled for CD31 (fetal endothelium) and cytokeratin-7 (villous trophoblast) for confocal imaging of the microanatomy. We present a detailed clarification and staining protocol augmented for imaging of nonhuman primate placental tissue. The image stacks generated by this refined staining method and our data acquisition parameters can be analyzed quantitatively to provide insights regarding the villous and vascular micro-anatomy of the placenta.
关键词： placenta,immunohistochemistry,tissue clarification,nonhuman primate,confocal microscopy
更新于2025-09-10 09:29:36
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MALDI Imaging for Proteomic Painting of Heterogeneous Tissue Structures
摘要： Purpose: To present matrix-assisted laser desorption/ionization (MALDI) imaging as a powerful method to highlight various tissue compartments. Experimental design: Formalin-fixed paraffin-embedded (FFPE) tissue of a uterine cervix, a pancreas, a duodenum, a teratoma and a breast cancer tissue microarray (TMA) were analyzed by MALDI imaging and by immunohistochemistry (IHC). Peptide images were visualized and analyzed using FlexImaging and SCiLS Lab software. Different histological compartments were compared by hierarchical cluster analysis. Results: MALDI imaging highlights tissue compartments comparable to IHC. In cervical tissue, normal epithelium could be discerned from intraepithelial neoplasia. In pancreatic and duodenal tissues, m/z signals from lymph follicles, vessels, duodenal mucosa, normal pancreas and smooth muscle structures could be visualized. In teratoma, specific m/z signals to discriminate squamous epithelium, sebaceous glands, and soft tissue were detected. Additionally, tumor tissue could be discerned from the surrounding stroma in small tissue cores of TMAs. Proteomic data acquisition of complex tissue compartments in FFPE tissue required less than one hour with recent mass spectrometers. Conclusion and clinical relevance: The simultaneous characterization of morphological and proteomic features in the same tissue section adds proteomic information for histopathological diagnostics, which relies at present on conventional Hematoxylin and Eosin staining, histochemical, IHC and molecular methods.
关键词： FFPE,immunohistochemistry,Tumor heterogeneity,MALDI imaging
更新于2025-09-10 09:29:36
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[IEEE 2018 International Symposium ELMAR - Zadar, Croatia (2018.9.16-2018.9.19)] 2018 International Symposium ELMAR - 3D Localization of Neurons in Bright-Field Histological Images
摘要： In this paper, we present a method for inferring the depth of neurons found in a bright-field microscopic image of a histological section of a human brain, digitized at high resolution in multiple planes along the z-axis. Individual neuron bodies are segmented and tracked throughout the depth of the whole image stack and placed at the appropriate z-level in the stack based on variations in image sharpness.
关键词： Digital Microscopy,Immunohistochemistry,High-resolution Imaging
更新于2025-09-09 09:28:46