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Published on: September 22, 2013
Image-Based Cell Profiling Enables Quantitative Tissue Microscopy in Gastroenterology
John W Wills1, Jack Robertson1, Huw D Summers2
1Biominerals Research, Cambridge University Department of Veterinary Medicine, School of Biological Sciences, Cambridge, UK.
Insights
This study introduces a user-friendly method for quantitative tissue microscopy, enabling detailed cell analysis in diverse gastrointestinal tissues across species. The approach enhances data reproducibility and offers new insights into gastroenterology research.
Area of Science:
- * Biomedical imaging
- * Cell biology
- * Gastroenterology
Background:
- * Immunofluorescence microscopy is crucial for tissue research but often yields only qualitative data.
- * Quantifying per-cell data in complex tissues like the gastrointestinal tract is challenging due to cellular diversity and species variation.
- * Existing methods struggle to provide location-specific, quantitative cellular information.
Purpose of the Study:
- * To develop an open, user-friendly methodology for quantitative tissue microscopy applicable to diverse gastrointestinal tissues, species, and preparation types.
- * To enable "flow cytometry-type" analyses with preserved spatial data for per-cell quantification.
- * To address the limitations of qualitative microscopy and improve data reproducibility in tissue-based research.
Main Methods:
- * Optimized antibody-independent cell labeling compatible with various tissue preparations and species.
- * Extracted per-cell data from confocal micrographs using semantic machine learning for dense tissues.
- * Performed flow cytometry-type analyses, visualization, and statistical mapping of cell locations, interactions, and microenvironments.
Main Results:
- * Successfully quantified rare events, such as *Escherichia coli* passage in human small bowel tissue.
- * Differentiated and quantified intraepithelial lymphocyte populations in rat jejunum relative to enterocytes.
- * Mapped mononuclear phagocyte-T cell interactions and spatial congregations in mouse Peyer's patches.
Conclusions:
- * Accessible quantitative tissue microscopy offers novel insights into gastroenterological research questions.
- * The developed methodology enhances data reproducibility and overcomes limitations of qualitative imaging and antibody reliance.
- * This approach facilitates detailed analysis of cell location, interactions, and microenvironments within complex tissues.
Abstract:
Immunofluorescence microscopy is an essential tool for tissue-based research, yet data reporting is almost always qualitative. Quantification of images, at the per-cell level, enables "flow cytometry-type" analyses with intact locational data but achieving this is complex. Gastrointestinal tissue, for example, is highly diverse: from mixed-cell epithelial layers through to discrete lymphoid patches. Moreover, different species (e.g., rat, mouse, and humans) and tissue preparations (paraffin/frozen) are all commonly studied. Here, using field-relevant examples, we develop open, user-friendly methodology that can encompass these variables to provide quantitative tissue microscopy for the field. Antibody-independent cell labeling approaches, compatible across preparation types and species, were optimized. Per-cell data were extracted from routine confocal micrographs, with semantic machine learning employed to tackle densely packed lymphoid tissues. Data analysis was achieved by flow cytometry-type analyses alongside visualization and statistical definition of cell locations, interactions and established microenvironments. First, quantification of Escherichia coli passage into human small bowel tissue, following Ussing chamber incubations exemplified objective quantification of rare events in the context of lumen-tissue crosstalk. Second, in rat jejenum, precise histological context revealed distinct populations of intraepithelial lymphocytes between and directly below enterocytes enabling quantification in context of total epithelial cell numbers. Finally, mouse mononuclear phagocyte-T cell interactions, cell expression and significant spatial cell congregations were mapped to shed light on cell-cell communication in lymphoid Peyer's patch. Accessible, quantitative tissue microscopy provides a new window-of-insight to diverse questions in gastroenterology. It can also help combat some of the data reproducibility crisis associated with antibody technologies and over-reliance on qualitative microscopy. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.
