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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 Peyers 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. (c) 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry. Funding Agencies|UK Medical Research CouncilMedical Research Council UK (MRC) [MR/R005699/1]; UK Engineering and Physical Sciences Research CouncilEngineering & Physical Sciences Research Council (EPSRC) [EP/H008683/1]; UK Biotechnology and Biological Sciences Research CouncilBiotechnology and Biological Sciences Research Council (BBSRC) [BB/P026818/1]; Girton College; University of Cambridge Herchel-Smith Fund

Daily oral exposure to vast numbers (>1013/adult/day) of micron or nano-sized persistent particles has become the norm for many populations. Significant airborne particle exposure is deleterious, so what about ingestion? Titanium dioxide in food grade form (fgTiO2) , which is an additive to some foods, capsules, tablets and toothpaste, may provide clues. Certainly, exposed human populations accumulate these particles in specialised intestinal cells at the base of large lymphoid follicles (Peyer’s patches) and it’s likely that a degree of absorption goes beyond this- i.e. lymphatics to blood circulation to tissues. We critically review the evidence and pathways. Regarding potential adverse effects, our primary message, for today’s state-of-art, is that in vivo models have not been good enough and at times woeful. We provide a ‘caveats list’ to improve approaches and experimentation and illustrate why studies on biomarkers of particle uptake, and lower gut /mesenteric lymph nodes as targets, should be prioritized., Medical Research Council [grant number MR/R005699/1], Brazilian Federal Agency for Support and Evaluation of Graduate Education - CAPES [grant number 88881.127953/2016-01], Girton College and the University of Cambridge Herchel-Smith Fellowships