title: Histo-cytometry: a method for highly multiplex quantitative tissue imaging analysis applied to dendritic cell subset microanatomy in lymph nodes.
abstract: Flow cytometry allows highly quantitative analysis of complex dissociated populations at the cost of neglecting their tissue localization. In contrast, conventional microscopy methods provide spatial information, but visualization and quantification of cellular subsets defined by complex phenotypic marker combinations is challenging. Here, we describe an analytical microscopy method, "histo-cytometry," for visualizing and quantifying phenotypically complex cell populations directly in tissue sections. This technology is based on multiplexed antibody staining, tiled high-resolution confocal microscopy, voxel gating, volumetric cell rendering, and quantitative analysis. We have tested this technology on various innate and adaptive immune populations in murine lymph nodes (LNs) and were able to identify complex cellular subsets and phenotypes, achieving quantitatively similar results to flow cytometry, while also gathering cellular positional information. Here, we employ histo-cytometry to describe the spatial segregation of resident and migratory dendritic cell subsets into specialized microanatomical domains, suggesting an unexpected LN demarcation into discrete functional compartments.
authors.affiliation: Lymphocyte Biology Section, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. gernermy@niaid.nih.gov
authors.forename: Michael Y
authors.initials: MY
authors.lastname: Gerner
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Kastenmuller
authors.forename: Ina
authors.initials: I
authors.lastname: Ifrim
authors.forename: Juraj
authors.initials: J
authors.lastname: Kabat
authors.forename: Ronald N
authors.initials: RN
authors.lastname: Germain
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D002678:Chimera / Q000276:immunology; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008198:Lymph Nodes / Q000166:cytology* / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism
chemical_list: D015703:Antigens, CD