Histo-Cytometry: A Method for Highly Multiplex Quantitative Tissue Imaging Analysis Applied to Dendritic Cell Subset Microanatomy in Lymph Nodes

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...

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Bibliographic Details
Published in:Immunity (Cambridge, Mass.) Mass.), 2012-08, Vol.37 (2), p.364-376
Main Authors: Gerner, Michael Y., Kastenmuller, Wolfgang, Ifrim, Ina, Kabat, Juraj, Germain, Ronald N.
Format: Article
Language:English
Subjects:
Animals
Antibodies
Antigens, CD - analysis
Chimera - immunology
Data processing
Dendritic Cells - cytology
Dendritic Cells - immunology
Flow cytometry
Flow Cytometry - methods
Humans
Immune system
Immunohistochemistry - methods
Laboratory animals
Lymph Nodes - cytology
Lymph Nodes - immunology
Lymphocyte Activation - immunology
Mice
Mice, Inbred C57BL
Mice, Transgenic
Microscopy
Microscopy, Confocal - methods
Microscopy, Fluorescence - methods
T-Lymphocytes - immunology
T-Lymphocytes - metabolism
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Summary: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. ► Histo-cytometry provides discrete intranodal localization of distinct DC subsets ► Histo-cytometry provides highly multiplex phenotyping of individual cells in tissue sections ► Histo-cytometry provides quantification and spatial fixation of immune cells in situ
ISSN:1074-7613
1097-4180
DOI:10.1016/j.immuni.2012.07.011