Mapping immune processes in intact tissues at cellular resolution
Christian Brede, … , Gregory S. Harms, Andreas Beilhack
Christian Brede, … , Gregory S. Harms, Andreas Beilhack
Published November 12, 2012
Citation Information: J Clin Invest. 2012;122(12):4439-4446. https://doi.org/10.1172/JCI65100.
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Technical Advance Immunology

Mapping immune processes in intact tissues at cellular resolution

Abstract

Understanding the spatiotemporal changes of cellular and molecular events within an organism is crucial to elucidate the complex immune processes involved in infections, autoimmune disorders, transplantation, and neoplastic transformation and metastasis. Here we introduce a novel multicolor light sheet fluorescence microscopy (LSFM) approach for deciphering immune processes in large tissue specimens on a single-cell level in 3 dimensions. We combined and optimized antibody penetration, tissue clearing, and triple-color illumination to create a method for analyzing intact mouse and human tissues. This approach allowed us to successfully quantify changes in expression patterns of mucosal vascular addressin cell adhesion molecule–1 (MAdCAM-1) and T cell responses in Peyer’s patches following stimulation of the immune system. In addition, we employed LSFM to map individual T cell subsets after hematopoietic cell transplantation and detected rare cellular events. Thus, we present a versatile imaging technology that should be highly beneficial in biomedical research.

Authors

Christian Brede, Mike Friedrich, Ana-Laura Jordán-Garrote, Simone S. Riedel, Carina A. Bäuerlein, Katrin G. Heinze, Tobias Bopp, Stephan Schulz, Anja Mottok, Carolin Kiesel, Katharina Mattenheimer, Miriam Ritz, Viktoria von Krosigk, Andreas Rosenwald, Hermann Einsele, Robert S. Negrin, Gregory S. Harms, Andreas Beilhack

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Figure 1

Principle of optical sectioning and computational 3D reconstruction by multicolor LSFM.

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Principle of optical sectioning and computational 3D reconstruction by m...
(A) The sample is placed into the chamber with clearing solution and illuminated by laser light sheets of different wavelengths to excite and detect fluorescence (beam path of emission and excitation is indicated by an arrow) in the labeled specimen in 3 detection channels by turning an optical filter wheel. To create optical z-stacks, the sample is moved by 1- to 5-μm increments through the light sheet. Assembling x-y-z planes allows computational 3D reconstruction. (B) Gut-associated PPs are important sites of mucosal immune reactions. (C) Imaging of tissue autofluorescence (green) allows for visualization of micro-anatomical features of intact organs such as the small intestinal tract containing a PP with high endothelial venules (MAdCAM-1, cyan) and CD4+ T cells (red) as single and color-merged single optical sections (objective, ×5; scale bar: 100 μm) and (D) after computational 3D reconstruction. (E) 3D reconstruction of the whole organ reveals co-localization of MAdCAM-1 expression and CD4+ T cells in a PP. Using a ×20 objective allowed a higher magnification of (F) single CD4+ T cells (z-projection scale bar: 100 μm) and (G) subcellular precision imaging single-cell nuclei (stained with DAPI) within lymph nodes (z-projection scale bar: 100 μm).