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