Immune Processes Mapped in Intact Tissues

Complex immune processes, as for instance in hematopoietic cell transplantation or in antitumor responses, can now be analyzed on a single-cell level in large tissue specimens or even entire organs.

A novel multicolor light sheet fluorescence microscopy (LSFM) approach has been used for deciphering immune processes in large tissue specimens on a single-cell level in three dimensions.


Scientists at the Würzburg University Hospital (Germany) combined and optimized antibody penetration, tissue clearing, and triple-color illumination to create a method for analyzing intact mouse and human tissues. An objective inverter (LSM Tech; Etters, PA, USA) on a commercial Axiovert 200 inverted microscope (Zeiss, Oberkochen, Germany) allowed a flexible horizontal positioning of the objective at a greater distance and range than typically possible with an inverted microscope.

The technique allowed the successful quantification of 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, the investigators employment of LSFM allowed them to map individual T cell subsets after hematopoietic cell transplantation and detected rare cellular events. The authors recommend reliance on fluorophores in the red or near-infrared color spectrum, as there is a strong autofluorescent background noise when green fluorophores are used for specific labeling, which poses certain limitations.

The scientists concluded that multicolor LSFM is a rigorous tool for effectively pinpointing and quantifying rare cellular events and cell-cell interactions within intact organs of mice and humans. This translates in scientific progress into clinical approaches, which should greatly benefit from the application of this high-resolution technology. The three dimensional multicolor LSFM holds the promise of complementing classical histopathological diagnosis. The study was published on November 12, 2012, in the Journal of Clinical Investigation.

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Würzburg University Hospital
LSM Tech
Zeiss