22 February 2013 An optimized two-photon method for in vivo lung imaging reveals intimate cell collaborations during infection
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Lung tissue motion arising from breathing and heart beating has been described as the largest annoyance of in vivo imaging. Consequently, infected lung tissue has never been imaged in vivo thus far, and little is known concerning the kinetics of the mucosal immune system at the cellular level. We have developed an optimized post-processing strategy to overcome tissue motion, based upon two-photon and second harmonic generation (SHG) microscopy. In contrast to previously published data, we have freed the lung parenchyma from any strain and depression in order to maintain the lungs under optimal physiological parameters. Excitation beams swept the sample throughout normal breathing and heart movements, allowing the collection of many images. Given that tissue motion is unpredictably, it was essential to sort images of interest. This step was enhanced by using SHG signal from collagen as a reference for sampling and realignment phases. A normalized cross-correlation criterion was used between a manually chosen reference image and rigid transformations of all others. Using CX3CR1+/gfp mice this process allowed the collection of high resolution images of pulmonary dendritic cells (DCs) interacting with Bacillus anthracis spores, a Gram-positive bacteria responsible for anthrax disease. We imaged lung tissue for up to one hour, without interrupting normal lung physiology. Interestingly, our data revealed unexpected interactions between DCs and macrophages, two specialized phagocytes. These contacts may participate in a better coordinate immune response. Our results not only demonstrate the phagocytizing task of lung DCs but also infer a cooperative role of alveolar macrophages and DCs.
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Daniel Fiole, Daniel Fiole, Pierre Deman, Pierre Deman, Yannick Trescos, Yannick Trescos, Julien Douady, Julien Douady, Jean-Nicolas Tournier, Jean-Nicolas Tournier, "An optimized two-photon method for in vivo lung imaging reveals intimate cell collaborations during infection", Proc. SPIE 8589, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XX, 85891H (22 February 2013); doi: 10.1117/12.2003708; https://doi.org/10.1117/12.2003708


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