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.