Abstract
By nature, confocal microscopy, which removes out of focus haze from fluorescent images, is hampered by a fixed point of view and limited depth information in individual image planes. We have developed a means for examining fixed infected host tissues in three-dimensions from multiple points of view. We analyzed events in a rat model of pneumococcal pneumonia using this virtual reality microscopy. Rats were "infectedλ via transtracheal instillation of fixed lucifer yellow-labeled type 3 pneumococci. Lung tissues from sacrificed rats were stained with rhodamine phalloidin to stain F-actin and Hoechst 33342 to stain host cell DNA. From digital confocal image stacks of fluorescent host cell cytoplasm, host-cell nuclei, and invading bacteria, tissue volumes were reconstructed three-dimensionally in a voxel-based rendering program. Polygonal surfaces from the objects in each of the three image stacks were extracted and imported into a ray-tracing surface rendering program and rendered with a 360 degree camera placed at various points inside the host cell objects. Rendered images were mapped to a sphere and viewed interactively using RealVR software. At 2h post-infection most pneumococci had exited the alveoli and entered the lung parenchyma. Occasional bacteria were seen within vascular spaces. At 6h many neutrophils had entered the interstitium. By 24h, alveolar air spaces appeared smaller due to inflammatory edema of the interstitium, and pneumococci clustered primarily in interstitial phagocytic cells. Virtual reality microscopy shows very early pneumococcal invasion of lung parenchyma. This contrasts with the widely held notion that the pathogenesis of pneumococcal pneumonia is due mainly to intra-alveolar events. Alveolar involvement with inflammatory exudate appears to be a relatively later event in the pathogenesis timeline.