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Figure 1. Panels A-C show images of z-section No. 39 from a stack of 74 0.2-µm z-sections (magnification 600X; the scale bar in Panel A also applies to Panels B and C). BEC in this field were double-labeled with the EUB338 universal probe (A) and the A. actinomycetemcomitans-specific probe (B). The cell in the center of Panel A contained a large mass of brightly fluorescent intracellular bacteria (red arrow). Other cells in the field contained smaller bacterial masses (not marked). Panel B shows that a portion of the large mass labeled with the universal probe also hybridized with the A. actinomycetemcomitans-specific probe (green arrow). The images from Panels A and B were superimposed (Panel C), confirming that bacteria labeled with both probes (yellow arrow) were adjacent to other bacteria labeled only with the universal probe (red arrow). Panel D presents a three-dimensional reconstruction of the same field. Bacteria recognized only by the universal probe are shown in solid red, while co-localization of the A. actinomycetemcomitans and universal probes is depicted by a green wireframe over a red interior. Reconstructed BEC surfaces are presented in blue. The red and green colors are muted when bacterial masses are intracellular, and brighter when bacteria appear to project out of the surface. The angle of view was rotated along the z-axis, and the image was zoomed. The large mass which appeared to have a lobular structure in z-section No. 39 was seen to be a cohesive unit containing A. actinomycetemcomitans in direct proximity to other species (red and green arrows).
