Fig. 5

Ultrastructural analysis of S. pneumoniae in human sputum. Sputum specimens were stained with Spn-FLUO (green), wheat germ agglutinin (WGA) labeled with Alexa 555 (red), and DNA was stained with DAPI (blue). Preparations were analyzed by confocal microscopy. Panel (A) shows middle XY, XZ (bottom), and YZ (side) optical sections. The white inset indicates an optical zoom shown in panel (B), while the yellow inset indicates the region shown in panel (C). The bottom panels in (B and C) show orthogonal XZ and YZ sections. Decapsulated pneumococci (D-CPS) are indicated by the arrows. (D-F) A mask was created in the green channel (D) to analyze the 3D structure of the pneumococcal (E) aggregate or (F) dispersed pneumococci. (G) Representative 3D images of aggregated pneumococci across the sputum specimen, showcasing their size, shape, and spatial distribution within the sputum. (H) The area of those aggregate pneumococci and that of dispersed bacteria was plotted. Student’s t-test was performed to assess statistical significance, ****p < 0.0001. (I) Orthogonal XY and YZ sections were created using the green (Spn-FLUO) and the red channel (WGA). WGA binds acetyl glucosamine and sialic acid residues (GlcNAc/SA). The top panel shows the masked green channel representing the area of the pneumococcal aggregate. The pneumococcal aggregate was removed from the middle panel to reveal the hollow spaces occupied by bacteria embedded in the sputum. The bottom panel shows the non-masked green channel and the red channel. All analyses were performed using the Imaris software