Electron microscopic detection of pathogens and neutrophil extracellular traps

Particulated structures of microbes are relatively resistant to paraffin embedding. Nuclear/cytoplasmic inclusions in paraffin-embedded H&E preparation or ethanol-fixed Papanicolaou-stained cytologic specimens can be targeted for EM observation to demonstrate the microbes within the cell [1-3]. The antigens of Chlamydia trachomatis, E. coli, cytomegalovirus and human papillomavirus have been localized in paraffin sections by applying pre-embedding immuno-EM [2, 4, 5]. Although the fine morphologic preservation is not good enough, this approach is useful to confirm the presence of microbes in the lesions and the specificity of the antibodies. The viral genomes are also identifiable in routine samples by in situ hybridization at the EM level [1, 2, 4, 5]. NETs represent an extracellular, spider’s web-like structure resulting from cell death of neutrophils to trap and kill microbes. NETs often co-exist with fibrin meshwork at the site of fibrinopurulent inflammation. Immuno-EM study and correlative light and electron microscopy (CLEM), employing confocal laser scanning microscopy and scanning EM, were performed for visualizing NETs and fibrin fibrils in autopsied legionnaire’s pneumonia [6-8]. Lactoferrin and fibrinogen gamma chain marked NETs and fibrin, respectively. Pre-embedding immuno-EM, immuno-scanning EM and CLEM indicated that NETs constructed thin fibrils with granular materials attached. Smooth-surfaced fibrin fibrils were much thicker than the NETs fibrils, and each fibril consisted of a cluster of fine filaments.