For confocal microscopy, procedures were as described previously (Ge et al., 2014b).
The immunofluorescent microscopy procedure was then performed as described above.
The two-photon intravital microscopy procedure was performed as described previously [ 22].
A standardized protocol for electron microscopy procedure was developed for ultrastructural analysis of lipid droplets.
The estimated percent recovery for enumerating G. duodenalis cysts using the immunofluorescent microscopy procedure was 36.2% (95% CI, 28.4% - 46.2%).
The fixing, staining and imaging procedures are described above in the " Confocal microscopy" section.
Such procedures are not only essential for the iLEM, but also of great value to other correlative microscopy approaches.
Fluorescence microscopy and image analysis procedures were adapted from previously published methods [ 25, 34, 35].
Especially for the diagnosis of E. histolytica, species differentiation based on alternative procedures is compulsory, since microscopy cannot readily distinguish between E. histolytica and the non-pathogenic E. dispar[ 66, 125, 126].
