Identifying Molecular Changes in Stages Using Hyperspectral Raman Microscopy.

Giardiasis is one of the oldest-reported infectious diseases worldwide. It affects individuals with weakened immune systems and progresses into chronic infection if untreated. Morphological analysis and visualization of cell shapes using unlabeled or fluorophore-labeled samples are commonly employed to identify the parasite. To distinguish molecular content variations between trophozoites and infectious type I cysts, the current research presents an alternative approach based on label-free Raman microscopy. Constituents responsible for plasma membrane thickening and cyst wall formation during encystation, such as N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc) were detected. Although these two coexisting compounds have similar molecular structures, their spectroscopic distinction and visual localization through Raman microscopy are achievable. While immature and non-viable cysts contain a larger amount of GlcNAc, a potential transition of this moiety to GalNAc might occur as the cysts mature and become infectious. Other Raman results revealed changes in the oxidation states of heme-binding proteins and in lipid-protein metabolism, each serving as an additional protection mechanism that the parasite employs for survival. Complementary bright field and confocal fluorescence microscopy results corroborate the Raman outcomes. The molecular-level findings of this work, which presents a detailed spectroscopic analysis of 's encystation and excystation stages, substantiate the need to use complementary methods for monitoring the parasite's dynamics and efficacy in terms of self-protection. This alternative method provides accurate insights for further understanding the multifaceted factors involved in 's encystation process and its acquired resistance to external stimuli.