M. Zelená

Advanced imaging of microorganisms, including protists, is challenging due to their small size. Specimen expansion prior to imaging is thus beneficial to increase resolution and cellular details. Here, we present a sample preparation workflow for improved observations of the single-celled eukaryotic pathogen Giardia intestinalis (Excavata, Metamonada). The binucleated trophozoites colonize the small intestine of humans and animals and cause a diarrhoeal disease. Their remarkable morphology includes two nuclei and a pronounced microtubular cytoskeleton enabling cell motility, attachment and proliferation. By use of expansion and confocal microscopy, we resolved in a great detail subcellular structures and organelles of the parasite cell. The acquired spatial resolution of 43 nm enabled novel observations of centrin localisation at Giardia basal bodies. Interestingly, non-luminal centrin localization between the Giardia basal bodies was observed, which is an atypical eukaryotic arrangement. Our protocol includes antibody staining and can be used for the localisation of epitope-tagged proteins, as well as for differential organelle labelling by amino reactive esters. This fast and simple protocol is suitable for routine use without a superresolution microscopy equipment.