Electron microscopy cytochemistry and three-dimensional reconstruction of labeled structures in Trypanosoma cruzi

Significant advances have occurred in the area of high-resolution scanning electron microscopy (SEM), especially related to methodologies that allow the observation of intracellular structures that are exposed either by successive abrasion with a gallium ion beam or by sectioning in epoxy-embedded cells. Images of series of successively exposed surfaces can then be rendered into 3D models. Here, we report our observations by combining this approach with classical cytochemical methods to facilitate the 3D reconstruction of labeled structures and organelles. We used epimastigotes of Trypanosoma cruzi whose endocytic pathway was labeled with horseradish peroxidase, followed by fixation and detection of the peroxidase activity using the classical diaminobenzidine-osmium method followed by incubation with thiocarbohydrazide, which increases the concentration of osmium at the sites where the enzyme is located as well as the contrast of lipid-containing structures. This procedure allows not only a better visualization of membranous structures and lipid inclusions but can also easily identify the endocytic tracer (HRP) inside the cell. All structures involved in the endocytic activity could be traced and reconstructed.