SBEM analysis of procyclic and metacyclic stages of Trypanosoma brucei

Abstract number
European Microscopy Congress 2020
Corresponding Email
[email protected]
LSA.9 - Applications of volume scanning electron microscopy in life sciences
Jiří Týč (1), Eva Doleželová (1), František Kitzberger (1), Alena Zíková (1), Jana Nebesářová (1)
1. Institute of Parasitology, Biology Centre, Czech Academy of Sciences
Abstract text

Trypanosoma brucei, the causative agent of Human and Animal Trypanosomiases, undergoes a striking cellular transformation during its digenetic life cycle. The transitions between the insect forms found in various tse-tse tissue to the mammalian life stages are accompanied by extensive remodelling of organelles, including among others mitochondria regression and repositioning of mitochondrial DNA and flagellum. Here we use serial block-face scanning electron microscopy (SBEM) to reconstruct two distinct insect life cycle forms, the midgut procyclic and salivary gland infective metacyclic trypomastigotes. Chemically fixed T. brucei cells were processed with „OTO“ protocol SBEM. Images were acquired in 3D using Apreo SEM equipped with Volumescope (Thermofisher Scientific) and variable pressure control. SBEM technology images regions of interest with dimensions up to several hundreds of µm yielding tens to hundreds of cells for analysis, thus allowing for statistical evaluation of obtained data. The final resolution at 6,5 nm (in X, Y axes) and 100nm (Z) or higher facilitated the distinction of fine ultrastructural features within the cell. Cells were segmented and visualized in 3D using Microscopy image browser and Amira software. This approach provided quantifiable results (volumes, lengths) and ultrastructural details of all essential organelles and allowed us to compare the two insect forms to the published reconstruction of T. brucei mammalian bloodstream form (Hughes et al., J. Cell Sci. (2017) 130(3):jcs.198887). Our study provides a deeper understanding of how organelles are remodelled during the complex life cycle of this medically and veterinary important parasite.