Optimization of Ultrastructural Expansion Microscopy (U-ExM) to study cytoskeletal structures and different organelles in Trypanosoma cruzi

ALONSO, VICTORIA LUCIA; DE HERNANDEZ, AZUL; MARTINEZ PERALTA, GONZALO; VENA, RODRIGO

Caxambu

Congreso; XXXVIII Meeting of the Brazilian Society of Protozoology; 2023

Brazilian Society of Protozoology

Ultrastructural Expansion Microscopy (U-ExM), is a method to improve the resolution of optical microscopy through the physical expansion of a sample. During U-ExM, a swellable polymer network (or hydrogel) is synthesized uniformly throughout a biological sample. The samples then undergo a process of mechanical softening and homogenization, followed by a process of isotropic three-dimensional physical expansion when water is added, causing the polymer and thus the sample in which the polymer is embedded to enlarge. As a result, the biomolecules of interest become spatially separated from each other and the effective resolution of the microscope increases. This technique es compatible with standard immunofluorescence protocols.We optimized an U-ExM protocol for Trypanosoma cruzi and quantifying the expansion factors of different subcellular compartments. We determine the localization patterns of different tubulin isoforms, such as α-tubulin and β-tubulin. Also we immunolocalized acetylated and tyrosinated α-tubulin isotypes in epimastigotes and use mitochondrial cell-permeable dyes to identify this organelle. Finally, U-ExM was also performed in trypomastigotes and amastigotes validating this technique in all life cycle stages of T. cruzi.We observed that the typical morphology of the epimastigotes is preserved during the process, achieving an expansion factor of 4.5 times. We analyzed the expansion of the nucleus, kinetoplast, and basal body to confirm the expansion by measuring their size with ImageJ software.These results allow us to corroborate the usefulness of this technique for T. cruzi, especially for the analysis of cytoskeletal proteins. Also, it is possible to appreciate within the nucleus areas with different fluorescence intensity, which allows differentiating areas with different chromatin compaction without having to use electron microscopy.