On the ultrastructural organization of Trypanosoma cruzi using cryopreparation methods and electron tomography
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The structural organization of Trypanosoma cruzi has been intensely investigated by different microscopy techniques. At the electron microscopy level, bi-dimensional analysis of thin sections of chemically fixed cells has been one of the most commonly used techniques, despite the known potential of generating artifacts during chemical fixation and the subsequent steps of sample preparation. In contrast, more sophisticated and elaborate techniques, such as cryofixation followed by freeze substitution that are known to preserve the samples in a more close-to-native state, have not been widely applied to T. cruzi. In addition, the 3D characterization of such cells has been carried out mostly using 3D reconstruction from serial sections, currently considered a low resolution technique when compared to electron tomography (ET). In this work, we re-visited the 3D ultrastructure of T. cruzi using a combination of two approaches: (1) analysis of both conventionally processed and cryofixed and freeze substituted cells and (2) 3D reconstruction of large volumes by serial electron tomography. The analysis of high-pressure frozen and freeze substituted parasites showed novel characteristics in a number of intracellular structures, both in their structure and content. Organelles generally showed a smooth and regular morphology in some cases presenting a characteristic electron dense content. Ribosomes and new microtubule sets showed an unexpected localization in the cell body. The improved preservation and imaging in 3D of T. cruzi cells using cryopreparation techniques has revealed some novel aspects of the ultrastructural organization of this parasite.
KeywordsElectron tomography High-pressure freezing Freeze substitution Trypanosoma cruzi Ultrastructure
We thank Lissa Catherine Reignault and Emile Barrias for the amastigote and trypomastigote forms of T. cruzi, Thiago Luiz de Barros Moreira for technical assistance, Dirceu Esdras for helping with the videos, and Ricardo Correia and Camilly Andrade for helping with the 3D models. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Financiadora de Estudos e Projetos (FINEP) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Brazil).
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