Electron microscopy cytochemistry and three-dimensional reconstruction of labeled structures in Trypanosoma cruzi
- PDF / 4,209,503 Bytes
- 5 Pages / 595.276 x 790.866 pts Page_size
- 40 Downloads / 184 Views
PROTOZOOLOGY - SHORT COMMUNICATION
Electron microscopy cytochemistry and three-dimensional reconstruction of labeled structures in Trypanosoma cruzi Wanderley de Souza 1,2,3
&
Carolina L. Alcantara 1,2,3 & Narcisa L. Cunha e Silva 1,2,3
Received: 16 April 2020 / Accepted: 6 July 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract 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. Keywords Trypanosoma cruzi . Scanning electron microscopy . Three-dimensional reconstruction . Peroxidase activity . Endocytosis . Lipids
Introduction Recent advances in high-resolution scanning electron microscopy (SEM) include methodologies that allow acquisition of “transmission-like” SEM images as they are able of showing intracellular structures that were exposed by the abrasion of the ion beam, resembling images that are obtained with transmission electron microscopy. These can (a) use smaller Section Editor: Hiroshi Sato * Wanderley de Souza [email protected] 1
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Av. Carlos Chagas Filho 373, Centro de Ciências da Saúde, Bloco G, Rio de Janeiro 21941-902, Brazil
2
Centro Nacional de Biologia Estrutural e Bioimages-CENABIO, Av. Carlos Chagas Filho 373, Centro de Ciências da Saúde, Bloco G, Rio de Janeiro 21941-902, Brazil
3
Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens-INBEB, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373, Centro de Ciências da Saúde, Bloco G, Rio de Janeiro 21941-902, Brazil
electron beam diameters; thus, signals generated from the specimen surface are probed with an electron beam of variable energy but also with a low accelerating voltage (1–5 kV) and (b) use highly sensitive secondary and backscattered electron detectors that are well positioned in relation to the sample. Such
Data Loading...
