Improvement on the visualization of cytoskeletal structures of protozoan parasites using high-resolution field emission scanning electron microscopy (FESEM).
The association of high resolution field emission scanning electron microscopy (FESEM), with a more efficient system of secondary electron (SE) collection and in-lens specimen position, provided a great improvement in the specimen's topographical contrast and in the generation of high-resolution images. In addition, images obtained with the use of the high-resolution backscattered electrons (BSE) detector provided a powerful tool for immunocytochemical analysis of biological material. In this work, we show the contribution of the FESEM to the detailed description of cytoskeletal structures of the protozoan parasites Herpetomonas megaseliae, Trypanosoma brucei and Giardia lamblia. High-resolution images of detergent extracted H. megaseliae and T. brucei showed the profile of the cortical microtubules, also known as sub-pellicular microtubules (SPMT), and protein bridges cross-linking them. Also, it was possible to visualize fine details of the filaments that form the lattice-like structure of the paraflagellar rod (PFR) and its connection with the axoneme. In G. lamblia, it was possible to observe the intricate structure of the adhesive disk, funis (a microtubular array) and other cytoskeletal structures poorly described previously. Since most of the stable cytoskeletal structures of this protozoan rely on tubulin, we used the BSE images to accurately map immunolabeled tubulin in its cytoskeleton. Our results suggest that the observation of detergent extracted parasites using FESEM associated to backscattered analysis of immunolabeled specimens represents a new approach for the study of parasite cytoskeletal elements and their protein associations.
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro CCS, Rio de Janeiro, bloco G, Cidade Universitária, 21949-900, Brazil.
Celso
C
Sant'Anna
Loraine
L
Campanati
Catarina
C
Gadelha
Daniela
D
Lourenço
Letícia
L
Labati-Terra
Joana
J
Bittencourt-Silvestre
Marlene
M
Benchimol
Narcisa Leal
NL
Cunha-e-Silva
Wanderley
W
De Souza
D000818:Animals; D003599:Cytoskeleton / Q000648:ultrastructure*; D056890:Eukaryota / Q000648:ultrastructure*; D005407:Flagella; D016829:Giardia lamblia / Q000166:cytology / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning / Q000379:methods* / Q000592:standards; D010271:Parasites / Q000648:ultrastructure*; D014346:Trypanosoma brucei brucei / Q000166:cytology / Q000648:ultrastructure; D014351:Trypanosomatina / Q000166:cytology / Q000648:ultrastructure