Bandeira Paula Terra, Ortiz Sharmila Fiama das Neves, Benchimol Marlene, de Souza Wanderley
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Centro de Pesquisa em Medicina de Precisão, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-901, Brazil; Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens e Centro Nacional de Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-901, Brazil.
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Centro de Pesquisa em Medicina de Precisão, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-901, Brazil.
Exp Parasitol. 2023 Dec;255:108629. doi: 10.1016/j.exppara.2023.108629. Epub 2023 Oct 4.
Light microscopy has significantly advanced in recent decades, especially concerning the increased resolution obtained in fluorescence images. Here we present the Expansion Microscopy (ExM) technique in two parasites, Trichomonas vaginalis and Tritrichomonas foetus, which significantly improved the localization of distinct proteins closely associated with cytoskeleton by immunofluorescence microscopy. The ExM techniques have been used in various cell types, tissues and other protist parasites. It requires the embedment of the samples in a swellable gel that is highly hydrophilic. As a result, cells are expanded 4.5 times in an isotropic manner, offering a spatial resolution of ∼70 nm. We used this new methodology not only to observe the structural organization of protozoa in more detail but also to increase the resolution by immunofluorescence microscopy of two major proteins such as tubulin, found in structures formed by microtubules, and costain 1, the only protein identified until now in the T. foetus's costa, a unique rod-shaped like structure. The individualized microtubules of the axostyle were seen for the first time in fluorescence microscopy and several other details are presented after this technique.
近几十年来，光学显微镜技术有了显著进步，特别是在荧光图像的分辨率提高方面。在此，我们展示了扩展显微镜（ExM）技术在两种寄生虫——阴道毛滴虫和胎儿三毛滴虫中的应用，该技术通过免疫荧光显微镜显著改善了与细胞骨架紧密相关的不同蛋白质的定位。ExM技术已应用于各种细胞类型、组织和其他原生生物寄生虫。它需要将样品嵌入高度亲水的可膨胀凝胶中。结果，细胞以各向同性的方式膨胀4.5倍，提供了约70纳米的空间分辨率。我们使用这种新方法不仅更详细地观察了原生动物的结构组织，还通过免疫荧光显微镜提高了两种主要蛋白质的分辨率，如在微管形成的结构中发现的微管蛋白，以及在胎儿三毛滴虫独特的杆状结构——肋（costa）中迄今唯一鉴定出的蛋白共染蛋白1。在荧光显微镜下首次看到了轴柱的单个微管，并且在该技术之后还展示了其他几个细节。
