Optimisation of freeze substitution protocols for the examination of malaria parasite structure by volumetric electron microscopy.

IF 1.9 4区 工程技术 Q3 MICROSCOPY
Rachel Rachid, Camila Wendt, Wanderley de Souza, Kildare Miranda
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引用次数: 0

Abstract

Malaria is one of the deadliest infectious diseases in the world, annually responsible for over 400,000 deaths. It is caused by parasites of the genus Plasmodium, which undergo remarkable structural changes during their development within different cells across various hosts. An important approach to understand the structural basis of biochemical and physiological processes during Plasmodium infection has been the quantitative measurement of dimensional parameters obtained by different microscopy techniques. In this regard, sample preparation, particularly electron microscopy protocols that rely on room-temperature chemical fixation, has posed significant challenges, as it is known to produce artefacts such as shrinking, swelling and displacement of structures and osmolytes. In contrast, specimen immobilisation by cryofixation followed by freeze substitution minimises these artefacts and provides better sample preservation. Nevertheless, the composition of the freeze substitution medium may vary depending on the cell type, making it a critical factor for achieving optimal sample preparation. In this work, we optimised a freeze substitution protocol for the structural analysis of intraerythrocytic stages of the murine malaria models Plasmodium chabaudi and P. berghei. We tested different freeze substitution recipes, considering the biochemical composition of malaria membranes, and compared the results with those obtained through conventional chemical fixation. Overall, the results showed a significant improvement on the preservation of cell morphology and haemozoin crystals. Establishing an efficient and reproducible freeze substitution protocol for murine malaria models provides an important tool for advancing our understanding of the structural organisation of Plasmodium spp.

体积电子显微镜检测疟原虫结构的冷冻替代方案优化。
疟疾是世界上最致命的传染病之一,每年造成40多万人死亡。它是由疟原虫属的寄生虫引起的,这些寄生虫在不同宿主的不同细胞内发育过程中经历了显著的结构变化。了解疟原虫感染过程中生化和生理过程的结构基础的一个重要方法是对不同显微镜技术获得的尺寸参数进行定量测量。在这方面,样品制备,特别是依赖于室温化学固定的电子显微镜方案,已经提出了重大挑战,因为已知它会产生诸如结构和渗透物的收缩、膨胀和位移等人工制品。相比之下,通过冷冻固定和冷冻替代来固定样品可以最大限度地减少这些人工制品,并提供更好的样品保存。然而,冷冻替代培养基的组成可能因细胞类型而异,使其成为实现最佳样品制备的关键因素。在这项工作中,我们优化了用于小鼠疟疾模型chabaudi疟原虫和P. berghei疟原虫红细胞内阶段结构分析的冷冻替代方案。考虑到疟疾膜的生化组成,我们测试了不同的冷冻替代配方,并将结果与常规化学固定获得的结果进行了比较。总体而言,结果显示细胞形态和血色素晶体的保存有显著改善。建立一种高效、可重复的小鼠疟疾模型冷冻替代方案,为我们进一步了解疟原虫的结构组织提供了重要的工具。
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来源期刊
Journal of microscopy
Journal of microscopy 工程技术-显微镜技术
CiteScore
4.30
自引率
5.00%
发文量
83
审稿时长
1 months
期刊介绍: The Journal of Microscopy is the oldest journal dedicated to the science of microscopy and the only peer-reviewed publication of the Royal Microscopical Society. It publishes papers that report on the very latest developments in microscopy such as advances in microscopy techniques or novel areas of application. The Journal does not seek to publish routine applications of microscopy or specimen preparation even though the submission may otherwise have a high scientific merit. The scope covers research in the physical and biological sciences and covers imaging methods using light, electrons, X-rays and other radiations as well as atomic force and near field techniques. Interdisciplinary research is welcome. Papers pertaining to microscopy are also welcomed on optical theory, spectroscopy, novel specimen preparation and manipulation methods and image recording, processing and analysis including dynamic analysis of living specimens. Publication types include full papers, hot topic fast tracked communications and review articles. Authors considering submitting a review article should contact the editorial office first.
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