Transmission Electron Microscopy of Coral Tissue

Erin Papke, Grace E. Kennedy, Elizabeth Elliott, Alison Taylor, Bradley B. Tolar, Blake Ushijima
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Abstract

Coral reefs are invaluable ecosystems that are under threat from various anthropogenic stressors. There has been a recent increase in the diagnostic tools utilized to understand how these threats impact coral reef health. Unfortunately, the application of diagnostic tools like transmission electron microscopy (TEM) is not as standardized or developed in coral research as in other research fields. Utilizing TEM in conjunction with other diagnostic methods can aid in understanding the impact of these stressors on the cellular level because TEM offers valuable insight into the structures and microsymbionts associated with coral tissue that cannot be obtained with a conventional light microscope. Additionally, a significant amount of coral tissue ultrastructure has not yet been extensively described, causing a considerable gap in our understanding of cellular structures that could relate to the immune response, cellular function, or symbioses. Moreover, additional standardization is needed for TEM in coral research to increase comparability and reproducibility of findings across studies. Here, we present standardized TEM sample fixation, embedding, and sectioning techniques for coral studies that ensure consistent ultrastructural preservation and minimize artifacts, enhancing the reliability and accuracy of TEM observations. We also demonstrate that these TEM protocols allow for the observation and quantification of bacterial and viral-like particles within the coral tissue as well as the endosymbiotic microalgae, potentially providing insight into their interactions within coral cells and how they relate to overall coral health and resilience. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Primary fixation

Basic Protocol 2: Decalcification

Basic Protocol 3: Sample dissection, secondary fixation, dehydration, and embedding

Basic Protocol 4: Sectioning and grid staining

Basic Protocol 5: Imaging

Abstract Image

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透射电子显微镜观察珊瑚组织
珊瑚礁是宝贵的生态系统,正受到各种人为压力因素的威胁。最近,为了解这些威胁如何影响珊瑚礁健康而使用的诊断工具越来越多。遗憾的是,在珊瑚研究领域,透射电子显微镜(TEM)等诊断工具的应用并不像其他研究领域那样标准化或发达。将透射电子显微镜与其他诊断方法结合起来使用,有助于了解这些压力因素对细胞水平的影响,因为透射电子显微镜可以深入了解与珊瑚组织相关的结构和微共生体,而这些是传统的光学显微镜无法获得的。此外,大量的珊瑚组织超微结构尚未被广泛描述,这导致我们对可能与免疫反应、细胞功能或共生有关的细胞结构的了解存在相当大的差距。此外,珊瑚研究中的 TEM 还需要进一步标准化,以提高不同研究结果的可比性和可重复性。在此,我们介绍了用于珊瑚研究的标准化 TEM 样品固定、包埋和切片技术,这些技术可确保一致的超微结构保存并最大程度地减少伪影,从而提高 TEM 观察结果的可靠性和准确性。我们还证明,这些 TEM 方案可以观察和量化珊瑚组织内的细菌和病毒样颗粒以及内共生微藻,从而深入了解它们在珊瑚细胞内的相互作用以及它们与珊瑚整体健康和恢复力的关系。© 2024 作者。基本规程 1:初步固定基本规程 2:脱钙基本规程 3:样本解剖、二次固定、脱水和包埋基本规程 4:切片和网格染色基本规程 5:成像
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