Ultrastructural observation of natural field phytoplankton cells by using rapid freezing and freeze substitution
Ultrastructural observation of natural field phytoplankton cells by using rapid freezing and freeze substitution
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DOI：
10.3800/pbr.7.126
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发表时间：
2012-08
影响因子：
0.6
通讯作者：
K. Kimura;Y. Tomaru;Keizo Nagasaki
中科院分区：
文献类型：
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作者：
K. Kimura;Y. Tomaru;Keizo Nagasaki
透射电子显微镜（TEM）是用于研究浮游植物超微结构的基本技术。然而，使用化学固定来制备 TEM 浮游植物样品既费力又费时。此外，化学固定通常会导致浮游植物细胞发生人为结构变化（伪影）。在本研究中，我们诱导并优化了快速冷冻和冷冻替代（RFS）方法，并配有过滤脱水系统，以制备海洋微藻的TEM样品，包括赤潮异形藻（Heterosigma akashiwo）、古董查顿氏菌（Raphidophyceae）、圆形异蚴藻（Dinophyceae）、细角毛藻（Chaetoceros tenuissimus）（硅藻纲）和Telaulax amphioxeia （隐藻纲）。整个过程比传统方法高效得多。此外，每个微藻细胞的精细结构都保存完好。例如，可以清晰地观察到核膜、核孔和多个囊泡的精细结构，这是使用化学固定方法相对难以观察到的。此外，这项新技术适用于赤潮红藻的自然开花细胞，并成功观察到了高分辨率的超微结构图像。这是第一份关于改良 RFS 方法在天然浮游植物群落中的应用的报告。该方法可用于天然水域中的多种浮游植物，高质量的观测有望为了解浮游植物的生理和生态提供有用的信息。
Transmission electron microscopy (TEM) is a basic technique used to study the ultrastructures of phytoplankton. However, the use of chemical fixation to prepare phytoplankton samples for TEM is laborious and timeconsuming. Furthermore, chemical fixation often causes artificial structural changes (artifacts) in phytoplankton cells. In this study, we induced and optimized a rapid freezing and freeze substitution (RFS) method, accompanied by a filter dehydration system, to prepare TEM samples of marine microalgae, including Heterosigma akashiwo, Chattonella antiqua (Raphidophyceae), Heterocapsa circularisquama (Dinophyceae), Chaetoceros tenuissimus (Bacillariophyceae), and Teleaulax amphioxeia (Cryptophyceae). The whole procedure was much more efficient than the traditional method. In addition, the fine structure of each microalgal cell was well preserved. For example, the fine structures of the nuclear membrane, nuclear pores, and several vesicles were clearly observed, which were comparatively difficult to observe using the chemical fixation method. In addition, this new technique was applicable to the natural blooming cells of H. akashiwo, and high-resolution ultrastructural images were successfully observed. This is the first report on the application of the modified-RFS method for natural phytoplankton assemblages. This method can be used for diverse phytoplankters in natural waters, and the high-quality observations are expected to provide useful information toward understanding phytoplankter physiology and ecology.