生物多样性 ›› 2016, Vol. 24 ›› Issue (7): 847-854.doi: 10.17520/biods.2015320

所属专题: 全球气候变化下的海洋生物多样性专辑

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颗石藻颗石粒形态的原子力显微观测方法: 以赫氏艾密里藻为例

王雨1, 张会勇2, 项鹏1, 叶又茵1, 林更铭1, 杨清良1, 林茂1, , A;*()   

  1. 1 .国家海洋局第三海洋研究所, 福建厦门 361005
    2 .泽泉科技有限公司开放实验室, 上海 200333
  • 收稿日期:2015-11-16 接受日期:2016-04-15 出版日期:2016-07-20
  • 通讯作者: 林茂 E-mail:linmao@tio.org.cn
  • 基金项目:
    国家自然科学基金(41506217, 41506136)、科技部基础专项(GASI-01-02-04)和科技部海洋公益性行业科研专项(201005015)

Observing the morphological features of Emiliania huxleyi coccoliths using atomic force microscopy

Yu Wang1, Huiyong Zhang2, Peng Xiang1, Youyin Ye1, Gengming Lin1, Qingliang Yang1, Mao Lin1, *()   

  1. 1 Third Institute of Oceanography, State Oceanic Administration, Xiamen, Fujian 361005
    2 Zealquest Open Laboratory of Zealquest Technology Limited Company, Shanghai 200333
  • Received:2015-11-16 Accepted:2016-04-15 Online:2016-07-20
  • Contact: Lin Mao E-mail:linmao@tio.org.cn

颗石藻(coccolithophore)作为一种模式生物, 在重建古海洋气候和环境以及预测未来全球气候变化中起着很重要的作用, 赫氏艾密里藻(Emiliania huxleyi)是颗石藻最为典型的代表种。钙质颗石粒(coccolith)是颗石藻形态分类的主要依据, 有着非常精细和复杂的结构, 在样品收集过程中很容易遭到破坏, 这是颗石藻鉴定中经常遇到的一个技术问题。国际上还没有统一的颗石藻定量采样和样品分析方法。本文采用原子力显微方法(atomic force microscopy, AFM)对赫氏艾密里藻的颗石粒形态进行了超显微观察研究, 获取不同扫描范围的高度图(height image)和形貌图(deflection image)以观测其形态结构, 并建立了针对颗石藻的原子力显微样品制备方法。通过离心与膜过滤两种方法收集赫氏艾密里藻, 比较后得出了一种简单、快速的适合于观测颗石藻在大气环境成像的样品处理、制备和图像采集方法: 3,000-4,000 rpm, 20℃离心5 min, 收集颗石藻, 去除有机杂质后取白色沉淀, 将沉淀物悬浮于0.05 M NH4HCO3溶液中, 悬浮液滴加于盖玻片表面, 20℃晾干后于样品台在AFM接触模式(contact mode)下原子级扫描, 扫描范围50 µm, 频率1 Hz, 可以得到优质的颗石粒形态图像, 有助于颗石藻的分类鉴别。该方法可用于室内不同环境梯度或参数下的颗石粒形态结构及颗石藻藻华的检测与研究。

关键词: Emiliania huxleyi, 颗石粒, 形态特征, 原子力显微法, 高度图, 形貌图

Coccolithophores are important components of the phytoplankton community that play a unique role in the global ocean biogeochemical cycle though carbon fixation and calcium carbonate production. In addition, coccolithophores are good indicators of climate change as they have indicative organic and inorganic remnants in sediments. Emiliania huxleyi is the most typical representative of coccolithophores. Classification of coccolithophores is mainly based on coccoliths, which have very fine and complex structures and are easily destroyed during the sample collection process. It becomes a technical problem to identify coccolithophores. In this study, atomic force microscopy (AFM) was employed to observe coccoliths of Emiliania huxleyi with the purpose of establishing an AFM sample preparation method for coccolithophores. Centrifugation was found to be an appropriate method to deal with the AFM samples compared with filtration. An optimized protocol was established: cells are centrifuged at 3,000-4,000 rpm, 20℃, over 5 minutes, a precipitate is suspended in 0.05 M NH4HCO3 solution, and then a droplet of solution containing suspended coccoliths is pipetted onto a glass attached to the sample holder. High resolution images had been captured under the contact mode of AFM in air conditioning with a scan range of 50 µm and frequency of 1 Hz. The method is applicable to analyze the morphological features of coccoliths under various environmental parameters or gradients in the laboratory and is also suitable for coccolithophore bloom detection.

Key words: Emiliania huxleyi, coccolith, morphological feature, atomic force microscopy (AFM), height image, deflection image

图1

藻液过滤制样条件下赫氏艾密里藻颗石粒的原子力20 µm×20 µm扫描范围高度图(a)和形貌图(b)"

图2

藻液过滤制样条件下赫氏艾密里藻颗石粒的原子力20 µm×20 µm(a)与5×5 µm2(b)扫描范围形貌图"

图3

离心收集条件下赫氏艾密里藻颗石粒的原子力100 µm×100 µm扫描范围高度图(a)与形貌图(b)"

图4

离心收集条件下赫氏艾密里藻颗石粒的原子力20 µm×20 µm(a)与5 µm×5 µm(b)扫描范围形貌图"

图5

离心收集条件下赫氏艾密里藻颗石粒的原子力2 µm×2 µm扫描范围正面(a)与反面(b)形貌图"

图6

离心收集条件下赫氏艾密里藻颗石粒(正面) 的原子力1 µm×1 µm扫描范围高度图(a)与形貌图(b)"

图7

离心收集条件下赫氏艾密里藻颗石粒(正面)的原子力500 nm×500 nm扫描范围高度图(a)与形貌图(b)"

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