Name: characterization of calcification events using live optical and electron microscopy techniques in a marine tubeworm
Uploaded: 2017-02-28T14:00:00
Description: Watch this Scientific Journal Video about Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm at JoVE.com

The authors would like to send a big thank you to Clemson Broadcast Productions, audio recording by J. Bright, Narration by A. D. McQuiston, Audio sweetening, K. Murphy, videography by G. Spake, Graphic arts by T. Messervy, Video editing by T. Messervy and E. Rodgers. Technical assistance and scientific advice was inspired by the advice of S. Kawada, S. Kubo, J. Hudson, T. Darroudi, D. Mulwee, H. Qian, Y. W. Lam, M. B. Johnstone, C. Campanati, A. C. Lane, and R. Dineshram. This study was funded by three GRF grants from the HKSAR-RGC (Grant Numbers: 705511P, 705112P, and 17304914).

1.筛选生命阶段和实时成像利益组织<ol><li>培养根据先前报道的方法6，7，9海洋幼虫能力。孵育tubeworm幼虫每毫升密度5幼虫用10μMSNARF-凌晨1点通宵过滤的海水。用铝箔覆盖该容器，以防止光致漂白的荧光探针。 </li><li>通过观察解剖显微镜的幼虫。主管tubeworm幼虫准备变态将在向前方向，而不是圆周运动游泳。 </li><li>转移主管?...

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活光学成像是一种用于在多细胞生物体观测细胞事件的有用方法。这里，内部pH和钙离子的指标被用于测量在矿化站点离子的通量。在这些区域中，活性离子泵送需要提高pH和钙离子浓度，以使钙化2,3。当施加荧光分子研究的有机体，重要的是确保所用的浓度具有可忽略毒性，使生物体在生理相关的方式来执行。较低浓度的染色是毒性较低，通?...

生物矿化是一个复杂的一系列事件，其中桥接一套导致产生精美有序矿物质1的细胞活动。面临的挑战是表征两个动态细胞过程和使用的光学和电子显微镜方法的组合的复杂的矿物结构。细胞内pH的正视利于碳酸钙晶体的形成，因此，鉴定具有增加的pH的生命阶段揭示当钙化可能被发生2,3的时间。 从家庭的龙介虫科管虫是海洋中的4种常见钙化。这也是海洋研究一种流行的无脊椎动物的模式，尤其是在生物污染5,6。在这项研究中，钙化成矿车厢的过程中杜里NG生物矿化观察。变态的快速过程包括碳酸钙结构7,8的出现。 我们展示了如何pH值内部测量可以在tubeworm进行，以及如何生活阶段，相关的钙化组织可筛选。感兴趣的生命阶段被识别后，负责钙化的组织可被表征在使用电子显微镜的方法更高的分辨率。使用荧光显微镜，我们确定变质诱导后出现需要碳酸钙的时间。人生的一个类似的阶段，随后用SEM-EDS对可视化元素组成的分布，并采用两种不同的电子显微镜方法，尤其是SEM-EBSD和FIB-TEM分析了沉积矿物中。

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characterization of calcification events using live optical and electron microscopy techniques in a marine tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

以下是tubeworm变态过程中钙化过程的一些看法。 图1示出了套环区域附近的pH值下比变态后的其他组织高。 图2I显示了钙的均匀分布，这表明没有大的钙化活动已经开始tubeworm; 图2II表明已钙化对于较长时间的tubeworm，表明钙化已经超越的感兴趣的时间点; 图2III示出了具有所感兴趣的钙化阶段，其选择用于进一步分析，以了?...

Watch this Scientific Journal Video about Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm at JoVE.com

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

钙化事件的表征在海洋Tubeworm使用实时光学和电子显微镜技术

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

Research

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相衬，微分干涉对比（DIC）显微镜

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

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Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

用化学干燥进行扫描电镜 (sem) 形态分析的原核生物和真核生物的制备

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...