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
Summary
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.
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| Hexamethyldisilazane | Electron Microscopy Sciences | 16700(EM) | |
| Osmium Tetroxide 2% Aqueous Solution | Electron Microscopy Sciences | 19192 | |
| IBMX 3-Isobutyl-1-methylxanthine | ThermoFisher Scientific | PHZ1124 | |
| Nigericin, Free Acid | ThermoFisher Scientific | N7143-5MG | |
| 35-mm-diam dish, hole size 27 mm, Glass No.0, Non-coat | ThermoFisher Scientific | D110400 | |
| 5-(and-6)-Carboxy SNARF-1, Acetoxymethyl Ester, Acetate | ThermoFisher Scientific | C-1271 | |
| BDH Potassium Chloride, ACS Grade | VWR | BDH0258-500G | |
| Paraformaldehyde reagent grade, crystalline |
Sigma | P6148 | |
| 1 M Hydrochloric Acid for Volumetric Analysis | Wako Pure Chemical Industries, Ltd | 083-01095 | |
| 0.05 M Sodium Hydroxide Solution for Volumetric Analysis | Wako Pure Chemical Industries, Ltd | 199-02185 | |
| Calcein | Sigma | C0875 | |
| FASW | Iwaki Co. Ltd. | Rei-sea Marine | |
| Mixed Cellulose Ester Membranes; 47 mm dia, 0.45 µm | ADVANTEC | A045A047A | |
| ethanol | Wako Pure Chemical Industries, Ltd | 051-00476 | |
| Artificial seawater for buffers | by SOP06 of DOE (1994), cdiac.ornl.gov/ftp/cdiac74/sop06.pdf | ||
| Sodium Chloride | Wako Pure Chemical Industries, Ltd | 191-01665 | |
| Potassium Chloride | Wako Pure Chemical Industries, Ltd | 163-03545 | |
| Magnesium Chloride Hexahydrate | Wako Pure Chemical Industries, Ltd | 135-00165 | |
| Calcium Chloride | Wako Pure Chemical Industries, Ltd | 039-00475 | |
| Sodium Sulfate | Wako Pure Chemical Industries, Ltd | 197-03345 | |
| Hydrochloric Acid | Wako Pure Chemical Industries, Ltd | 089-08415 | |
| 2-amino-2-hydroxymethyl-1,3-propanediol (tris) | Wako Pure Chemical Industries, Ltd | 207-06275 | |
| 2-aminopyridine | Wako Pure Chemical Industries, Ltd | 011-02775 | |
| Orion 5-star Plus pH meter | Thermo Scientific | ||
| PrpHecT ROSS Micro Combination pH Electrode 8220BNWP | Thermo Scientific | ||
| Axiovision, Version 4.6, Axio Observer Z1 | Zeiss | ||
| ImageJ | NIH, Bethesda, MD, USA | ||
| HRTEM H500 | Hitachi | ||
| SU6600 VPSEM | Hitachi | ||
| NB5000 Focused Ion and Electron Beam (FIB-SEM) system | Hitachi |
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