Chan Vera B S, Toyofuku Takashi, Wetzel George, Saraf Laxmikant, Thiyagarajan Vengatesen, Mount Andrew S
Department of Biological Sciences, Clemson University;
Department of Marine Biodiversity Research (BioDive), Japan Agency for Marine-Earth Science and Technology (JAMSTEC).
J Vis Exp. 2017 Feb 28(120):55164. doi: 10.3791/55164.
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.
表征碳酸钙生物生成的首个事件需要多种显微镜方法相结合。首先，可以通过实时显微镜随时间观察细胞内pH分布和钙离子。这有助于识别生命阶段以及具有感兴趣特征的组织，以便进行进一步的电子显微镜研究。感兴趣的生命阶段和组织通常具有较高的pH和钙信号。在此，我们以秀丽隐杆线虫为例，展示一种在扫描电子显微镜（SEM）上表征生物样本中碳酸钙结构存在情况的方案，使用能量色散X射线光谱（EDS）来可视化元素组成，使用电子背散射衍射（EBSD）来确定晶体结构的存在，并使用透射电子显微镜（TEM）来分析材料的组成和结构。在本方案中，聚焦离子束（FIB）用于分离尺寸适合TEM分析的样本。由于FIB是一种位点特异性技术，我们展示了如何利用先前技术的信息来识别感兴趣的区域，即钙信号最高的区域。
