Name: characterization of calcification events using live optical and electron microscopy techniques in a marine tubeworm
Uploaded: 2017-02-28T14:00:00.000+00:00
Duration: 15 min 39 s
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>转移主管幼虫到60微米的网格。按网格对培养皿中，提供了良好的密封留住液体。快速释放密封释放并丢弃含有荧光染料的海水，保留幼虫。 </li><li>洗净用过滤人工海水（FASW）幼虫两次，取注意不要干出过程中的幼虫。 </li><li>将10至20幼虫薄玻璃底菜用5毫升含有变态过程观察10 -4 M异丁基（IBMX）FASW的。 </li><li>插入滤波器立方体检测SNARF-1信号（通道1:实施例二十五分之五百十EM三十零分之五百八十○;通道2:实施例二十五分之五百十EM三十五分之六百四十○）和幼虫的DIC图像。 </li><li>在20X物镜下的变态幼虫的位置，那么，优化快门时间（100-300毫秒）足够快，以确保活体动物的清晰图像被捕获。 </li><li>置1微米的距离来捕获所有三个通道的Z堆叠，成像活体动物时，在移动到z方向的下一层将使信道之间的更好的相关性之前，成像所有三个通道的每一层。 </li><li>对所有通道和图层作为从显微镜软件TIF文件导出灰度图像:文件|出口|文件类型TIF |开始。 </li><li> 使用ImageJ，即时通讯端口各通道的图像序列:文件|进口|图像序列。 <ol><li>要导入通道1λ1 EM，输入起始影像3和增量:4。 </li><li>要导入通道2λ2 EM，输入起始影像4和增量:4。 </li></ol></li><li>通过将λ2 EM由λ1 EM每一层逐个像素生成的合成图像（流程|图片计算器... |图像文件640纳米的"鸿沟"的图像文件580纳米），即五百八分之六百四纳米比。 </li><li>选择图片|查找表| 16种颜色。 </li><li>选择分析|工具|校准条。 </li><li>检查不同的层，识别包含最异质层。这提供了关于内部pH值分布最有用的信息。 </li><li>使用之间五百八十零分之六百四十〇纳米比和pH值之间的关系，建立一个情节信息可视化内部pH值的分布。 </li></ol><ol><li> 10μMSNARF-凌晨1点20左右幼虫隔夜染色后，加入尼日利亚和氯化钾库存解决方案，以弥补50μM尼日利亚和150毫米氯化钾进行校准。 </li><li>调整用稀的NaOH或HCl校准AFSW的pH值，直到pH值周围5.9实现。记下确切的pH值。测量pH值用pH计用玻璃电极的基于已经校准由海水2-氨基-2-羟甲基-1,3-丙二醇（TRIS）和2-氨基吡啶9。 </li><li>一起用液体具有已知pH值在640 nm和580nm处传送一个染色tubeworm幼虫一个干燥和清洁盘为信号强度的测量。 </li><li>重复步骤2.2和2.3四个点的pH范围为5.9至9.9。它们代表了一系列生理相关的值。 </li><li>检查以确保信号出现在整个动物体内均匀。这表明，细胞内pH一直ëquilibrated与环境海水。 </li><li>图像在五个不同的海水的pH环境中tubeworm幼虫，记录示为在640纳米"灰度值"，并在580纳米，即，λ2 EXC，λ1 EXC的强度。 </li></ol> 3.比例式成像数据分析<ol><li>从五胡乱地点或从上一试算表感兴趣的区域测量输入灰度值。 </li><li>检查看看，五点校准呈线性关系，表明细胞内pH（ 例如 ，像方程Y = 0.30x - 1.47; Y =灰度值; X = pH值; R = 0.934）。 </li><li>从使用测量五百八十零分之六百四十〇纳米强度比在步骤1.13的方程式计算细胞内pH值。 </li></ol> 4.样品保鲜，脱水和安装电子显微镜<ol><li>保留的用4％多聚甲醛感兴趣的生命阶段。在这个研究中，修复管虫2-4天附着后，保持动物固定液直至分析。 </li><li>在通风橱中工作时，后固定，用1％四氧化锇水溶液试样30分钟期间脱水过程，以减少组织的收缩。 </li><li>脱水用梯度乙醇系列试样:50％的乙醇5分钟，70％的乙醇5分钟，85％的乙醇5分钟，95％乙醇进行5分钟，和5分钟最后无水乙醇两次。 </li><li>在通风橱中工作，则添加1:乙醇和六甲基二硅氮烷（HMDS）1溶液向试样，允许液体完全蒸发。 </li><li>在通风柜，100％的HMDS添加到样品，允许液体完全蒸发。 </li><li>使用钻石刀，介绍几个切到培养皿中，周围一些管虫。盖上盖子，按住底部的菜对铝存根打破菜。 </li><li>在解剖镜下，发现包含一个完整的tubeworm骨折片。 </li><li>应用银漆以铝存根和碎片小心地固定在存根。漆围绕塑料碎片的边缘，以减少充电。 </li></ol> 5.定位钙丰富的地区运用SEM-EDS <ol><li>使试样存根到样品保持器。 </li><li>衡量设置有显微镜的计整个组件的高度，通过松开锁紧垫圈和旋转后调整高度直到试样是在标准高度，或输入的高度所记录。 </li><li>允许空气进入显微镜交换室和摆动室门打开。样品架滑动到交换杆的端部。然后关闭并撤离室。 </li><li>打开闸阀和朝向显微镜室中滑动的交换杆。在滑动杆交换一路样品架完全固定在显微镜阶段。删除Exchange杆和关闭闸阀。 </li><li>输入采样他样品尺寸和发送阶段初始位置放置电子柱下方的样本。 </li><li>坐落在VP-SEM模式室真空度为20-30帕。设置电子加速电压20千伏，并打开高压。 </li><li>提高阶段为10毫米的样品的工作距离。收购活饲料，并找到标本。优化图像调整散光，并根据需要聚焦。 </li><li>打开SEM-EDS程序，并选择EDS-SEM模式选项。选择菜单选项来运行的EDS图。 </li><li>变更聚光透镜和光圈设定在3的处理时间内实现的〜30％的死区时间通过在SEM-EDS节目的速率计监测。使得梁设置进行任何更改后运行光束和光圈调整程序。 </li><li>选择一个放大率使得单个生物体填充的整个视场。启用漂移校正选项，并在SEM-EDS程序捕获图像。 </li><li>收购马与生物体填充的整个视野​​，使用50-100毫秒的停留时间数p的数据。跑地图，直到数据显示有机体（约15分钟）内钙的独特定位。 </li><li>以获得点量化数据，改变工艺时间为6，并调整电子探针获得的〜30％的死区时间。运行调整的步骤和优化图像是必要的。 </li><li>选择在SEM-EDS程序指向和ID选项，并获得另一幅图像。使用单点工具，请单击上出现钙丰富的EDS图，以及钙缺乏的地区进行比较的区域。扫描每个点为30秒的生存时间。 </li></ol> 6.确定钙丰富的地区的晶体信息使用SEM-EBSD <ol><li>从扁试样存根消除利益塑料含有片段标本并加盖用导电银胶45°的预倾斜存根的倾斜面。 </li><li>拧存根到样品H老年人和朝向样本保持器的正面位置存根的倾斜面（用试样）。插入样品架到使用交换室显微镜的腔室。 </li><li>设置室真空至30Pa和电子加速电压20千伏。发送电子柱下方的样品和倾斜级25°到样品表面从正常大约70°放置于电子束。开启高电压。 </li><li>提高阶段的〜18mm的工作距离。使用轨迹球移动舞台，找到一个标本。增加放大倍数帧感兴趣的特定功能。对准光束和优化图像是必要的。 </li><li>打开该电子背散射衍射模式的SEM-EDS计划。选择方解石和文石为感兴趣相。将相机EBSD在154毫米的距离。将相机条件为1×1分档和一个100毫秒的帧时间。使用快速束光栅，收购的背景。 注:不同的帧速率可以根据电子探针需;调整探头或相机帧速率达到约90％的信号。 </li><li>捕获在SEM-EDS节目的图像。使用现场分析工具，然后单击某处图像扫描该点的光束。观察摄像头窗口，看是否检测到任何菊池模式。 </li><li>点击图像的不同区域进行扫描电位钙化位点，观察照相机窗口在每个点，以查看是否菊池频带都存在。如果图案是本和匹配任何数据库中的所选阶段的，它们将自动地被索引。 </li></ol>透射电镜7.样品制备使用FIB-SEM <ol><li> 溅射涂层制备的扫描电镜样品用铂或类似材料以产生导电层。 <ol><li>将以前固定，脱水，并安装到标本溅射镀膜机的外壳和接通电源，开始抽水室了。 </li><li>一旦该腔室真空读取40毫乇或更少，打开煤气开关并增加氩气流量达到200毫托的室压。调节气体流量，以达到80毫托的最终腔室压力。 </li><li>开启电压切换开关，并增加电压达到15毫安的电流。设置定时器和涂层中的时间过长（〜10分钟），以创建从离子辐照损坏保护样品表面的厚层（〜60纳米）。调节，以保持一个稳定的15毫安电流的电压。 </li><li>一旦完成后，涂布器以释放真空并除去样品掉电。 </li></ol></li><li>螺钉所制备的基底，溅射涂覆的样品到仪器样品架的M4螺钉讯息。直到拧紧手，松开锁紧螺母，然后旋转样品架后改变组件的高度。使用激光高度计和标准与供应的（如在视频显示= 0.04英寸）调整高度为1mm以内ð与仪器。 </li><li>关闭在FIB-SEM所有枪阀门，然后让空气进入eucentric阶段气闸。一旦压力与环境均衡，滑动空气锁打开并加盖样本保持器到交换杆的尖头。打开交换杆的端部的旋钮的"锁定"位置。关闭空气锁并撤离气闸室。 </li><li>一旦eucentric级空气锁已被疏散和压力相匹配的FIB-SEM室，打开闸阀，并在交流中杆推插入样品。在滑动交换杆一路在仪器eucentric阶段样品架座，则交换杆旋转到"解锁"位置上。滑动杆交换背出，关闭闸阀。 </li><li>移动台到所述离子束列下的样本位置。打开闸阀，并获得FIB的活离子引起的二次电子图像。使用低倍率和快速光栅扫描，以尽量减少离子损伤。重置普通观察光束的聚焦，提高阶段的Z高度，直到所述样品是在焦点。在这一点上，样品是在其中两个所述离子和电子束聚焦在同一位置的交叉点的位置。 </li><li>从扫描电镜使用二次电子图像，定位在标本感兴趣的区域，使用轨迹球来移动样品。感兴趣的领域是先前由EDS图谱测定钙丰富的地区。旋转作为必要得到的符合窗口的框架感兴趣的特征的阶段。 </li><li>上的FIB窗口接口，捕获样品的快照在1,000X的与另外的2X数字变焦的倍率和在感兴趣的区域绘制〜8微米×2微米的沉积模板。沉积箱的倍率尺寸可以调整，以适应不同尺寸的特征。设定溅射条件下，使用一个40千伏至淀积碳，0.09 nA的光束，采用0.5微秒的停留时间为5分钟。 </li><li>碳的沉积后，修改沉积条件使用40千伏，0.7 nA的光束，并沉积5分钟，以产生一〜2-3微米钨帽以沉积钨。 </li><li>捕获使用观察光束FIB快照，并使用微量取样溅射工具绘制围绕钨帽四个框的模式。设置的上，下框为约15μm×8微米;右箱10微米×5微米，和左框6微米×5微米，或大到足以包围感兴趣的区域。 <ol><li>修改的制造条件下，使用一个40千伏，19 nA的光束，和50微秒，以及每个框3-4扫描帧的驻留时间，以切断。然后制作图案。制造，感兴趣的区域，以保护Ç和W层之后，将类似于一个岛，在顶部，底部，和右边除去所有相邻的材料。 </li></ol></li><li>倾斜级58°把SAMP勒表面法线到电子柱中，在一个陡峭的角度，以在离子柱。找到样本"孤岛"使用FIB的背面，并在捕捉放大倍数1000倍和2-4X数码变焦快照。 </li><li>在其中，它是可见的最末端绘制在样品的背面的底部上的〜15微米×2微米的溅射图案"岛"。制造使用4 nA的光束的框3分钟，或直到光束已经穿过样品的底切"岛"。 </li><li>倾斜eucentric阶段从当前位置（回零）-58°。通过点击"探头"，在FIB接口将钨微量取样探头。选择"MS"的舞台控制面板上，并使用轨迹球在试样的顶部移动探头。 </li><li>以1 KX的倍率获取来自FIB活饲料，并且使得尖端是直接的微量样品的钨盖的右侧上方的探针的位置。同时观察活从SEM SE图像，使用阶段面板的Z控制旋钮降低探针，直到它接触钨帽。蜂鸣器发声，一旦接触已经取得进展。 </li><li>捕获使用FIB使用40千伏，0.01 nA的束快照，在1000倍和2倍数码变焦的放大倍数。使用沉积工具在它已接触微量样品的钨帽的探针的末端画出2微米×2微米的框。 <ol><li>设定的条件使用40千伏，0.09 nA的光束以沉积钨，2分钟和0.5微秒的停留时间。然后制作图案。 </li></ol></li><li>绘制〜2微米×5微米的溅射图案在微量样品的左端"臂"（其中它仍然连接到散装样品的）。使用40千伏制造的图案，0.7 nA的光束，进行2分钟，或直至蜂鸣表明微量样品已经从散装样品分离。 </li><li>使用Z控制旋钮附加到微量样品提高探头它清除了大样，然后缩回探头。发送eucentric阶段主页或交换位置。 </li><li>放置一个铜半格成侧面入口支架和支架加载到侧入口阶段清洗室。疏散室，并插入侧进持有人。将持有人的FIB位置。 </li><li>台上按控制面板上侧，并使用轨迹球带来的半格眼帘的FIB监视器上。移动以清洁半格的区域和使用Z旋钮表面带入焦点。 </li><li>按探针在插入阶段控制面板上的探针，按MS和使用轨迹球和Z旋钮在半格到微量样品的位置。降低探头，直到接触微量样品格。 </li><li>捕获的FIB图像在放大1000倍（2X变焦）。使用沉积工具，并通过微量样品的下侧绘制〜10微米×3微米的图案。存款钨为40千伏，0.7 nA的光束，利用停留Ť0.5微秒3分钟IME。 </li><li>使用溅射工具和在探针的末端画小1微米×3微米的图案。使用40千伏，0.7 nA的光束在3毫秒的停留时间，以切割探针从微量样品远离制造的图案。缩回探头一次免费的。 </li><li>捕获在5000倍放大倍率的FIB图像，并绘制两个溅射图案在微量样品的顶部和底部7微米×4微米，而使箱之间的〜1微米的间隙。居中的图案，以便不切割微量样品的左，右两侧。制造使用40千伏，4 NA光束，和3微秒的停留时间，这两个图案2分钟，设置朝向微量样品的中心光栅方向。 </li><li>使用捕捉在5000倍的FIB观察梁另一个图像。调整之前的溅射框〜6.5微米×1微米，靠拢在一起，留下〜差距0.5微米，并制造使用40千伏，0.7 nA的光束。 </li><li>倾斜侧入门阶段0.5°和瓶盖TURE另一FIB图像。调整溅射箱6微米宽。在微量样品的顶侧的顶部图案的位置，并用40千伏，0.7 nA的光束制造。倾斜-0.5°并且与微量样品的下溅射图案侧和底侧重复。 </li><li>进一步降低由〜0.5微米的图案宽度。倾斜0.5°，并用40千伏，0.09 nA的光束溅射微量样品的顶侧。在-0.5°倾斜的底侧重复。 </li><li>倾斜的舞台2.2°，在1万倍获取FIB图像。调整溅射模式，以5微米，宽光束改变条件，5千伏，0.03呐。上部图案在微量样品的上侧，放置直到顶边，和制造。倾斜-2.2°，与下侧和底部图案重复。 </li><li>重复5千伏铣步骤，直到微量样品变成电子透明（约100纳米厚）。一旦完成后，关闭gunvalves并卸下侧入座。 </li></ol> 8.Øbtaining选区衍射图样上的TEM <ol><li>为了准备分析仪器，请在两个杜瓦液氮瓶和加速电压为300千伏。 </li><li>使用FIB样品制备后，取出从FIB-SEM的侧入架和调整脚位置，使得持有人的长度相匹配的300千伏TEM的。旋转支架到正确的观察位置的提示，再次比较它与标准的TEM持有人，以确保正确的采样方向。 <ol><li>另外，卸下半格与标准TEM持有人的FIB-SEM座和地方附加片状结晶。 </li></ol></li><li>加载样本保持器插入TEM的交换室和排空腔室。确保每当空气被允许进入交换室仪器枪阀关闭。一旦交换室已抽了下来，一个发出报警声。样品架顺时针旋转，让VACU微米拉样品架中的第一停止位置。 </li><li>让仪器真空收回，然后打开阀门枪。重新聚焦和变焦至约1万倍的放大倍数。 </li><li>使用对准旋钮光束转移到荧光屏的中心。收缩和扩张的光束，并确保所有光束移动同心。调整聚光散光是必要的。 </li><li>逆时针旋转样品架，并允许该真空在支架拉完全插入柱。使用台移动旋钮导航和定位的样本。 </li><li>增加样品上放大和调整的Z高度，直到所述样品是在焦点。使用光学目镜是必要的。 </li><li>插入相机，取出荧光屏。展开必要的光束，以避免过饱和相机。调整焦距和摄像机参数，然后开始采集捕获的图像。 </li><li>为了获得一个衍射图样，第一中心感兴趣的功能。除去客观光圈和插入衍射光圈。 </li><li>按控制面板上的按钮，DIFF更改为衍射镜片模式，并使用DIFF控制旋钮缩小光束。 </li><li>插入必要的消隐，以防止衍射图案的中心从燃烧相机或荧光屏。开始采集的相机捕捉到图案的图像。 </li></ol>

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The authors have nothing to disclose.

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，通常配上较长的染色时间10。它保持在温育时间幼虫密度低，在染色期间最小化氧气剥夺和废物积累是重要的。 此前追求FIB / TEM方法，它涉及到本地化感兴趣区域，它是通过生命阶段和使用较低分辨率的方法，如SEM-EDS和SEM-EBSD兴趣组织筛选关键。在反向散射模式观察标本的地方能够对比较轻的关联到重原子量11的相对元素组成的可视化。因此，SEM-BSE图像也提供的，其中，如钙离子和锇染色的脂质球较重元件位于的估计。 SEM-EDS分析允许对样品的钙离子的更具体的映射。然而，它不确认结晶的CaCO 3的存在。 SEM-EBSD允许晶体结构的检测，虽然常规EBSD方法需要一个抛光表面12。这项研究演示了如何在未抛光的样品SEM-EBSD可以提供有用的信息。一个20千伏射束电流使在样品表面分析更加深入。因此，EBSD是检测一个理想的方法生物矿物的存在下，特别是在小的，完整的生物样品，如海洋幼虫。虽然当矿物质不面向检测器在约70℃，看到了菊池图案被认为是在感兴趣13的区域中的矿物相的明确的证据未抛光的样品将给出假阴性。 透射电子显微镜（TEM）允许更宽范围的材料14的结构表征。聚焦的离子束（FIB）允许位点特异性提取和制备与用于TEM分析15典型尺寸的样品。因此，FIB / TEM技术是分析新沉积的生物材料在一个局部区域的合适方法。结合FIB-SEM工具还允许通过使用一个集成的电子束柱的样品无损伤的观察。检体的钨溅射涂覆增加的导电性和限制离子我样品16 mplantation和辐射损伤。细聚焦离子束具有低的停留时间的使用使有机材料铣削。的FIB-SEM内，感兴趣的特定功能可以从散装样品中提取并减薄至电子透明度用于TEM分析17。由于离子束的能量高，晶体材料可能呈现无定形的。这可制备用于TEM分析的薄片层，并且可以使用低加速电压的离子束以除去非晶体层来减轻时发生。

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。

<table><tbody><tr><td>Hexamethyldisilazane&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>16700(EM)</td><td></td></tr><tr><td>Osmium Tetroxide 2% Aqueous Solution</td><td>Electron Microscopy Sciences</td><td>19192</td><td></td></tr><tr><td>IBMX 3-Isobutyl-1-methylxanthine</td><td>ThermoFisher Scientific</td><td>PHZ1124</td><td></td></tr><tr><td>Nigericin, Free Acid</td><td>ThermoFisher Scientific</td><td>N7143-5MG</td><td></td></tr><tr><td>35 mm diameter dish, hole size 27 mm, Glass No.0, Non-coat</td><td>ThermoFisher Scientific</td><td>D110400</td><td></td></tr><tr><td>5-(and-6)-Carboxy SNARF-1, Acetoxymethyl Ester, Acetate</td><td>ThermoFisher Scientific</td><td>C-1271</td><td></td></tr><tr><td>BDH Potassium Chloride, ACS Grade</td><td>VWR</td><td>BDH0258-500G</td><td></td></tr><tr><td>Paraformaldehyde&lt;br/&gt; reagent grade, crystalline</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>1 M Hydrochloric Acid for Volumetric Analysis</td><td>Wako Pure Chemical Industries, Ltd</td><td>083-01095</td><td></td></tr><tr><td>0.05 M Sodium Hydroxide Solution for Volumetric Analysis</td><td>Wako Pure Chemical Industries, Ltd</td><td>199-02185</td><td></td></tr><tr><td>Calcein</td><td>Sigma</td><td>C0875</td><td></td></tr><tr><td>FASW</td><td>Iwaki Co. Ltd.</td><td>Rei-sea Marine</td><td></td></tr><tr><td>Mixed Cellulose Ester Membranes; 47 mm dia, 0.45 &amp;micro;m</td><td>ADVANTEC</td><td>A045A047A</td><td></td></tr><tr><td>ethanol</td><td>Wako Pure Chemical Industries, Ltd</td><td>051-00476</td><td></td></tr><tr><td>Artificial seawater for buffers</td><td></td><td></td><td>by SOP06 of DOE (1994), cdiac.ornl.gov/ftp/cdiac74/sop06.pdf</td></tr><tr><td>Sodium Chloride</td><td>Wako Pure Chemical Industries, Ltd</td><td>191-01665</td><td></td></tr><tr><td>Potassium Chloride</td><td>Wako Pure Chemical Industries, Ltd</td><td>163-03545</td><td></td></tr><tr><td>Magnesium Chloride Hexahydrate</td><td>Wako Pure Chemical Industries, Ltd</td><td>135-00165</td><td></td></tr><tr><td>Calcium Chloride</td><td>Wako Pure Chemical Industries, Ltd</td><td>039-00475</td><td></td></tr><tr><td>Sodium Sulfate</td><td>Wako Pure Chemical Industries, Ltd</td><td>197-03345</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Wako Pure Chemical Industries, Ltd</td><td>089-08415</td><td></td></tr><tr><td>2-amino-2-hydroxymethyl-1,3-propanediol (tris)</td><td>Wako Pure Chemical Industries, Ltd</td><td>207-06275</td><td></td></tr><tr><td>2-aminopyridine</td><td>Wako Pure Chemical Industries, Ltd</td><td>011-02775</td><td></td></tr><tr><td>Orion 5-star Plus pH meter</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>PrpHecT ROSS Micro Combination pH Electrode 8220BNWP</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Axiovision, Version 4.6, Axio Observer Z1</td><td>Zeiss</td><td></td><td></td></tr><tr><td>ImageJ</td><td>NIH, Bethesda, MD, USA</td><td></td><td></td></tr><tr><td>HRTEM H500</td><td>Hitachi</td><td></td><td></td></tr><tr><td>SU6600 VPSEM</td><td>Hitachi</td><td></td><td></td></tr><tr><td>NB5000 Focused Ion and Electron Beam (FIB-SEM) system</td><td>Hitachi&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

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，以了解负责矿化组织一个tubeworm。 图3显示了较高的pH值与来自钙黄绿素染色和SEM-EDX测绘较高钙离子信号相关。从这些观察，从感兴趣的生活阶段的组织在一喜了检查采用更加本地化的技术，SEM-EBSD gher分辨率。在矿物相的发现， 图4示出了聚焦离子束的应用解除出来的用于TEM兴趣和选定的区域的衍射分析中的材料，以确认矿物的结晶。 <img alt="图1" src="/files/ftp_upload/55164/55164fig1.jpg" /> 图1:tubeworm的细胞内pH映射（由Chan 等人 2015年）。在IBMX治疗后71小时，一tubeworm， 盘管虫线虫 ，它代表变态的慢的速率表明在细胞内pH分布的极大异质性。 DIC图像（左上）和pseudocolorized在640nm在580nm处通过灰度值除以从灰度值生成的合成图像（右上）被示出。从五百八十零分之六百四十〇纳米比所计算的灰度值成比例的细胞内pH值。该PROFI从合成图像的灰度值的莱斯，分别通过沿所述tubeworm（底部面板）的纵向主体轴线的距离换算成细胞内pH值。在发射细胞内pH和五百八十零分之六百四十〇纳米比之间的关系是由在体外校准建立（Y = 0.30x - 1.47; R 2 = 0.934; Y，灰度值; X，细胞内pH值）。与细胞内pH高于pH 8.5（红色）区域相应于被发现钙化结构的区域。比例尺= 100微米; C，领; B，鳃瓣​​;蚂蚁，anteior;后，后路。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55164/55164fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55164/55164fig2.jpg" /> 图2: 确定使用SEM-EDS感兴趣的生命阶段。 在不同的管虫变态的时间点进行了筛选捕捉到新的钙化阶段作进一步的分析（一）每日1后变质tubeworm显示钙信号的均匀分布（ii）在快速增长后第2天变质tubeworm显示钙信号的短环（三）的速度成长后第2天变质tubeworm显示钙信号的斑点。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55164/55164fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55164/55164fig3.jpg" /> 图3: 用活显微镜（ 从 Chan 等人 2015年）钙信号的表征。 （I-IV），SEM-EDS（V-VII）和SEM-EBSD（VIII-X）。钙离子截至31 H和盘管虫线虫的变态后53ħ相关的钙黄绿素信号的分布。 DIC的图像（左面板，3i和III）和T他的信号钙黄绿素（左图，2II和iv）所示。在第2天后变质盘管虫线虫相关电子显微镜检测钙化结构斑点，如图较低电压（5千伏）SEM图像（中间面板，3V）。 SEM-EDS分析的映射，在20kV进行，显示了钙的不均匀分布（中间面板，3vi，CA;在黄色）。 （分析摩尔重量％，小数点对齐，以显示点的位置）从点量化得到的钙含量表示为号码重叠较低电压（5千伏）的更大的表面细节的tubeworm的图像，钙含量（用SEM-EDS在20kV）（中间面板; 3vii）。与Ca含量大于15摩尔％（重量）的区域很可能是钙化的结构。钙丰富的地区的SEM-EBSD分析2viii-x的右侧面板illuastrated。在SEM-EDS结果发现，钙丰富的地区（白盒）与电子背散射衍射进一步分析（右图; 3viii）（二）圆（右图;3ix）表示EBSD分析部位;一个菊池图案（右图; 2倍）表明该网站是从EDS / EBSD微观分析软件的界面文石。 C，领; B，鳃瓣​​;蚂蚁，前;后，后路。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55164/55164fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55164/55164fig4.jpg" /> 图4:使用FIB-TEM晶体结构的表征（从Chan 等人 2015年）。 （ⅰ）表现出从EBSD一个菊池图案的区域被切除，以制备TEM样品，（ii）由聚焦离子束（FIB）技术（顶视图）切下除去周围材料，（ⅲ）的材料，使用一提升钨探针（侧视图），（iv）与〜200纳米，这是在一个较低的电压而获得的最终厚度的样品。 （V）中分别为圆圈区域分析所选区域衍射图案中的TEM（ⅵ）的存在下，呈现出文石单晶图案。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55164/55164fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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 ...

characterization-calcification-events-using-live-optical-electron

Nanyang Technological University

Research

JoVE Journal

Biology

7.4K 次观看.  Clemson University. 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. 

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

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps&#160;and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter&#160;scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation,&#160;(2) superior optical sectioning ability,&#160;and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.

An integrated suite of imaging techniques has been applied to determine polyp morphology and tissue structure in the Caribbean corals Montastraeaannularis and M. faveolata. Fluorescence, serial block face,&#160;and two-photon confocal laser scanning microscopy have identified lobate structure, polyp walls, and estimated chromatophore and zooxanthellae densities and distributions.

多模态光学显微镜方法在加勒比海礁珊瑚显示息肉组织形态和结构

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues ...

科研

环境科学

Microfocus X-ray CT (microCT) Imaging of Actinia equina (Cnidaria), Harmothoe sp. (Annelida), and Xenoturbella japonica (Xenacoelomorpha)

Traditionally, biologists have had to rely on destructive methods such as sectioning in order to investigate the internal structures of opaque organisms. Non-destructive microfocus X-ray computed tomography (microCT) imaging has become a powerful and emerging protocol in biology, due to technological advancements in sample staining methods and innovations in microCT hardware, processing computers, and data analysis software. However, this protocol is not commonly used, as it is in the medical and industrial fields. One of the reasons for this limited use is the lack of a simple and comprehensible manual that covers all of the necessary steps: sample collection, fixation, staining, mounting, scanning, and data analyses. Another reason is the vast diversity of metazoans, particularly marine invertebrates. Because of marine invertebrates&#8217; diverse sizes, morphologies, and physiologies, it is crucial to adjust experimental conditions and hardware configurations at each step, depending on the sample. Here, microCT imaging methods are explained in detail using three phylogenetically diverse marine invertebrates: Actinia equina (Anthozoa, Cnidaria), Harmothoe sp. (Polychaeta, Annelida), and Xenoturbella japonica (Xenoturbellida, Xenacoelomorpha). Suggestions on performing microCT imaging on various animals are also provided.

Microfocus X-ray CT microCT Imaging of Actinia equina Cnidaria, Harmothoe sp. Annelida, and Xenoturbella japonica Xenacoelomorpha

Here, protocols for performing microfocus X-ray computed tomography (microCT) imaging of three marine invertebrate animals are explained in detail. This study describes steps such as sample fixation, staining, mounting, scanning, image reconstruction, and data analyses. Suggestions on how the protocol can be adjusted for different samples are also provided.

微聚焦 X 射线 CT (微CT) 阿克蒂尼亚 equina (Cnidaria),哈莫特霍sp. (安妮利达) 和Xenoturbella japonica (西纳科洛莫卡) 成像

Traditionally, biologists have had to rely on destructive methods such as sectioning in order to investigate the internal structures of opaque organisms. ...

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

生物化学

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. Animals cope with these stimuli by modifying their behavior. Understanding the mechanisms underlying such behavioral modification requires quantification of neural activity during the behavior of interest. Here, we report a method for calcium imaging in freely behaving Caenorhabditis elegans with nonlocalized vibration of specific frequency, displacement, and duration. This method allows the production of well-controlled, nonlocalized vibration using an acoustic transducer and quantification of evoked calcium responses at single-cell resolution. As a proof of principle, the calcium response of a single interneuron, AVA, during the escape response of C. elegans to vibration is demonstrated. This system will facilitate understanding of neural mechanisms underlying behavioral responses to mechanical stimuli.

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Reported here is a system for calcium imaging in freely behaving Caenorhabditis elegans with well-controlled, nonlocalized vibration. This system allows researchers to evoke nonlocalized vibrations with well-controlled properties at nano-scale displacement and to quantify calcium currents during responses of C. elegans to the vibrations.

自由行为 秀丽隐杆线虫 的钙成像，具有良好控制，非局部化振动

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. ...

行为学

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence of calcification inhibitors, that concomitantly lead to a loss of vessel elasticity and function. Vascular calcification is an important contributor to morbidity and mortality in many pathologies, including chronic kidney disease, diabetes mellitus, and atherosclerosis. Current research models to study vascular calcification are limited and are only viable at the late stages of calcification development in vivo. In vitro tools for studying vascular calcification use end-point measurements, increasing the demands on biological material and risking the introduction of variability to research studies. We demonstrate the application of a novel fluorescently labeled probe that binds to in vitro calcification development on human vascular smooth muscle cells and determines the real-time development of in vitro calcification. In this protocol, we describe the application of our newly developed calcification assay, a novel tool in disease modeling that has potential translational applications. We envisage this assay to be relevant in a broader spectrum of mineral deposition research, including applications in bone, cartilage, or dental research.

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Cardiovascular disease is the leading cause of death worldwide. Vascular calcification contributes substantially to the burden of cardiovascular morbidity and mortality. This protocol describes a simple method to quantify vascular smooth muscle cell-mediated calcium precipitation in vitro by fluorescent imaging.

一种半自动且可重现的基于生物学的体外定量钙沉积的方法

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

琼脂糖微室的长期钙成像<em&gt;秀丽隐杆线虫</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

神经科学

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

秀丽线虫纵向成像的微流控平台

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

生物工程

Tracking Calcium in the Early Developmental Phases of C. elegans

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode Caenorhabditis elegans provides several unique advantages for the study of the OET as it is transparent and has an ordered gonad that produces one mature oocyte every ~23 min at 20 &#176;C. We have modified the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization within a living organism. We have termed this reporter &#34;CaFE&#34; for Calcium during Fertilization in C. elegans. The CaFE reporter was&#160;engineered into a safe harbor locus in single copy, has no significant impact on physiology or fecundity, and produces a robust signal upon fertilization. Here, a series of protocols is presented for utilizing the CaFE reporter as an in vivo tool for dissecting the OET and embryogenesis. We include methods to synchronize worms, examine the effects of RNAi knockdown, mount worms for imaging, and to visualize calcium in oocytes and embryos. Additionally, we present the generation of additional worm strains to aid in this type of analysis. Demonstrating the utility of the CaFE reporter to visualize the timing of fertilization, we report that double ovulation occurs when ipp-5 is targeted by RNAi and that only the first oocyte undergoes immediate fertilization. Furthermore, the discovery of single-cell calcium transients during early embryogenesis is reported here, demonstrating that the CaFE reporter persists into early development. Importantly, the CaFE reporter in worms is simple enough to use for incorporation into course-based undergraduate research (CURE) laboratory classes. The CaFE reporter, coupled with the ordered gonad and ease of RNAi in worms, facilitates inquiry into the cell-cell dynamics required to regulate internal fertilization and early embryogenesis.

Here, we present protocols and tools for visualizing calcium during fertilization and early embryogenesis using a genetically encoded calcium reporter expressed in the germline of the model nematode C. elegans.

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode ...

发育生物学

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

体内神经元钙处理的亚细胞成像

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf can measure PSII absorption cross-section (&#963;PSII), maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq&#8242;/Fm&#8242;), and non-photochemical quenching (NPQNSV) for various eukaryotic algae and cyanobacteria, almost all FRRf studies to date have focused on phytoplankton. Here, the protocol describes how to measure PSII photophysiology of an epizoic alga Colacium sp.&#160;Ehrenberg 1834 (Euglenophyta), in its attached stage (attached to zooplankton), using cuvette-type FRRf. First, we estimated the effects of substrate zooplankton (Scapholeberis mucronata O.F. M&#252;ller 1776, Cladocera, Daphniidae) on baseline fluorescence and &#963;PSII, Fv/Fm, Fq&#8242;/Fm&#8242;, and NPQNSV of planktonic Colacium sp. To validate this methodology, we recorded photophysiology measurements of attached Colacium sp. on S. mucronata and compared these results with its planktonic stage. Representative results showed how the protocol could determine the effects of calcium (Ca) and manganese (Mn) on Colacium sp. photophysiology and identify the various effects of Mn enrichment between attached and planktonic stages. Finally, we discuss the adaptability of this protocol to other periphytic algae.

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II photophysiology and primary productivity. Here we describe a protocol to measure PSII photophysiology of epizoic alga, Colacium sp. on substrate zooplankton using cuvette-type FRRf.

使用比色皿型快速重复率荧光计测量 Colacium sp.附着阶段的光生理学

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf ...

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

摘要

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多模态光学显微镜方法在加勒比海礁珊瑚显示息肉组织形态和结构

微聚焦 X 射线 CT (微CT) 阿克蒂尼亚 equina (Cnidaria),哈莫特霍sp. (安妮利达) 和Xenoturbella japonica (西纳科洛莫卡) 成像

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

自由行为秀丽隐杆线虫的钙成像，具有良好控制，非局部化振动

一种半自动且可重现的基于生物学的体外定量钙沉积的方法

琼脂糖微室的长期钙成像

秀丽线虫纵向成像的微流控平台

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

体内神经元钙处理的亚细胞成像

使用比色皿型快速重复率荧光计测量 Colacium sp.附着阶段的光生理学

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

摘要

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多模态光学显微镜方法在加勒比海礁珊瑚显示息肉组织形态和结构

微聚焦 X 射线 CT (微CT) 阿克蒂尼亚 equina (Cnidaria),哈莫特霍sp. (安妮利达) 和Xenoturbella japonica (西纳科洛莫卡) 成像

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

自由行为 秀丽隐杆线虫 的钙成像，具有良好控制，非局部化振动

一种半自动且可重现的基于生物学的体外定量钙沉积的方法

琼脂糖微室的长期钙成像

秀丽线虫纵向成像的微流控平台

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

体内神经元钙处理的亚细胞成像

使用比色皿型快速重复率荧光计测量 Colacium sp.附着阶段的光生理学

自由行为秀丽隐杆线虫的钙成像，具有良好控制，非局部化振动