We thank Donna Epps, histologist at Institute for Genomic Biology, University of Illinois Urbana-Champaign (UIUC), for her capable technical assistance in sample preparation and sectioning. This work was supported by a research grant to B.W. Fouke from the Office of Naval Research (N00014-00-1-0609). In addition, C.A.H. Miller received grants from the UIUC Department of Geology Wanless Fellowship, UIUC Department of Geology Leighton fund and UIUC Department of Geology Roscoe Jackson fieldwork fund. Interpretations presented in this manuscript are those of the authors and may not necessarily represent those of the granting institutions. We also thank the Caribbean Research and Management of Biodiversity (Carmabi) laboratory on Cura&#231;ao for their support and collaboration in collecting the coral tissue biopsy samples. We thank Claudia Lutz, IGB Media Communication Specialist for her able language correction.

被珊瑚样品的连续块面成像试剂准备:注意 1 Preinfiltration蜡<ol><li>熔融3.6克硬脂薄片在玻璃烧杯中。在热板（60-70℃）拌匀。 </li><li>添加400毫克苏丹红四号的（最小化蜡背景荧光）。搅拌均匀，等到红色半透明的解决方案来实现的。 </li><li>新增96毫升热熔融石蜡（100％），搅拌均匀。 </li></ol> 1.2）嵌入蜡<ol><li>熔体7.2克硬脂精片的玻璃烧杯中搅拌均匀的热板（60-70℃）。 </li><li>添加0.8克苏丹四。搅拌均匀，等到红色半透明的解决方案来实现的。 </li><li>添加石蜡粒（161克），搅拌至融化石蜡完全。 </li><li>加入30克白色粒状VYBAR并在相同的烧杯中完全熔化;一旦融化，混匀。 </li><li>松散关闭有盖的玻璃瓶。将玻璃瓶在60℃对流OV烯以保持各组分在液体状态。开展这一炉的所有渗透。 </li><li>分裂200毫升红蜡的总体积中，以两片玻璃瓶每次100毫升。使用一个等分试样进行渗透，另一个用于最终嵌入。 </li></ol> 1.3）嵌入珊瑚组织的串行块面成像<ol><li> （在4-5℃，在多聚甲醛3-6个月）在磷酸盐缓冲盐水（3×5分钟）洗涤收集在字段（SI视频1），并存储在珊瑚虫和脱钙当准备进行成像。除垢在ExCal解决方案息肉24小时，或者直到为息肉是完全没有的CaCO 3。培育若干脱钙珊瑚虫为单块的25，50，75，和100％的乙醇系列，其次是1X二甲苯替代品脱水的样品。 </li><li>将处理过的息肉含100％二甲苯代替预热65℃的烘箱。樟宜孵育30分钟两次ng到新鲜的解决方案。定向成这样的息肉的息肉的顶端朝下，顶表面是尽可能平坦。 </li><li>使2解决方案:1，1:1和1:2二甲苯替代品和preinfiltration蜡（步骤1.1）在50毫升猎鹰管。 </li><li>孵育珊瑚虫与这三个浓度增加preinfiltration蜡中，随后3次温育在100％preinfiltration蜡，每次30-60分钟。 </li><li>注意:根据样品的厚度，步骤1.3.1-1.3.5可以随时间较长。 </li><li>将样品包埋蜡（见步骤1.2.6）在100％preinfiltration蜡3×孵育后。 </li><li> 30分钟后，取出嵌入蜡。更换新鲜嵌入蜡，继续培养至少4小时在65℃。 </li></ol> 1.4）在红蜡嵌入<ol><li>拍摄所述块（不锈钢托盘，其中的白色蜡放置并在其顶部的SAMPLE放置）。这是必要的，因为一旦嵌在蜡中，样品的位置将变得不可见的嵌入红蜡是不透明的。 </li><li>放置小滴的高熔点蜡围绕新的预热的不锈钢包埋模具并使其冷却。如步骤1.2.6声明倒入少量从第二个容器新鲜融化的石蜡包埋的。 </li><li>位置朝下在白蜡点很快的珊瑚虫，然后放置一个塑料的样品架的不锈钢托盘上，倒更嵌入蜡，使得蜡过来对塑料模具的表面上。 </li><li>以整个安装了65℃烘箱中，并允许它在长凳或阴凉表面冷却，直到蜡完全变硬。 </li><li>这可能需要6小时，最多两天。放置在4℃下干燥处理的冰箱挡，避光，长期储存。 </li></ol> 1.5）切片的串行块面设置<ol><li> TR即时通讯块切成1微米的部分用切片机。不收的部分，因为它们会变成粉末。请记住，我们只是成像的块面。样品在白色蜡开始消失出现。 </li><li>捕捉到光滑的块面，其中包含的样本部分被删除，每次的图像。继续下去，直到珊瑚虫消失在SI视频2。 </li><li>用FITC标记的荧光滤镜拿起自动荧光色素细胞/珊瑚虫的录制/撷取用黑白摄像机的图像。图片来自每个品种3-4脱钙核心。 </li></ol> 2，成像珊瑚在双光子荧光显微镜<ol><li>修复珊瑚虫核心，以收集（海边）网站每个包含大约10-12息肉直径约一英寸，在4％多聚甲醛，只要它们在水中收获。 </li><li>保持样品在4°C，直到成像。洗涤芯被放置在第ê同一个解决方案中的玻璃盖底部盘倒挂（0.17毫米厚）。 </li><li>利用双光子激光在使用10X（0.3 NA）的物镜780 nm激发，像3-4息肉两种不同的放大倍率（数码变焦）。珊瑚息肉形状和高度的样品（通常为1-2毫米），该成像深度之间的差异也只限于通过成像物镜的工作距离。 </li><li>使用瓦片扫描模式收集约25-100（5×5或10×10瓦）到z轴中的xy每焦平面图像和50-100的图像10或20微米的间隔，总数约为5,000-10,000的图像/珊瑚虫。 </li><li>图片三时五十七息肉领域，代表了核心和珊瑚的物种。注:2-5小时/息肉区域之间的图像采集时间不同。 </li><li>存储原始数据格式的所有图像在系统的硬盘作为LSM 5渲染3D在3D图像的分析和渲染软件。 </li></ol> 3，三维体绘制与可视化Ñ​​SBFI和双光子荧光光谱数据<ol><li>裁剪二维数据通过专注于一个单一的息肉用在程序中广场裁剪工具，以减少文件大小和编译成一个单一的TIFF文件（减小文件的大小也通过保存在8位格式的文件）的采集软件。 </li><li>在程序中了Imaris超越模块下的体积算法打开SBFI数据的汇编文件（收集步骤1.5.1）或平铺多个z栈的双光​​子光学部分（收集步骤2.1.4）。 </li><li>项目中使用了一层阴影投射在3D渲染的SBFI数据。创造一个体素是阈值化创造了坚实的表面图案（SI视频3）的等值面模式。 </li><li>使用可视化的剪取平面算法在XY，XZ的3D投影和YZ正交模式，揭示珊瑚三维结构和形状。 </li><li>在相同了Imaris程序使用关键帧动画模块的动画投影（SI六DEOS 3-7）。 </li><li>生成的视频文件，在5％的压缩和使用量生成AVI格式的影片剪辑（SI影片4和6），三维等值面和方式（SI视频5和7）。 </li></ol>

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The authors declare no conflict of interests.

珊瑚礁研究是一个高度跨学科的研究工作，包括分析，同时物理，化学和生物现象工作在海洋环境。复杂的珊瑚礁生态系统的研究中上下文框架（ 图10）中的"十国"，因此最好的完成。此图编制说明，珊瑚生态系统涵盖了广泛的空间尺寸（10-9至10 5米）。此外，这次演习表明地球生物学在中间长度比例为1毫米1厘米分析是结合现场和实验室分析所需的桥梁。十大框架这是权力的背景下进行的实验，分析，建模和综合的物理，化学，以及控制库拉索岛珊瑚礁生态系统的生物参数。 本研究是首次应用完全集成套件Ø˚F调频，SBFI和激光共聚焦显微镜技术来跟踪表面健康的珊瑚，以增加水深的响应。可将图像的全部组织或厚的生物样品光学显微镜工具已经因各种各样的由样品本身，它包括吸收，散射，以及其他现象所造成的光学条件的限制。在刚刚过去的十年中，SBFI扫描电子显微镜使用几种生物样品的基于宽视场成像SBFI用于超高分辨率31以及光显微成像。这包括了从脑到整个心脏组织甚至含有骨骼进行研磨，抛光，标记的样品的分析，并成像在之后的每个部分被制成23-29在同一时间。最近，荧光和共聚焦显微镜用于揭示的轮廓，形状，以及蛋白质和颜料分配生物样品32-33中。然而，个别的珊瑚虫公顷的三维结构ð以前没有解决。珊瑚样品SBFI前提是脱钙，如碳酸钙，必须使服从渗透的样本，并利用常规切片机切片之前被删除。 这是其中的双光子荧光显微镜变为必不可少的生物组织的分析，由于其独特的双光子激发的现象。这导致了珊瑚组织在本研究中，其中脱钙步骤被有效地消除了扩展深度渗透33-34由于非线性双光子激发的现象进行34。此外，用双光子激光的预补偿模块提供更短的脉冲宽度，其还改善了渗透深度。在样品上侧，由于碳酸钙不会在780nm处吸收的近红外光，穿透深度是由物镜的工作距离通常限制。还有一个缺点Ø˚F脱钙和去除珊瑚骨架是的珊瑚组织，这使组织体积的估计更为困难后的样品已经通过多个脱水和再水合步骤消失，在不含水的蜡包埋的结构完整性的丧失。这强调前和处理后确定的珊瑚组织体积的重要性。 珊瑚荧光蛋白的共焦特性已结束对大堡礁珊瑚和保护的光谱特性已经被关联以珊瑚栖息地32的深度。然而，在本研究中，我们表现出的第一次使用双光子显微镜，有一个显着减少色素细胞的无处不在深水珊瑚除了在口腔组织。此外，不同的变化，虫黄相分布的色素细胞已穿过深度断面观察到M。 annularis，wherE中的浅水珊瑚组织被完全覆盖色素细胞。与由高倍率成像（ 图8C和图8D）中提供的分辨率，现在我们可以精确地量化由共生藻所占用的面积和体积，以及它们的组织密度比可以相对于其它细胞组分来确定。两者合计，SBFI的整合和双光子荧光光谱成像在本研究中已经取得了非常重要的新的见解的珊瑚组织的形态和结构。这个数据将帮助量化珊瑚组织的各个部件，无论是在单个息肉水平或在殖民息肉之间相互作用对整个珊瑚头的上下文级别。这种情况下，现在将允许珊瑚holobiont对海平面变化的水深和光照的适应性反应进行量化跟踪和预测。

全球变暖和相应的环境变化都直接影响热带海洋珊瑚1-4的健康和分布。多重影响正在被观察到，包括珊瑚白化和传染病5-6的出现。然而，未来的珊瑚应对这些环境威胁更准确的预测将需要进行组织学"基线"成立，它定义了组织形态和细胞的组成和分布"看起来健康"的珊瑚。反过来，"影响"珊瑚然后可以定量比较。此外，该基线应建立在外表健康的珊瑚在各种环境条件下，使"健康反应"也可以跨越环境梯度来衡量。由于对建立这一基础的第一步，高分辨率3D已开展研究如何看起来健康的珊瑚虫组织形态和细胞成分响应增加水深（WD）和随附的跌幅在阳光辐照度。结果可以用来确定珊瑚适应更全面的机械理解，以及深入了解珊瑚共生演进和捕光增强。 石珊瑚（ 石珊瑚 ）是殖民地的海洋无脊椎动物中发挥主机到复杂的组合等微生物，统称为珊瑚holobiont 7-10。在本研究中所进行的研究旨在利用一套先进的成像技术，同时跟踪随水深组织颜料和看起来健康的宿主珊瑚共生的虫黄藻的变化。这将建立所需要的比较组织细胞的"基线"跨越水深梯度看起来健康的珊瑚，并作为珊瑚老天指标LTH 10。珊瑚色素，称为色素细胞 ，起到吸收，反射，散射，折射，衍射，或与入射太阳辐射11以其他方式干扰。虫黄藻，色素细胞内共生关系，使中高屋建瓴捕光优化和骨骼成长战略的协同进化，以及营养的可塑性（移喂养策略回来的往复从自养到异养）的珊瑚动物12。 库拉索岛南部的加勒比海岛国（荷属安的列斯以前部分）位于委内瑞拉以北约为65公里，东西走向的阿鲁巴岛香格里拉Blanquilla群岛（ 图1A）内。库拉索岛的70公里长的南海岸包含一个连续的现代化和中新世-上新世-更新世-全新世古岸珊瑚礁道13,14。年平均海温的变化库拉索岛约3℃的nually，从一月下旬最低26℃，最高29℃，在九月初，有27.5±0.5ºC的年平均温度（NOAA海温资料集2000-2010）。珊瑚礁的Playa Kalki（12°22&#39;31.63"N，69°09&#39;29.62"W），躺在库拉索（ 图1A）的西北尖端，被选为抽样，因为它之前已经充分研究和在这个位置上的海洋生态系统正沐浴在清新无污染海水中7,15-19。 。两个密切相关的石珊瑚物种，M. annularis和M faveolata，被选择用于实验和分析在本研究中，因为每个物种:（1）相对于所述货架断和上礁道呈现明显不同的和不重叠的等深线分布相关的碳酸盐沉积的沉积环境（M. annularis范围= 0-10米的WD; 米faveolata范围= 10-20米WD 20; 图1B，2A和2B）; （2）在整个加勒比海21一 ​​个共同的珊瑚礁框架构建;和（3）具有充分研究的生态，生理和进化关系22。 现场取样本研究采用离岸滩Kalki库拉索岛的标准水肺潜水技术进行的。浅到深的水中测深断面成立跨架子跑了，过了保质突破，并进入深水区前礁环境。显然，健康的珊瑚头，然后确定了采样沿着这条测深断面，其中包括:（1）三个人约1米直径的珊瑚M的头annularis，所有这些都是以5m水深度（WD）; （2）三个独立的约1米直径的珊瑚M的头所有这些faveolata，是在12米WD。光合有效辐射（PAR）的测定为33-36％的PAR为5m的WD和18-22％的PAR以10米的WD。采样是在1月​​进行，当海温为26℃时的水深均5米和12米每六珊瑚头进行采样，一式三份，在同等空间位置（ 即约45°北纬每六个半球状珊瑚头）。每个单独的样本包括收集与清洗拱冲，一个直径为2.5厘米的珊瑚组织构架核心活检。三珊瑚组织，骨骼活检取样标准潜水用戴手套的手从每个珊瑚头（从9 米annularis为5m的WD殖民地，从9 米faveolata在12米WD）。后立即收集在深度，每个活检核心样品置于无菌50毫升聚丙烯离心管中，螺旋盖密封，并返回到表面。海水从每个离心管中倾出，每个核心活检后，浸渍，储存，并在4％多聚甲醛输送。 23-30的进行。这些研究大多利用光/光镜带任何荧光或明场技术。然而，研究已经在利用扫描电子串行块面成像，在过去的31超高放大倍率进行。在本研究中，修饰SBFI协议已被开发用于与施加到珊瑚首次。由于M。 annularis和M。 faveolata珊瑚虫是1-2毫米的厚度，没有任何的常规光显微技术将能够穿透珊瑚息肉组织的整个厚度。因此，我们有专门为珊瑚样本设计SBFI样品制备协议。此外，我们的定制设计了立体支架，该机动化地在X和Y两个方向上移动。该装置采用的样本的块面的图像，而不是使用常规的切片在显微镜的前收集的部分。我们还介绍了另一种非线性光学双光子显微技术将图像的同一珊瑚虫整个珊瑚组织的整个厚度。这克服了由SBFI在脱钙的术语，并且可以由样品制备（脱水）和处理协议来诱导组织的形态和容积（收缩）的变化的可能性，所施加的限制。此外，从珊瑚排放剖面，光谱分辨识别色素细胞和虫黄藻光合作用的高峰排放量和变化。这些结果在使用该方法的情况下和其有关的采集时间个别的优点，分析时间，并解决精细结构细节不损害str中的能力进行了评价珊瑚组织uctural完整性。

<table><tbody><tr><td>Coral Tissue Skeleton</td><td></td><td></td><td>2.5 cm Biopsy from natural habitat</td></tr><tr><td>Arch Punch Coring Device</td><td>C.S. Osborne and Company</td><td>No. 149</td><td>For Coral biopsy collection</td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>RT 15700</td><td>16% Pre-diluted</td></tr><tr><td>Histoclear/Safeclear II</td><td>Electron Microscopy Sciences</td><td>RT 64111-04</td><td>Non-Toxic alternate to Xylene, Dehydration and Deparafinization</td></tr><tr><td>Xylene and Ethanol</td><td>Fisher Scientific</td><td></td><td>Dehydration</td></tr><tr><td>Paraffin Wax</td><td>Richard Allen Scientific</td><td>Type H REF 8338</td><td>Infiltration solution</td></tr><tr><td>Vybar</td><td>The Candle Maker</td><td></td><td>Component of Red Wax</td></tr><tr><td>Stearin</td><td>The Candle Maker</td><td></td><td>Component of Red Wax</td></tr><tr><td>Sudan IV</td><td>Fisher Chemical</td><td>S667-25</td><td>Red Wax-Opaque background</td></tr><tr><td>Wheat Germ Agglutinin (WGA)</td><td>Life Technologies</td><td>W32466</td><td>For labeling&amp;nbsp; Coral Mucus</td></tr><tr><td>Prolong Gold</td><td>Life Technologies</td><td>P36095</td><td>Anti-fade mounting media</td></tr><tr><td>Fluoro Dish</td><td>World Precision Instruments</td><td>FD-35-100</td><td>For two-photon imaging</td></tr><tr><td>XY Motor, Driver and Controller</td><td>Lin Engineering</td><td>211-13-01R0, R325, R256-RO</td><td>XY Translational Movement</td></tr><tr><td>Hot Plate</td><td>Corning</td><td>DC-220</td><td>Melting all wax</td></tr><tr><td>Convection Oven</td><td>Yamato</td><td>DX-600</td><td>Infiltration and Embedding</td></tr><tr><td>Tissue Processor</td><td>Leica</td><td>ASP 300</td><td>Dehydration, Infiltration</td></tr><tr><td>Microtome</td><td>Leica</td><td>RM2055</td><td>Disposable knifes</td></tr><tr><td>Stereo Microscope</td><td>Carl Zeiss</td><td>Stereolumar V 12</td><td>1.5x (30 mm WD) Objective</td></tr><tr><td>Fluorescence Microscope with ApoTome</td><td>Carl Zeiss</td><td>Axiovert M 200, ApoTome I System</td><td>Imaging thin section of a polyp: Zooxanthellae</td></tr><tr><td>Axiocam camera</td><td>Carl Zeiss</td><td>MRm</td><td>Monochrome camera 1388x1040 pixels</td></tr><tr><td>Axiovision Software</td><td>Carl Zeiss</td><td>Version 4.8</td><td>Image acquisition program</td></tr><tr><td>Two-Photon Laser</td><td>Spectraphysics</td><td>Maitai eHP, pulsed laser (70 fs)</td><td>With DeepSee module</td></tr><tr><td>Laser Scanning Microscope</td><td>Carl Zeiss</td><td>LSM 710 with Spectral Detector</td><td>34 channel PMT detection</td></tr><tr><td>Zen Software</td><td>Carl Zeiss</td><td>2010 or above</td><td>for two-photon and spectral image acquisition</td></tr><tr><td>Imaris Suite Software</td><td>Bitplane, Inc.,</td><td>Version 7.0 or above</td><td>3D Volume, Iso-surface Rendering, Visualization</td></tr></tbody></table>

multimodal optical microscopy methods reveal polyp tissue morphology and structure in caribbean reef building corals

一个集成的成像技术套件已经被应用来确定三维（3D）的形态学和细胞息肉组织结构，包括加勒比礁珊瑚Montastraea annularis和M. faveolata。这些方法包括荧光显微技术（FM），串行块面成像（SBFI），和双光子激光扫描共聚焦显微镜（TPLSM）。 SBFI提供物理切片后深层组织成像;它详细说明了组织表面纹理和三维可视化到组织的深度超过2毫米。组织细胞结构的互补性调频和TPLSM产量的超高分辨率图像。结果:（1）确定的个别珊瑚虫的外壁和先前未报告叶形组织的形态（2）创建的色素细胞和藻类状藻虫黄内共生体的三维分布和组织密度的第一表面上的地图。光谱吸收豌豆500 nm和675 nm的KS，分别提示M. annularis和M。 faveolata包含相似类型的叶绿素和色素细胞的。然而，M annularis和M。 faveolata呈现的组织密度，对这些关键细胞成分的三维分布显著差异。本研究着重于影像学方法表明SBFI是对脱钙珊瑚组织大型毫米级样品的分析非常有用。免费FM和TPLSM揭示nondecalcified珊瑚组织样本中的细胞分布和密度微妙的亚毫米尺度的变化。该TPLSM技术能提供:（1）微创样品制备，（2）优良的光学切片的能力，以及（3）最小的光的吸收和散射，同时仍允许深部组织成像。

Watch this Scientific Journal Video about Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals at JoVE.com

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

一个集成的成像技术套件已经被应用于确定息肉形态学和组织结构在加勒比海珊瑚Montastraea annularis和M. faveolata。荧光，串行块面，和双光子激光扫描共聚焦显微术已经确定叶形结构，息肉墙壁，和估计的色素细胞和虫黄藻密度和分布。

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 ...

multimodal-optical-microscopy-methods-reveal-polyp-tissue-morphology

Nanyang Technological University

Research

JoVE Journal

Environment

12.2K 次观看.  University of Illinois at Urbana-Champaign. 一个集成的成像技术套件已经被应用于确定息肉形态学和组织结构在加勒比海珊瑚Montastraea annularis和M. faveolata。荧光，串行块面，和双光子激光扫描共聚焦显微术已经确定叶形结构，息肉墙壁，和估计的色素细胞和虫黄藻密度和分布。 

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

A Field Primer for Monitoring Benthic Ecosystems Using Structure-From-Motion Photogrammetry

Structure-from-motion (SfM) photogrammetry is a technique used to generate three-dimensional (3D) reconstructions from a sequence of two-dimensional (2D) images. SfM methods are becoming increasingly popular as a noninvasive way to monitor many systems, including anthropogenic and natural landscapes, geologic structures, and both terrestrial and aquatic ecosystems. Here, a detailed protocol is provided for collecting SfM imagery to generate 3D models of benthic habitats. Additionally, the cost, time efficiency, and output quality of employing a Digital Single Lens Reflex (DSLR) camera versus a less expensive action camera have been compared. A tradeoff between computational time and resolution was observed, with the DSLR camera producing models with more than twice the resolution, but taking approximately 1.4-times longer to produce than the action camera. This primer aims to provide a thorough description of the steps necessary to collect SfM data in benthic habitats for those who are unfamiliar with the technique as well as for those already using similar methods.

We provide a detailed protocol for conducting underwater structure-from-motion photogrammetry surveys to generate 3D models and orthomosaics.

利用运动结构摄影测量法监测底栖生态系统的野外入门书

Structure-from-motion (SfM) photogrammetry is a technique used to generate three-dimensional (3D) reconstructions from a sequence of two-dimensional (2D) ...

科研

环境科学

Coral Reef Arks: An In Situ Mesocosm and Toolkit for Assembling Reef Communities

Coral reefs thrive and provide maximal ecosystem services when they support a multi-level trophic structure and grow in favorable water quality conditions that include high light levels, rapid water flow, and low nutrient levels. Poor water quality and other anthropogenic stressors have caused coral mortality in recent decades, leading to trophic downgrading and the loss of biological complexity on many reefs. Solutions to reverse the causes of trophic downgrading remain elusive, in part because efforts to restore reefs are often attempted in the same diminished conditions that caused coral mortality in the first place. 
Coral Arks, positively buoyant, midwater structures, are designed to provide improved water quality conditions and supportive cryptic biodiversity for translocated and naturally recruited corals to assemble healthy reef mesocosms for use as long-term research platforms. Autonomous Reef Monitoring Structures (ARMS), passive settlement devices, are used to translocate the cryptic reef biodiversity to the Coral Arks, thereby providing a "boost" to natural recruitment and contributing ecological support to the coral health. We modeled and experimentally tested two designs of Arks to evaluate the drag characteristics of the structures and assess their long-term stability in the midwater based on their response to hydrodynamic forces. 
We then installed two designs of Arks structures at two Caribbean reef sites and measured several water quality metrics associated with the Arks environment over time. At deployment and 6 months after, the Coral Arks displayed enhanced metrics of reef function, including higher flow, light, and dissolved oxygen, higher survival of translocated corals, and reduced sedimentation and microbialization relative to nearby seafloor sites at the same depth. This method provides researchers with an adaptable, long-term platform for building reef communities where local water quality conditions can be adjusted by altering deployment parameters such as the depth and site.

Coral Reef Arks: An In Situ Mesocosm and Toolkit for Assembling Reef Communities

Moored midwater geodesic structures called Coral Arks provide a modular, scalable, and vertically adjustable research platform that can be used to build, monitor, and perturb coral reef communities in previously inoperative areas, including offshore.

珊瑚礁方舟:用于组装珊瑚礁群落的 原位 介观和工具包

Coral reefs thrive and provide maximal ecosystem services when they support a multi-level trophic structure and grow in favorable water quality conditions ...

Measuring the Structure, Composition, and Change of Underwater Environments with Large-area Imaging

Digital imaging and processing technology have evolved to facilitate the expansion of large-area imaging surveys, which increase our capacity to study the status, trends, and dynamics of organisms living in subtidal habitats. By creating photo-realistic digital twins for ex situ analyses, these approaches allow small field teams to collect substantially more data than was previously possible. Here we present a four-step, large-area imaging survey pipeline and analysis methodology, including image collection, model construction, ecological analysis, and data curation, that has been developed and refined through experimentation over the past decade. Each step described has a consistent focus on the unique value of the original source imagery. While types of data extracted from large-area image surveys are vast, we include here workflows to extract ecological data for structural complexity, community composition, and demographic analyses valuable for monitoring and hypothesis-driven efforts. We additionally include recommendations for metadata standards, which complement the collection of large-area imaging data and support archival efforts facilitating transparency and collaboration between research groups.

This protocol covers a four-step large-area imaging survey methodology used to extract metrics of structural complexity, community composition, and population demographics for coral reef communities. The quality of imagery collected and integrated access to the source imagery are prioritized within each step of the protocol.

Digital imaging and processing technology have evolved to facilitate the expansion of large-area imaging surveys, which increase our capacity to study the ...

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

多峰成像和光谱学纤维束显微内窥镜纲要非侵入性，<em&gt;在体内</em&gt;组织分析

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

生物工程

用于促进和监测珊瑚生长的组合微型器件系统

An Integrated Micro-Device System for Coral Growth and Monitoring

Corals are fundamental organisms in marine and coastal ecosystems. With the advancement of coral protection research in recent years, precise control of the coral culture environment is highly in demand for coral conservation and study. Here, we developed a semi-closed coral culture micro-device system as a multi-functional platform, which can provide accurate and programmable temperature control, a sterile initial environment, long-term stable water quality, an adjustable dissolved oxygen concentration, and a customized light spectrum for corals. Owing to the modular design, the coral culture system can be upgraded or modified by installing desirable new modules or removing existing ones. Currently, under appropriate conditions and with proper system maintenance, the sample corals can survive for at least 30 days in a healthy state. Furthermore, due to the controllable and sterile initial environment, this coral culture system can support research into the symbiotic relationship between corals and associated microorganisms. Therefore, this micro-device system can be applied to monitor and investigate sea corals in a relatively quantitative manner.

作者聚焦：推进珊瑚文化 - 创建半定量控制微环境系统以应对当前限制 

This protocol describes the development of a modular controllable micro-device system that can be applied for the long-term culturing and monitoring of sea corals.

用于珊瑚生长和监测的集成微型设备系统

Corals are fundamental organisms in marine and coastal ecosystems. With the advancement of coral protection research in recent years, precise control of ...

Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting factor(s) for success of deep anterior lamellar and penetrating keratoplasty. These are surgical procedures that involve replacing part or all of the diseased corneal layers, respectively, by donated tissue, the graft, taken from a recently deceased individual. However, means to evaluate stromal quality of corneal grafts in eye banks are limited and lack the capability of high-resolution quantitative assessment of disease indicators. Full-field optical coherence microscopy (FF-OCM), permitting high-resolution 3D imaging of fresh or fixed ex vivo biological tissue samples, is a non-invasive technique well suited for donor cornea assessment. Here we describe a method for the qualitative and quantitative analysis of corneal stroma using FF-OCM. The protocol has been successfully applied to normal donor corneas and pathological corneal buttons, and can be used to identify healthy and pathologic features on both the macroscopic and microscopic level, thereby facilitating the detection of stromal disorders that could compromise the outcome of keratoplasty. By improving the graft quality control, this protocol has the potential to result in better selection (and rejection) of donor tissues and hence decreased graft failure.

We describe use of full-field optical coherence microscopy as a method for high quality assessment of corneal donor stroma. This protocol can be used to identify features indicative of health or disease, and is aimed at improving the screening and selection of donor tissues, and hence the results of keratoplasty.

全场光学相干显微镜，用于角膜移植物基质特征的组织学样分析

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting ...

免疫与感染

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.

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 ...

生物学

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

荧光激活细胞分拣，用于分离瘦杆菌细胞群

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

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

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利用运动结构摄影测量法监测底栖生态系统的野外入门书

珊瑚礁方舟:用于组装珊瑚礁群落的原位介观和工具包

Measuring the Structure, Composition, and Change of Underwater Environments with Large-area Imaging

多峰成像和光谱学纤维束显微内窥镜纲要非侵入性，

用于珊瑚生长和监测的集成微型设备系统

用于珊瑚生长和监测的集成微型设备系统

用于珊瑚生长和监测的集成微型设备系统

全场光学相干显微镜，用于角膜移植物基质特征的组织学样分析

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

荧光激活细胞分拣，用于分离瘦杆菌细胞群

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

摘要

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此视频中的章节

利用运动结构摄影测量法监测底栖生态系统的野外入门书

珊瑚礁方舟:用于组装珊瑚礁群落的 原位 介观和工具包

Measuring the Structure, Composition, and Change of Underwater Environments with Large-area Imaging

多峰成像和光谱学纤维束显微内窥镜纲要非侵入性，

用于珊瑚生长和监测的集成微型设备系统

用于珊瑚生长和监测的集成微型设备系统

用于珊瑚生长和监测的集成微型设备系统

全场光学相干显微镜，用于角膜移植物基质特征的组织学样分析

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

荧光激活细胞分拣，用于分离瘦杆菌细胞群

珊瑚礁方舟:用于组装珊瑚礁群落的原位介观和工具包