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
Summary
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。
Abstract
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。
Introduction
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°22'31.63“N，69°09'29.62”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。
<p class="“jove_content”"> SBFI成像之前已在广泛的生物样品，包括全脑和全心脏的人体组织，完整的小鼠胚胎中，斑马鱼胚胎中，并且多种类型的动物样品的具有完好骨头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。Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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 Curaçao 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.
Materials
| Coral Tissue Skeleton | None | None | 2.5 cm Biopsy from natural habitat |
| Arch Punch Coring Device | C.S. Osborne and Company | No. 149 | For Coral biopsy collection |
| Paraformaldehyde | Electron Microscopy Sciences | RT 15700 | 16% Pre-diluted |
| Histoclear/Safeclear II | Electron Microscopy Sciences | RT 64111-04 | Non-Toxic alternate to Xylene, Dehydration and Deparafinization |
| Xylene and Ethanol | Fisher Scientific | Fisher Scientific | Dehydration |
| Paraffin Wax | Richard Allen Scientific | Type H REF 8338 | Infiltration solution |
| Vybar | The Candle Maker | None | Component of Red Wax |
| Stearin | The Candle Maker | None | Component of Red Wax |
| Sudan IV | Fisher Chemical | S667-25 | Red Wax-Opaque background |
| Wheat Germ Agglutinin (WGA) | Life Technologies | W32466 | For labeling Coral Mucus |
| Prolong Gold | Life Technologies | P36095 | Anti-fade mounting media |
| Fluoro Dish | World Precision Instruments | FD-35-100 | For two-photon imaging |
| XY Motor, Driver and Controller | Lin Engineering | 211-13-01R0, R325, R256-RO | XY Translational Movement |
| Hot Plate | Corning | DC-220 | Melting all wax |
| Convection Oven | Yamato | DX-600 | Infiltration and Embedding |
| Tissue Processor | Leica | ASP 300 | Dehydration, Infiltration |
| Microtome | Leica | RM2055 | Disposable knifes |
| Stereo Microscope | Carl Zeiss | Stereolumar V 12 | 1.5x (30 mm WD) Objective |
| Fluorescence Microscope with ApoTome | Carl Zeiss | Axiovert M 200, ApoTome I System | Imaging thin section of a polyp: Zooxanthellae |
| Axiocam camera | Carl Zeiss | MRm | Monochrome camera 1388×1040 pixels |
| Axiovision Software | Carl Zeiss | Version 4.8 | Image acquisition program |
| Two-Photon Laser | Spectraphysics | Maitai eHP, pulsed laser (70 fs) | With DeepSee module |
| Laser Scanning Microscope | Carl Zeiss | LSM 710 with Spectral Detector | 34 channel PMT detection |
| Zen Software | Carl Zeiss | 2010 or above | for two-photon and spectral image acquisition |
| Imaris Suite Software | Bitplane, Inc., | Version 7.0 or above | 3D Volume, Iso-surface Rendering, Visualization |
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