The authors would like to acknowledge the effort of Sandro Lucchinetti (Schenkung Dapples, Z&#252;rich) for constructing the devices needed to guarantee progress in sample preparation.

1.抽样技术<ol><li>岩芯取样<ol><li>对于抽样树茎，用增量取样器从每干，分析其生长发育提取至少两个核心。变化的采样的位置上的研究课题， 例如 ，对于普通的气候重建采取平行于斜坡的芯。当热带种类的工作，至少需要三个或更多的内核。 </li><li>使用削尖增量取样器（5，10或12毫米直径），然后将取样管在垂直于杆的生长轴。 注:要具备取样器的控制和稳定的位置，使用推进器时，钻成树，以避免除噪比其旋转等的运动。钻入茎一路进入并通过髓。核心非常致密的木材（ 即热带种），使用特殊的重新配置电锯机构与增量内核，允许核心甚至茂密的树林作为Diospyrus ebenum的适配器（乌木）。&lt;/ li&gt; <li>使用的提取，并从树中删除所得到的芯。转出取样器，并从干中删除。 </li><li>添加特定的ID来使用软铅笔写在磁带上，然后将其直接放置在芯所提取的核心。重复该过程在阀杆的相反侧。 </li><li>存储的两个样品的树中的塑料或纸管或专用箱，以防止任何损害。 注:纸张秸秆是在热带环境中尤其有益，因为他们阻止核的成型。 </li><li>最后，安装在木支撑梁的内核（纤维方向竖直）。用冷水耐木胶，并让他们干。 注意:不要安装在木支架的核心，如果研究的目的包括进一步的化学，同位素或密度分析。为了这些目的，固定在开放式电缆导管或一个单面瓦楞板的芯。 </li></ol></li><li>微芯取样<ol><li>使用一种特殊的冲压设备或针以2mm的开口。取决于树皮厚度用凿子以除去树皮的一个必要部分。 </li><li>将设备垂直于上述阀杆的生长轴，并使用锤穿透茎的木质部，以在深度约2厘米。 </li><li>转断针刀具内的所得微芯和从杆中取出。存储微内核的离心瓶和标签他们。 </li></ol></li><li>圆盘取样<ol><li>在特殊情况下（尤其是在热带地区）， 如采样树桩和倒下的树木，或尽可能的时候，拿光盘用链锯（垂直于轴线增长横截面）。简单地标记光盘，并将其存储，直到进一步的分析。 注:此采样过程中最常采用的全干死，历史，考古和亚化石材料，以及非常密集的硬木，不允许使用增量取样器中。 </li></ol&gt; </li></ol> 2.样品制备<ol><li>打磨盘<ol><li>放置光盘砂光机上，并使用越来越细的磨料的序列，开始用砂纸的80号粒度，随后120，220，300号粒度磨的表面上。与400砂砾最终抛光足以满足大多数针叶树。对于硬木，特别是热带树种，用砂纸打磨糁高达1,200区分增长型环的边界。 </li></ol></li><li>切割面表面上的核心<ol><li>修复增量内核在一个新开发的核心切片从而能够削减核平板面在其整个范围内的核心持有人。 注:更正夹持器内芯的取向有样品直立，这是在一个合适的角度，以切片机的刀的纤维方向。 </li><li>抬起核心，直到它略微触及刀刃。拉叶片，其固定在滚珠轴承指导在芯的程度成Cu吨假顶的第一部分。 </li><li>推回刀的核心背后，抬起样品架与核心约10微米，并重复切割程序。为此，直到大约三分之一的核心的直径被减少从而产生连续的平面表面。 </li></ol></li><li>加入玉米淀粉溶液制备微部分。 <ol><li>对于解剖结构的详细的图像分析，固定芯或其它样本从光盘分离出来的切片机的支架上。定位切片机刀片，通过在样品的上边缘一新开发的雪橇切片机的滚珠轴承指导引导并拉过它。 </li><li>推回刀片抬起样品由约20至30微米，再拉刀片在样品。直到在样品的顶部的连续表面被创建重复此过程。 </li><li>盖住得到的用玉米淀粉溶液表面（10克玉米淀粉，8ml水和7克100％甘油基的升）与一个共同的刷子。 注:淀粉粒填满样品的开放单元以稳定结构的薄切片的随后切割。 </li><li>通过15微米提起样品，放置一个刷子将样品的表面上，并慢慢地将叶片朝向开始切断的薄部（下面刷）的样品。切割时，所得到的部分上滑动的电刷导引刀片。 </li><li>当整个部分被切断，去除用刷子和水在叶片的微部分（以减少摩擦）并放置部上用于进一步制备载玻片。以防止它们干燥，用少量的甘油（33％）=（1部分的100％的甘油和水的2份）覆盖它们。 </li></ol></li></ol> 3.准备的MicroSlide <ol><li>永久滑梯的下面准备，首先从微观部分用吸管和水冲洗甘油而去。 </li><li>覆盖湿SEction用几滴番红的（1克番红粉末+ 100ml水）和Astrablue（0.5克阿斯特拉蓝色粉+2毫升100％乙酸+ 100ml水）的木质化和非木质化区分结构，也能提高对于随后的图像分析的对比度。 </li><li>让部其余为覆盖的染料5分钟，然后用移液管和水再次冲走。 </li><li>一旦多余的染料被除去，通过用序列的75％的稀释，96％和最后的100％乙醇洗涤它们脱水部用移液器。 </li><li>使用移液器冲洗载玻片上，而不是将所述样品中的浴，以减少处理时间，以大约2至3分钟，在整个脱水过程中的样品。 注:脱水用乙醇防止脆弱的样品断裂。 </li><li>此后冲洗部分用100％二甲苯。 注:二甲苯是致癌和使用时的通风是必须在实验室中。其他产品存在更换二甲苯，但如果有意向存储超过一个潜在的再分析更长的时间段（年，数十年）的样品中，加拿大香脂（步骤3.5.2）是保持不改变颜色随时间明确的唯一灌封介质。不幸的是加拿大香脂需要与二甲苯组合使用。 </li><li>只要二甲苯变为乳白色（白色），重复脱水过程，因为它是不充分脱水。 </li><li>只要二甲苯撑清楚，覆盖部分用一滴100％加拿大香脂和与所述部分的至少大小的玻璃盖覆盖。 注:请注意轻轻按下玻璃盖拆卸下来的气泡。 </li><li>放置所得到的微滑动两个条的耐热性的塑料之间，并将其放置在金属板上。放置在滑动的顶部的重量（ 例如 ，磁体）向下按，在随后的干燥过程中，保持部平坦。 </li><li>将载玻片在烘箱中在60℃下进行约12小时。 12小时后，以幻灯片出炉，并把它们放在一个架子，让他们失望冷却至室温（约20℃）。冷却时，除去重量和塑料条，并清洁用刀片以除去多余的加拿大香脂的幻灯片。 </li></ol> 4.可视化单元格内容<ol><li>准备Nawashin解决方案25。 <ol><li>制备溶液（A）与5g铬酸，50毫升96％乙酸，和320毫升去离子水。制备另一溶液（B）用200毫升福尔马林175毫升去离子水。 </li><li>混合溶液A和溶液B以1:1的比例以创建Nawashin溶液。 </li></ol></li><li>为了显现单元格内容，修正与Nawashin溶液的样品10分钟，以停止该小区内的所有降解过程。 注:固定保留细胞核允许电池寿命（电池寿命=当前核/核缺席的估计，存在Øř没有细胞核的指示细胞是否是活的或死的）。 </li><li>固定后，冲洗部分用水染色它们与番红阿斯特拉蓝色和之前另外染色它们与苦味酸 - 苯胺蓝（1份饱和水溶性苯胺蓝+ 4份10％的苦味酸）之前完全卸下Nawashin溶液。 </li><li>小心加热样品至约80℃（在下面沸点任何情况下），以加速染色过程。用于脱水的样品，并包埋遵循协议的步骤3.4至3.8。 </li></ol> 5.准备解剖特点数字图像<ol><li>对于血管分析创建核心表面的数字图像。 <ol><li>使用核心切片机切芯表面平面，简单地用毛毡标记染色产生的表面为黑色。 </li><li>只要染料被干燥时，擦用白粉笔芯的表面上。按单元格中的粉笔通过简单地擦粉笔在表面使用手指。还删除了此过程粉笔的盈余。 注意:作为一个结果细胞壁是黑色的，血管的内腔部分是白色的。这种对比是一个先决条件的自动图像分析。 </li><li>放置下面双目显微镜配备有数码相机的制备核心。取的稍微重叠的序列（约10％）开始在芯的一侧的图像，直到整个表面被捕获。缝合的单图像来创建芯表面的一个完整的图像。 </li></ol></li><li>创建数字图像从微观幻灯片<ol><li>放置在显微镜下的微观清洗幻灯片，并采取从整个部分重叠的图像。 </li><li>定义取决于结构的特定倍率待分析，40X和1,000X放大倍数之间的范围内。 </li><li>拼接单个图像创建部分的一个完整的图像。为了避免在拼接过程中可能扭曲，用";计划"型物镜的显微镜。 </li></ol></li></ol> 6.量化解剖特点<ol><li>为了检测使用图像分析工具ROXAS18或类似的软件单元和环形边框，装入一个微段和相关联的空间校准，以获得公制单位的定量结果的一个完整的图像。通过测量在相同的放大倍数下拍摄，并通过以公制单位的刻度间距除以所得到的值的微米级的图像的尺度之间的像素数（ 例如 1000微米）计算出空间校准。 </li><li>选择从提供的列表（在软件中提供的）开始分析的自动部分前一个合适的构造。 注:配置的程序设置一个先前的优化组，例如，考虑到要被检测的样品，将细胞的大小和形状范围的染色的颜色。量身定制的配置从而允许产生最佳的识别结果不同的物种和图像质量。 <ol><li>选择进一步的选项，如要被包括或排除，避免，区域的图像中的使用例如 ，样品中的空白图像边缘或裂缝，如果需要的话。 </li></ol></li><li>通过按下分析按钮开始分析。如果选择了，定义了将被包括或使用多边形，矩形或圆形工具排除区域的图像中。以下自动分析使用灵活的算法纠正一些图象缺陷（ 例如 ，对比度差），并提高对比度的基础上的图像的质量。 </li></ol>

<ol>
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C., Walls, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory techniques in Botany. , (1966).</li><li>Schneider, L., G&#228;rtner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+advantage+of+using+non-Newtonian+fluids+to+prepare+micro+sections.">The advantage of using non-Newtonian fluids to prepare micro sections.</a> Dendrochronologia. 31 (3), 175-178 (2013).</li><li>De Ridder, M., Vanden Bulcke, J., Van Ackera, J., Beeckman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tree-ring+analysis+of+an+African+long-lived+pioneer+species+as+a+tool+for+sustainable+forest+management.">Tree-ring analysis of an African long-lived pioneer species as a tool for sustainable forest management.</a> Forest Ecology and Management. 304, 417-426 (2013).</li><li>De Ridder, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tree-ring+based+comparison+of+Terminalia+superba+climate&#8211;growth+relationships+in+West+and+Central.">A tree-ring based comparison of Terminalia superba climate&#8211;growth relationships in West and Central.</a> Trees. 27 (5), 1225-1238 (2013).</li><li>Redband, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ImageJ. , (1997).</li></ol>

The authors declare that they have no competing financial interests.

木材解剖的成功和可持续一体化的挑战转化为dendroecological的研究是，除了多方面的分析问题，主要是由于技术方面的问题。这些挑战包括从原则抽样方法，以创造高品质的微型部分及其后续分析19。 乍一看，核心甚至光盘的采样是已经知道了很多年，现在一个简单的过程。有迹象表明，可以做错误和一个小的不准确的采样会导致在随后的制备和分析阶段严重问题很多事情。小的不准确，如核化是不完全垂直于杆轴或使用不完全削尖取样器不是一个问题，如果该研究的目的是限制在环宽度的测量。然而，目标是样品的显微分析时，不正确的采样方向可能导致光学畸变细胞壁，而使用钝取样器的结果在芯内的微裂纹。其结果是，试图削减这些内核的微部分时，该薄片只是土崩瓦解和高效的准备不再保证。这同样适用于微芯取样。钝尖会造成高压，当冲床被钉进干木材。因此，该形成层层将被压缩。在形成层的细胞（ 图5）的结果挤压而不能进行分析。 圆盘取样的确是在分析增长的变化将它们与环境变化的最佳策略。不幸的是这是根本不可能采取从光盘拟取样作进一步分析所有的树。然而，特别是在壳体热带树木年轮的，需要在与增量芯组合一定量的干盘。磁盘被用作基础以限定环的界限，为此，以支持boundarIES定义了基于分析的增量核心12,27,28。 打磨与切割的优点和缺点都经常讨论1,11,21。因为它是上面提到的，最好的程序总是取决于所研究的问题和参数进行分析（宏观或微观）。如果同位素或化学分析被投影在一个进一步的加工步骤，这是最重要的是通过砂磨可填入细胞管腔在整个样品中创建的磨料尘埃，小心地抽真空或压力空气除去。 切削微切片是所有微观分析制备样品用于进一步分析的最适当的方式。首先，该部分被切断的样本，然后可以被保持而没有任何污染的潜在进一步分析。其次，这些路段允许单晶胞参数高分辨率测量。此外，避免了耗时的嵌入技术通过使用玉米淀粉溶液26稳定细胞是在微切 ​​片的一大优势。 微切片的缺点仍然是导致长的制备时间有限的样本大小。对于真正的时间序列分析时光倒流了几百年甚至上千年，有必要进一步发展现有的切割装置17,19，也图像处理和分析18。第一步到这个方向是核心切片机21的发展，最初制造成削减芯平面的表面（ 图1）。最近的试验表明，以削减使用这种装置（ 图1）整个芯的微切片的能力。 高品质的微型部分提供了基本原则的有效图像分析。服用显微镜下的图像是一种常见的程序19中 ，但它们的有效的分析仍然需要一个任务得到进一步的发展17。所有现有的图像分析系统是半自动，也就是说 ，它们需要更多或更少的强烈的技术人员来控制。在许多情况下，该图像需要被纠正或甚至新的图像必须完成，以提高对比度更好的登记的结构由软件无图像内改变细胞壁的厚度。 专门的图像分析工具，如ROXAS 18 WinCell或ImageJ的29特定脚本能够提供基本解剖学数据，诸如细胞数目，细胞尺寸，细胞壁厚与年轮内细胞的位置。许多额外的解剖度量是​​相关于一个dendroecological上下文可以从这些基本的测量被计算，如最大导管的尺寸，导管的尺寸分布，早材或导管中的第一行的大小，（光）密度木材，帧内年度导管的大小和细胞壁的剖面厚度，和导管（孤，倍数等 ）的分组模式。 使用软件ROXAS 18，导管管腔（ 即 ，水导细胞）和年轮边框的轮廓被自动识别和视觉表示为叠加在原始图像。检测算法对于导管是基于颜色，大小和形状的信息，检测算法对每个导管的局部上下文环边界。工具箱使我们能够通过直接编辑叠加功能， 即 ，删除，添加和修改环边界和管道概述手动改善这些结果。编辑之后，最终的数据输出，包括细胞壁厚度（针叶树），自动生成并保存到电子表格。完全自动化的系统目前无法使用，甚至没有对于针叶树示出一个比较简单的结构，但是这是为将来的发展的一个目标。这将有力地支持了福木材解剖参数在时间序列LL整合分析。

树木，以及灌木，侏儒灌木，甚至草药，显示与环境变化的歧管的反应模式。中期以来，19 世纪这些模式一直受到植物学和植物生理学。在那时，对木本植物研究主要集中在树的结构和年轮变异性的生态环境1的描述性分析。当安德鲁埃利科特道格拉斯发明了树木年轮研究2交叉测年技术，这种生态环境中或多或少地受到精确迄今为止发现的木考古的新功能抑制。跨约会，第一次使树木年轮历年的准确约会，并一直到现在视为树轮研究中的应用1的各个领域的中坚力量。 与此同时，自19世纪末，木材解剖演变成一个重要的研究学科相关d可自然与应用科学3等诸多领域。两个主要的结构域被建立:系统木材解剖，这是基础识别木材在考古学4，并且施加木材解剖，涉及到木技术，生理，病理，和生态学3,5。 在树轮研究，dendroecology现今被定义为一个主题涵盖年轮相关的研究集中在环境研究如地貌过程（dendrogeomorphology），温度和降水重建（dendroclimatology），水位变化（dendrohydrology）或甚至冰川波动（ dendroglaciology）6。作为该定义表示，年轮分析已成为在约会和重建的环境工艺领域日益重要，如（ⅰ）过去的气候条件下，通过分析在环宽度7,8，木材密度9年变化或同位素10，或（二）T他复发地貌过程11的区间。这些关于环宽度的变化和它们的同位素含量非常详细的研究表明，有必要分析环中更详细地说， 也就是 ，要研究的环的解剖结构。然而，有关环境变化的年轮内木材解剖学特征的详细研究并不多见12,13。虽然这些微观特征是已知的14，它们很少被应用在微观水平dendroecological研究。此外，在自然生长的树木，进行精确测的目的必不可少的，这些增长反应的精确定时，很少被最近15记录。 关于全球变暖16的影响，现有的和新的技术发展的提高，以记录和量化是必需的过去和目前的环境过程，特别是在气候影响研究11表示。通过扩大传统的木材解剖学研究的基于生态木材解剖17，dendro科学家可以分析新的参数，并开发新的方法，以了解具体的环境因素对木本植物18的解剖结构的短期和长期影响。有关变化与特定的驱动程序（ 例如 ，机械力，气候变化）个人五环内不同的细胞参数的详细知识是理解年轮形成变异的基本要求。相比普通环宽度的测量，确定木材解剖变异要求需要大量的劳力和时间的更复杂和更广阔的制备技术。的试样切割，染色，和嵌入的详细过程是多方面的和总是依赖于研究19的目的。 对于环宽的针叶树，甚至结构数量，规模还是颇宏观分析船舶在硬木ibution，样品的表面用细砂纸或专用磨床20通常抛光。这个程序的一个缺点是各个单元与灰尘，防止进一步的半自动显微分析21的填充。对于宏观样品制备的最好的结果是当样品表面被用剃刀刀片或其他锋利的刀切割实现。 而对于小样本，刀片是一个完美的工具;更大的样本作为核所需要的平面表面在芯的整个程度的切割。在对比砂光，将细胞没有填充尘埃，这使进一步制备为连续图像分析。此外，开孔管腔，所述适当切细胞壁，并且将整个样品的平面表面使高频密度22到芯的整个范围中的应用。为图像分析，将样品的表面（小区壁）可以使用深色墨水进行染色和开孔管腔随后可以填充有白粉笔增强细胞壁和管腔面积19,23之间的对比度。这个相当简单的技术实现了较大的细胞结构容器三围尺寸基本宏观评估。 这些技术用于切割平面的表面是足够用于宏观分析。一个详细的木材解剖（ 即微观）分析，透射光显微术是在dendro科学应用中最常用的方法。通过复杂的过程包含细胞类型测定，细胞分裂，细胞分化木质部细胞分化，和程序性细胞死亡24。由于在发生这些过程确定细胞​​解剖特点的定时和速率，影响这些过程的环境条件下能产生在环结构中解剖偏差。作为这些分析的重要先决条件S，微段需要被用切片机19制得。当制备样品切片，管胞或纤维方向的可见性是至关重要的。利用手驱动的滑动切片机的建议削减微型部分，因为这种技术有利于高品质的路段根据需要进行图像分析19。根据某研究的具体目的，微切片切成垂直于或平行于电池的纵向范围。这些部分再拍下显微镜下面，并使用专门的图像测量单元的尺寸分析软件。 直到最近，以制备微切片的能力被限制在小样本大小只（约1cm×1厘米）。这是可以接受的，分析单个事件在特定年紊乱，但这种技术不允许所需环境重建扩展时间序列分析。这项工作只能实现通过对新型，高效，经济的准备过程和分析技术的发展ð。近年来，在瑞士联邦研究所WSL瑞士树木年轮实验室的成员已经开始对这个话题深入细致的工作。其结果是，新的设备和分析技术已被开发以支持整合木材解剖特征来广泛的环境研究课题的想法。

<table border="0" cellpadding="0" cellspacing="0" width="1096">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:147px;">Increment corer</td>
			<td style="width:147px;"></td>
			<td style="width:147px;"></td>
			<td style="width:659px;">http://www.haglofinc.com/index.php?option=com_content&#38;view=article 
			&#38;id=57&#38;Itemid=88&#38;lang=en</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Core-Microtome</td>
			<td></td>
			<td></td>
			<td>http://www.wsl.ch/dendro/products/microtomes/index_EN</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laboratory microtome</td>
			<td></td>
			<td></td>
			<td>http://www.wsl.ch/dendro/products/microtomes/index_EN</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trephor micro corer</td>
			<td></td>
			<td></td>
			<td>http://intra.tesaf.unipd.it/Sanvito/trephorEn.asp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nawashin solution</td>
			<td></td>
			<td></td>
			<td>Ten parts 1% chromic acid, four parts 4% formaldehyde and one part acetic acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Picric-Anilin blue</td>
			<td></td>
			<td></td>
			<td>One part saturated aniline blue and four parts Trinitrophenol dissolved in 95% ethanol</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Safranin</td>
			<td></td>
			<td></td>
			<td>Empirical Formula (Hill Notation) C20H19ClN4&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Astra-blue</td>
			<td></td>
			<td></td>
			<td>Empirical Formula (Hill Notation) C47H52CuN14O6S3&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Ethanol</td>
			<td></td>
			<td></td>
			<td>Linear Formula CH3CH2OH&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Xylol (Xylene)</td>
			<td></td>
			<td></td>
			<td>Linear Formula C6H4(CH3)2&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Canada Balsam</td>
			<td></td>
			<td></td>
			<td>Embedding solution for microscopy</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Roxas Software</td>
			<td></td>
			<td></td>
			<td>http://www.wsl.ch/dienstleistungen/produkte/software/roxas/index_EN</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ImageJ Software</td>
			<td></td>
			<td></td>
			<td>http://imagej.nih.gov/ij/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">WinCell</td>
			<td></td>
			<td></td>
			<td>http://imagej.nih.gov/ij/</td>
		</tr>
	</tbody>
</table>

a technical perspective in modern tree-ring research - how to overcome dendroecological and wood anatomical challenges

Dendroecological研究使用存储在树木年轮资料，以了解单棵树，甚至整个森林生态系统应对环境变化，并最终重建这种变化。这是通过分析生长变化回在时间和相关联的各种植物的具体参数（例如）温度记录完成。集成在这些分析的木材解剖参数将加强重建，甚至是内部年度决议。因此，我们提出，关于如何进行采样，准备，和分析木试样为普通宏观分析，而且还用于随后的微观分析的协议。此外，我们还引进了分析，从常见的小型和大型标本产生的数字图像，支持时间序列分析潜在的解决方案。该协议提出了基本步骤目前可以使用它们。除此之外，有一个持续的需要对现有技术的提高，以及新吨发展echniques，记录和量化过去和目前环境的进程。传统的木材解剖学研究需要扩大到包括生态资料，这一领域的研究。这将支持dendro科学家谁打算来分析新的参数，并开发新的方法，以了解具体的环境因素对木本植物的解剖结构的短期和长期影响。

所有dendroecological分析依赖于准确的样本，无论光盘，内核，或微型内核拍摄。对于这一点，该设备必须在最佳状态（准确削尖）避免木制样品内的微裂纹。当增量芯制备曲面，采用一个芯切片机是必不可少的。有开放单元，其可以进一步处理以增强对比度的图像分析和容器尺寸的测量（ 图1）的能力是对解剖结构的适配成时间序列分析的第一个重要步骤。有时一个硬木样品的密度可防止使用一个切片机。在这种情况下，一个适当的抛光和随后除去过量的木屑从与压缩机或真空容器是最好的选择。 为更小的单元结构如早材和针叶树晚材管胞更详细的分析，需要高品质的微部分。在这里，势的人工件，例如二次细胞壁被剥去主壁需要被避免（ 图2）。如果发生在数字图像，这些伪影，细胞尺寸的自动分析不再可能。的工件然后需要手动校正，这是耗时的，并且在大多数情况下，导致细胞的尺寸的不正确的测量。非牛顿流体， 即玉米淀粉溶液，在样品的顶部的简单应用程序支持结构的稳定性，同时降低伪像的发生为最小（ 图2）26。玉米淀粉，适用于所有的木样本，包括热带树种此应用程序，使得嵌入程序应用到样品切割多余之前。 微部分启用年轮更安全的决心。这是特别为针叶树生长在其自然的限制， 即该情况下&lt;/ em&gt;的，在高山地区的林木线。极窄环是频繁和难以检测宏观（ 图3）。在极端情况下，所述环包含一个或两行早材细胞和扁平晚材细胞，其缺少（相对于共同晚材细胞）加厚细胞壁的一行。为此，他们利用微部分时，有更好的甚至才可见。此外，密度波动可以从环的边界更清楚地区别，从而简化了的年轮尤其是在地中海和热带（ 图3）的检测。 当以40倍或更高的放大率分析图像，单细胞是可见的，它们的细胞壁的厚度也可检测的。半自动分析软件使沿其下面的时间和空间的发展（ 图4）的方向上定义的路径特定的参数进行测量。有了这个，成龙的单个参数GES诸如蜂窝管腔或细胞壁厚度可以在年度环（ 图4）的整个范围来确定。这可以在图像中可见的所有环来完成，这充分支持需要延长的时间序列分析。 图像分析也可以被用来确定所述的植被期间（ 图5）内年轮的发育阶段。当分析染色的番红和阿斯特拉蓝色使用偏振光，木质化的甚至在不同的阶段，开始在细胞壁直到次级细胞壁的完整木质化的最外拐角截面的图像，变得可见。这是因为，木质化（成熟）细胞壁闪耀在偏振光（ 图5）。详细信息可以与记录在各自植被期间的环境数据，以确定例如一个更详细的气候生长relati<html> onship。 <img alt="图1" src="/files/ftp_upload/52337/52337fig1.jpg" /> 图微段1例和橡木的准备平面包括环宽度和容器大小的测量左:橡树增加核心的最外层部分。使用芯切片的直径5mm芯被割断。然后，表面被染成黑色用毛毡标记和细胞内充满白色粉笔后，污渍是干的。右:该图表指示在左边示出的芯的表面上完成的环的宽度和容器的大小的测量。不需要任何微切片做这些测量由于通过核心切片机（21后修饰）中创建的清晰的表面。下图:微部分切断的增量核心，染色，脱水和固定在加拿大香脂。厚度:20微米，长度:25厘米<html> G"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/52337/52337fig2.jpg" /> 图微部分2图像切不稳定性与使用玉米淀粉溶液的一段剪左:微显示部分切割工件的针叶树的早材管胞。将样品切成不嵌入，其结果的早材细胞的相当薄的次级墙壁被分离出来的主壁（蓝色箭头）。右:没有在针叶树的早材管胞任何文物微部分。本部分（如左图所示相同的样品）应用与样品表面的顶部刷玉米淀粉溶液后切断。 <a href="https://www.jove.com/files/ftp_upload/52337/52337fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ove_content"FO:保持together.within页="总是"&gt; <img alt="图3" src="/files/ftp_upload/52337/52337fig3.jpg" /> 图几乎检测不到年轮边界3.实施例左:表示环边界（黑色箭头），而细胞壁的任何增稠剂由扁平晚材细胞的单个行中表示:一个增量芯（ 落叶松这里）的微部分。这枚戒指将是不可见的宏观。右:内部年度密度波动（白色箭头）是地中海种常见（微部分: 冬青栎 ）。细胞结构的逐渐变化对晚材和回早材结构（白色箭头）允许区分从实际环边界密度波动（黑色箭头）。 <a href="https://www.jove.com/files/ftp_upload/52337/52337fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图中的针叶树的年轮4.插图管腔面积和壁厚测量前:切出图像显示ROXAS分析在树木年轮樟子松 （欧洲赤松）的示范效果。环边框显示为黄色和青色概述管胞管腔。对于一个径向文件（蓝色管胞流明）测得的细胞壁厚度是用红色圆圈表示。黑色比例尺= 100微米。下图:在内部年度变化管胞管腔面积和管胞细胞壁厚度为整个年轮<a href="https://www.jove.com/files/ftp_upload/52337/52337fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52337/52337fig5.jpg" /> 图5.为例E A形成年轮的该图像已经在光学显微镜用偏振光从微芯取样7月7日日 ，2007从落叶松的生长而获得的番红和阿斯特拉蓝染色的微部分下被捕获Lötschental在1300米以上的海拔。在此微部形成层的细胞，将细胞在放大阶段，将细胞在壁增厚相和成熟细胞的识别。图像的切向宽度占地约1毫米木质部截面。 <a href="https://www.jove.com/files/ftp_upload/52337/52337fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Technical Perspective in Modern Tree-ring Research - How to Overcome Dendroecological and Wood Anatomical Challenges at JoVE.com

A Technical Perspective in Modern Tree-ring Research - How to Overcome Dendroecological and Wood Anatomical Challenges

Here we present a protocol outlining how to sample wooden specimens for the overall assessment of their growth structures. Macro- and microscopic preparation and visualization techniques necessary to generate well-replicated and highly resolved wood anatomical and dendroecological dataset, are described are described.

现代树轮研究技术的角度 - 如何克服Dendroecological和木解剖挑战

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to ...

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Research

JoVE Journal

Environment

29.0K 次观看.  Swiss Federal Research Institute WSL. Here we present a protocol outlining how to sample wooden specimens for the overall assessment of their growth structures. Macro- and microscopic preparation and visualization techniques necessary to generate well-replicated and highly resolved wood anatomical and dendroecological dataset, are described are described.

视频: A Technical Perspective in Modern Tree-ring Research - How to Overcome Dendroecological and Wood Anatomical Challenges

Design and Construction of an Urban Runoff Research Facility

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m2 facility was constructed which consists of 24 individual 33.6 m2 field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO4-P, K+, Mg2+, and Ca2+ had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.

This paper describes the design, construction, and function of a 1,000 m2 facility containing 24 individual 33.6 m2 field plots equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated home lawns.

设计一个城市径流研究设施的建设

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use ...

科研

环境科学

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。病毒样本的收集I.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

Preparation and Testing of Plant Seed Meal-based Wood Adhesives

Recently, the interest in plant seed meal-based products as wood adhesives has steadily increased, as these plant raw materials are considered renewable and environment-friendly. These natural products may serve as alternatives to petroleum-based adhesives to ease environmental and sustainability concerns. This work demonstrates the preparation and testing of the plant seed-based wood adhesives using cottonseed and soy meal as raw materials. In addition to untreated meals, water washed meals and protein isolates are prepared and tested. Adhesive slurries are prepared by mixing a freeze-dried meal product with deionized water (3:25 w/w) for 2 hr. Each adhesive preparation is applied to one end of 2 wood veneer strips using a brush. The tacky adhesive coated areas of the wood veneer strips are lapped and glued by hot-pressing. Adhesive strength is reported as the shear strength of the bonded wood specimen at break. Water resistance of the adhesives is measured by the change in shear strength of the bonded wood specimens at break after water soaking. This protocol allows one to assess plant seed-based agricultural products as suitable candidates for substitution of synthetic-based wood adhesives. Adjustments to the adhesive formulation with or without additives and bonding conditions could optimize their adhesive properties for various practical applications.

To facilitate the effort in seeking more economic and environment-friendly formulations of natural product-based wood adhesives, this work demonstrates the preparation and testing of plant seed-based wood adhesives. This protocol allows one to assess plant seed-based agricultural products as suitable candidates for the substitution of synthetic-based wood adhesives.

准备和植物种子膳食为基础的木材胶粘剂的测试

Recently, the interest in plant seed meal-based products as wood adhesives has steadily increased, as these plant raw materials are considered renewable ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。第三部分。病毒检测用RT-qPCR的

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ecological interactions. Arthropods are usually tracked in nature by tagging them with a unique mark and then re-collecting them over time and space to determine their dispersal capabilities. In addition to actual physical tags, such as colored dust or paint, various types of proteins have proven very effective for marking arthropods for ecological research. Proteins can be administered internally and/or externally. The proteins can then be detected on recaptured arthropods with a protein-specific enzyme-linked immunosorbent assay (ELISA). Here we describe protocols for externally and internally tagging arthropods with protein. Two simple experimental examples are demonstrated: (1) an internal protein mark introduced to an insect by providing a protein-enriched diet and (2) an external protein mark topically applied to an insect using a medical nebulizer. We then relate a step-by-step guide of the sandwich and indirect ELISA methods used to detect protein marks on the insects. In this demonstration, various aspects of the acquisition and detection of protein markers on arthropods for mark-release-recapture, mark-capture, and self-mark-capture types of research are discussed, along with the various ways that the immunomarking procedure has been adapted to suit a wide variety of research objectives.

Proteins are often used to mark arthropods for dispersal research. Methods for administering internal and external protein marks on arthropods are demonstrated. Protein mark detection techniques, either through indirect or sandwich enzyme-linked immunosorbent assays (ELISAs), are also illustrated.

管理和检测蛋白标记的节肢动物的扩散研究

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ...

How to Create Conditioned Taste Aversion for Grazing Ground Covers in Woody Crops with Small Ruminants

Conditioned taste aversion (CTA) is a learning behavior process where animals are trained to reject certain feed after gastrointestinal discomfort has been produced. Lithium chloride (LiCl) is the preferred agent used in livestock to induce CTA because it specifically stimulates the vomit center. In addition, LiCl is commercially available, and easy to prepare and administer using a drenching gun. Nevertheless, some factors have to be considered to obtain an effective long-lasting CTA, which allows small ruminants to graze during the cropping season. A key aspect is to use animals with no previous contact with the target plant (the plant chosen to be avoided; new feed). Due to their native neophobic feeding behavior, small ruminants can easily associate the negative feedback effects with the new feed, resulting in a strong and persistent CTA. The recommended doses are 200 and 225 mg LiCl/kg body weight (BW) for goats and sheep, respectively. To induce CTA, 100 g of the target plant should be individually offered for at least 30 min, and LiCl administered thereafter if the intake is greater than 10 g. Each time the animal eats the target plant without negative consequences, the CTA becomes weaker. Consequently, to minimize the risk of target plant consumption, it is essential to have sufficient palatable ground cover available. The presence of an alternative feed (of quality and quantity) prevents the accidental consumption of the target plant. A close monitoring of the flock is recommended to remove and re-dose any animal consuming more than 4 bites or 10 g of the target plant. At the beginning of each grazing season, check the CTA status of each animal before moving them to the crop.

Feeding behavior preferences in livestock can be modified to implement a grazing management plan in woody crops. Here, we present a protocol to show a lithium chloride dose after eating a new plant that induces conditioned taste aversion.

如何创建条件性味觉厌恶放牧在地面木本作物与小反刍动物封面

Conditioned taste aversion (CTA) is a learning behavior process where animals are trained to reject certain feed after gastrointestinal discomfort has ...

Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive species known to redox regulate biological processes in health and disease. Pre-clinical and clinical evidence points to beneficial effects of plasma treatment in the healing of chronic ulcer of the skin. Other emerging topics, such as plasma cancer treatment, are receiving increasing attention. Plasma medical research requires interdisciplinary expertise in physics, chemistry, and biomedicine. One goal of plasma research is to characterize plasma-treated cells in a variety of specific applications. This includes, for example, cell count and viability, cellular oxidation, mitochondrial activity, cytotoxicity and mode of cell death, cell cycle analysis, cell surface marker expression, and cytokine release. This study describes the essential equipment and workflows required for such research in plasma biomedicine. It describes the proper operation of an atmospheric pressure argon plasma jet, specifically monitoring its basic emission spectra and feed gas settings to modulate reactive species output. Using a high-precision xyz-table and computer software, the jet is hovered in millisecond-precision over the cavities of 96-well plates in micrometer-precision for maximal reproducibility. Downstream assays for liquid analysis of redox-active molecules are shown, and target cells are plasma-treated. Specifically, melanoma cells are analyzed in an efficient sequence of different consecutive assays but using the same cells: measurement of metabolic activity, total cell area, and surface marker expression of calreticulin, a molecule important for the immunogenic cell death of cancer cells. These assays retrieve content-rich biological information about plasma effects from a single plate. Altogether, this study describes the essential steps and protocols for plasma medical research.

A high-throughput cold physical plasma treatment protocol of liquids and cells is shown. It involves setting up different feed gas compositions for plasma ignition, measuring the plasma&#39;s emission spectra, and the subsequent analysis of liquids and cellular activity after plasma treatment.

等离子体医学基础研究--从液体到细胞的吞吐量方法

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive ...

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to &#946;-hexachlorocyclohexane (&#946;-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with &#946;-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

检测暴露于有机氯农药的肝细胞线粒体功能的实验方案

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

Sampling Soils in a Heterogeneous Research Plot

Soils are highly heterogeneous. In general, the number of soil samples required for soil research has always been determined arbitrarily and the associated accuracy is unknown. Here, we present a detailed protocol for efficient and clustered soil sampling in a research plot and, relying on a pilot sampling using this design, for demonstrating soil spatial heterogeneity and informing reasonable sample sizes and associated accuracy for future study. The protocol mainly comprises four steps: sampling design, field collection, soil analysis, and geostatistical analysis. The step-by-step procedure is modified according to former publications. Two examples will be presented to demonstrate contrasting spatial distributions of soil organic carbon (SOC) and soil microbial biomass carbon (MBC) under different management practices. In addition, we present a strategy to determine the sample size requirement (SSR) given a certain level of accuracy based on the plot-level coefficient of variation (CV). The field sampling protocol and the quantitative determination of the sample size will assist researchers in seeking feasible sampling strategies to meet research needs and resources' availability.

The traditional soil-sampling procedure determines the number of soil samples arbitrarily. Here, we provide a simple yet efficient clustered soil-sampling design to demonstrate soil spatial heterogeneity and quantitatively determine the number of soil samples required and the associated sampling accuracy.

在异构研究图中取样土壤

Soils are highly heterogeneous. In general, the number of soil samples required for soil research has always been determined arbitrarily and the ...

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water distribution in plant tissues in combination with freeze fixation techniques using liquid nitrogen (LN2). For woody species, however, preparations for observing the xylem transverse-cut surface involve some difficulties due to the orientation of wood fibers. Additionally, higher tension in the water column in xylem conduits can occasionally cause artifactual changes in water distribution, especially during sample fixation and collection. In this study, we demonstrate an efficient procedure to observe the water distribution within the xylem of woody plants in situ&#160;by using a cryostat and cryo-SEM. At first, during sample collection, measuring the xylem water potential should determine whether high tension is present in the xylem conduits. When the xylem water potential is low (&#60; ca. &#8722;0.5 MPa), a tension relaxation procedure is needed to facilitate better preservation of the water status in xylem conduits during sample freeze fixation. Next, a watertight collar is attached around the tree stem and filled with LN2&#160;for freeze fixation of the water status of xylem. After harvesting, care should be taken to ensure that the sample is preserved frozen while completing the procedures of sample preparation for observation. A cryostat is employed to clearly expose the xylem transverse-cut surface. In cryo-SEM observations, time adjustment for freeze-etching is required to remove frost dust and accentuate the edge of the cell walls on the viewing surface. Our results demonstrate the applicability of cryo-SEM techniques for the observation of water distribution within xylem at cellular and subcellular levels. The combination of cryo-SEM with non-destructive in situ observation techniques will profoundly improve the exploration of woody plant water flow dynamics.

Observing the water distribution within the xylem provides significant information regarding water flow dynamics in woody plants. In this study, we demonstrate the practical approach to observe xylem water distribution in situ&#160;by using a cryostat and cryo-SEM, which eliminates artifactual changes in the water status during sample preparation.

木本植物的赛莱默水分布,用低温扫描电子显微镜可视化

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water ...