LeafJ是由JNM，而他是在休假中的凯瑟琳·波拉德博士的实验室在格拉德斯通研究所。 这项工作是由美国国家科学基金会的资助（批准号IOS-0923752）。

1。植物材料请注意，该的植物生长协议的目的是为避荫反应检测。你最喜欢的条件下可以生长的植物。 <ol><li>洒在9厘米培养皿和存储（分层）在4°C的水浸泡滤纸四天在黑暗中的拟南芥种子。 </li><li>将这些培养皿中传输模拟太阳光条件:80〜100μE光合有效辐射（PAR）和远红光的补充带来的R:FR比为1.86。漫长的一天（16小时光照/ 8小时黑暗）和恒定的温度为22°C。在此条件下培养三天，让种子发芽。 </li><li>发芽的种子，土壤和阳光条件下保持植物。对于大规模的实验，我们建议您准备的小标签来标记每个工厂使用数据合并管理器在Microsoft Word 2004（或更高版本）制作的标签。 </li><li> 11天后TRansfer土壤，将有一半的植物遮荫条件:同样的阳光下，但补充远红光带来的R / FR比0.52。 </li><li>一个额外的12天之后，植物叶成像。在这个阶段，老叶已经完全成熟，而年轻的叶子还在不断扩大，让你捕获快照的发展。您可能需要根据您的需要选择不同的发育时间。 </li></ol> 2。叶片图像捕捉解剖<ol><li>准备与植物的基因型和生长状况与5个矩形框标记的投影胶片。一帧对应于从一种植物的叶子。 Microsoft Excel可以用来打印标签一致的网格。 </li><li>解剖叶，26天老厂。 </li><li>扫描分辨率为600 dpi离开床一台扫描仪。需要注意的是从一种植物叶应垂直放置在透明的薄片三明治，一个黑色的窗口内。避免接触的叶子一个黑色的窗口框架和重叠的树叶，这将给下列程序中的错误。 </li></ol> 3。叶图像分析LeafJ <ol><li>下载ImageJ的拖动LeafJ.jar文件到plugins文件夹中ImageJ的。 </li><li>打开ImageJ的1.45s或更高版本14中的图像文件。 </li><li>图像分割成三个颜色通道（红色，绿色和蓝色）的"图像"&gt;"颜色"&gt;"拆分通道"和应用在蓝色通道的图像的阈值。 </li><li>选择所有的叶子从一个工厂的矩形工具（ 图1A）。 </li><li>选择的插件菜单的"LeafJ"。 </li><li>从出现的对话框中选择"注释信息，为这家工厂。您可以出现在这里点击"编辑选项为:"编辑默认值。 </li><li>运行后LeafJ插件，然后单击"OK"按钮，编辑跟踪线，从该地区的利益（投资回报率）管理器"窗口（如果有必要， 图1B）。在此过程中是很有用的触摸屏平板电脑（如iPad的）。 ipad公司可以连接到一台计算机，外部显示器，使用空气显示软件。 </li><li>导出测量结果和相关信息（文件名，开花时间，解剖，测量等）到Microsoft Excel或相同的软件。 </li></ol> 4。 ImageJ的叶细胞图像分析<ol><li>修复解剖离开扫描（步骤2）后，如参考文献15中描述的。至少6个月，FAA固定叶片可以保存在4°C。 </li><li> FAA固定液，通过改变水合氯醛溶液，并培育叶1〜2显微镜观察15小时前，清除落叶。 </li><li>山叶英里croscope滑动皮毛朝上。使用复合式显微镜，图像叶肉层的每一片叶子的中心主脉两侧的40倍的放大倍率，避免皮毛或静脉附近的细胞。 </li><li>微量叶细胞概述ImageJ的投资回报率管理工具，借助触摸屏的平板电脑和手写笔（如在步骤3）。细胞图像分析使用ImageJ的内置功能，但不需要LeafJ。 </li></ol>

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没有利益冲突的声明。

我们的"LeafJ"的插件，使测量叶柄长半，自动比手动测量吞吐量提高了近6倍。叶柄长是SAS的一个重要指标，也是一个具有里程碑意义的现象，如淹没性与偏下的增长17。因此，这个插件可能是有用的范围广泛的植物研究。 我们的插件来实现在一个完善的基于Java的免费软件，ImageJ的。这使得可以轻松跨平台的安装。易于修改程序也是一个优势的LeafJ插件的ImageJ的，因为已经有一个大的库写的插件，Java和ImageJ的宏语言（ <a href="http://imagejdocu.tudor.lu/doku.php?id=tutorial:start" target="_blank">http://imagejdocu.tudor.lu/doku.php?id=tutorial :启动</a> ）。目前，我们只测试了拟南芥的叶子，但我们的叶柄/叶片边界检测的算法，可申请吨o其他双子叶植物的叶子做一些修改后的插件。 LeafJ插件在测试过程中，我们发现大多数的14个错误来自人为错误，如乱放复制结果数据表和/或贴错标签的植物基因型。在极少数情况下，叶柄/叶片边界被称为不正确，需要手动校正和创造更多的复制和粘贴错误的风险。后，我们可以发现这样的错误数据（a）阈值的值（ 例如，叶柄长度大于叶片长度）和（二）发现重复的样本条件（ 例如 "太阳"或"阴影"），基因型，或叶片的位置。 我们的触摸屏平板法有利于测量的精度和速度。我们的方法的局限性是，无线局域网络（LAN）的速度依赖于主计算机之间的通信和在触摸屏平板。

<html><table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td></td></tr><tr><td>远红光LED </td><td> Orbitec </td><td>客户定制</td><td></td></tr><tr><td>透明度</td><td> IKON </td><td> HSCA / 5 </td><td></td></tr><tr><td>扫描器</td><td>爱普生</td><td>爱普生Perfection V700 PHOTO </td><td></td></tr><tr><td>图像J </td><td>美国国立卫生研究院</td><td></td><td> <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> </td></tr><tr><td> LeafJ </td><td>习惯</td><td></td><td> <a href="http://www.openwetware.org/wiki/Maloof_Lab" target="_blank">http://www.openwetware.org/wiki/Maloof_Lab</a> </td></tr><tr><td>空气显示</td><td> Avatron软件公司</td><td></td><td>空白的"http://avatron.com/ </td></tr><tr><td> iPad2的</td><td>苹果公司</td><td></td><td> <a href="http://www.apple.com/" target="_blank">http://www.apple.com/</a> </td></tr></tbody></table>

leafj: an imagej plugin for semi-automated leaf shape measurement

高通量分型（组学）是一个功能强大的工具，用于连接的基因，其功能（见综述1，最近的例子2-4）。叶片是主要的光合器官，并且它们的大小和形状各不相同发育和工厂内的环境。出于这些原因，叶片形态研究需要从众多的叶片，这最好是通过半自动化组学工具5,6的多个参数的测量。遮荫环境是一个重要的线索，影响植物的建筑和生活的历史套件的反应统称为避荫综合症（SAS）7。在SAS响应，遮阳诱导叶柄伸长和叶片面积的变化作为指标8是特别有用的。至目前为止，叶形方案（ 如形状层10，LeafAnalyzer 11，LEAFPROCESSOR 12）可以测量叶片轮廓和分类 ​​，叶形，但不能输出叶柄长度。叶柄缺乏大型测量系统的抑制SAS组学的方法来研究。在本文中，我们将介绍一种新开发的ImageJ的插件，称为LeafJ，它可以快速测量模式植物拟南芥叶柄长，叶片参数。偶尔叶的叶柄/叶片边界，需要手动校正我们使用的是触摸屏平板。此外，叶片细胞的形状和，叶细胞数量是重要的决定因素，叶片大小13。从LeafJ分离，我们也提出为测量细胞的形状，面积和尺寸的触摸屏平板电脑使用的协议。我们的叶性状测量系统不限于阴凉处，避免研究，并会加速叶片的许多突变体的表型筛选植物叶表型。

1。叶片图像显示的叶柄和叶片边界的估计，其测量窗口的LeafJ最有用的功能之一是自动检测的叶片/叶柄边界的（ 图1）。 LeafJ算法的工作原理如下:内置ImageJ的ParticleAnalyzer功能中被用来寻找并确定内的用户选择的叶子的取向。对于每片叶子的叶的宽度确定沿叶片的整个轴。使用运行的窗口（为7近端到焦点位置的位置的平均宽度的平均宽度为7到焦点位置的远侧位置减去），然后沿该轴的每个位置处，宽度变化的确定。叶柄/刀片边界被定义为所述第一位置以外的叶片基部，宽度变化的是大于90％的所有计算的宽度差。 LeafJ额外的检查，以提高reliabi附录一般资料的此呼叫;具体LeafJ也要求1）的宽度超过的最窄的5％的长度的位置（这可防止异常呼叫在叶柄基部）; 2）近端到焦点位置的区域不会有重大的变化的宽度，以及3）到焦点位置的远侧叶的20％的宽度，比在焦点位置（真宽是至少为150％，如果是焦点位置的边界，因为20％的近端边界应该是叶片和因此更广泛的）。 一旦LeafJ已定义的叶柄/刀片边界，内置在ImageJ的类和方法被用于确定叶片的面积，周长，和圆形。内置ImageJ的方法也用于以适合一个椭圆形的叶片，并计算（然后用作叶片的长度和宽度），椭圆的长轴和短轴。叶柄长度确定由一条线，跟踪沿叶柄区域的中心。 2。检测的阴影引起的叶柄伸长率&lt;/ P&gt; 要问，是否LeafJ测量用于测定避荫，我们采用了混合效应模型的治疗和叶片数作为随机效应为固定效应和复制。我们发现，叶柄长，叶片面积，叶片长，叶片宽，叶柄长/叶片长度比显着影响的遮光处理，而刀片圆刀片长度/刀片宽度比（P &lt;0.05）。我们的数据表明，LeafJ插件是用于叶避荫反应的研究（ 图2）。 3。精度和速度的LeafJ插件要确定的LeafJ插件在更大的数据集的性能，我们测量手册和插件的运行时间和精度之间比较。对于手工测量，我们叶柄/刀片边界定义为叶宽的地方出现了迅速增加。花了一个经验丰富的研究员平均水平的1<html> 9分3秒来衡量一个透明度与五家工厂（约50叶）由手工测量，而与LeafJ只用了3分20秒。平均5透明胶片，测量与插件5.7倍的速度比手工测量。手工测量是由一个有经验的研究人员在使许多手工测量;，初学者将大大慢于手工测量，从而在更大的优势LeafJ。我们评估精度的数据进行比较，从两种方法;高度相关的所有叶参数数据（ 图3）。 3,532个数据点有172（4.9％），表现出较强的方法（证明的相关图上的异常值）之间的差异。我们分析了这些异常的原因。 172离群，有29人由于在LeafJ插件测量误差和143名由于手工测量误差。这个错误的分析结果也显示插件测量的准确性。 jove_step"&gt; 4。细胞大小和细胞数测定独立的LeafJ，我们还开发了一种高效的工作流程，为测量细胞的数量和规模。理论上，细胞的数量和大小可以被用来分类成九类突变体植物时，与野生型相比，（1）更小的细胞大小与细胞数减少，（2）更小的细胞与正常细胞的数量，（3）增加细胞的小细胞号码，（4）细胞的正常细胞数下降的大小与正常细胞与正常细胞的数量的大小（5），（6）细胞数下降的大小与正常细胞，（7）细胞数减少单元尺寸较大，（8）放大细胞与正常细胞的数量的大小，（9）较大的细胞的细胞数增加的大小与15,16。我们测量了叶细胞在67个基因型的拟南芥（Arabidopsis thaliana）片剂为基础的细胞大小和细胞数测定方法的参数。我们测量了877树叶的224家工厂的8629细胞的细胞大小。乘细胞密度bŸ叶面积（测量LeafJ），我们估计438从219种植物叶的叶总细胞数的数据。我们分析这些基因型的可能九大类（F IG 4）6。 （5）:正常细胞的大小和数量最大的一类;第二大（8）:较大的细胞与正常细胞的数量规模。虽然后续工作是必要的，这表明，我们的片剂为基础的方法可以用于进行分类，根据叶细胞的大小和数量的突变体。 <img alt="图1" fo:src="/files/ftp_upload/50028/50028fig1highres.jpg" src="/files/ftp_upload/50028/50028fig1.jpg" /> 图1。叶柄/的叶片边界检测和用户界面的一个例子。需要注意的是LeafJ是能够自动定义叶柄/叶片边界（ 图1B）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50028/50028fig1large.jpg" target="_blank">点击这里查看大figu的</a>重。 <img alt="图2" fo:src="/files/ftp_upload/50028/50028fig2highres.jpg" src="/files/ftp_upload/50028/50028fig2.jpg" /> 图2。LeafJ的插件可以检测到避荫反应在各种叶参。叶3到叶片6从7（阳光和阴影）的各条件下的野生型植物（ 拟南芥 Columbia生态型）进行了研究。从左上方到右下方，y轴的单位是毫米，毫米，毫米，2毫米，比叶柄长叶片长度，叶片长度，叶片宽度的比例。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50028/50028fig2large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图3" fo:src="/files/ftp_upload/50028/50028fig3highres.jpg" src="/files/ftp_upload/50028/50028fig3.jpg" /> 图3。LeafJ插件是非常准确的。 Ç从手工测量和LeafJ的插件测量从3,532个数据点的数据orrelation。每个点代表一个叶。绿色圆点表示170此图中的离群值。轴的叶面积为mm 2所 ​​有的人都毫米。每个参数前面的数字代表叶位置（ 即 "3"是第三片叶子）。 <img alt="图4" fo:src="/files/ftp_upload/50028/50028fig4highres.jpg" src="/files/ftp_upload/50028/50028fig4.jpg" /> 图4。67基因型的拟南芥叶细胞面积及叶细胞数量之间的散点图。每个点代表每个遮荫条件下生长的基因的表型。植物分为九大类，根据他们的从野生型（Col）所确定的线性混合效应模型和多个测试校正P值的差异:（1​​）较小的细胞大小，细胞数量减少，（2）小细胞规模与规范人细胞数，（3）更小的细胞大小与细胞数量增加，（4）细胞数下降的大小与正常细胞，（5）正常细胞与正常细胞的数量的大小，（6）细胞数下降的大小与正常细胞，（7 ），较大的细胞大小与细胞数降低，较大的细胞与正常细胞的数量的大小（8），（9）细胞数增加较大的细胞大小与。 "*"表示野生型（Col）的表型。

Watch this Scientific Journal Video about LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement at JoVE.com

LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement

示范的高吞吐量叶测量的关键方法。这些方法可以用来加速学习时，许多植物叶表型的突变体或以其他方式筛选植物叶表型。

LeafJ:ImageJ的插件半自动叶形状测量

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the ...

leafj-an-imagej-plugin-for-semi-automated-leaf-shape-measurement

Nanyang Technological University

Research

JoVE Journal

Biology

28.2K 次观看.  University of California Davis. 示范的高吞吐量叶测量的关键方法。这些方法可以用来加速学习时，许多植物叶表型的突变体或以其他方式筛选植物叶表型。

视频: LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

自制的网站定向诱变整个质粒

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

科研

生物学

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

半自动小红桃光学心跳分析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (&Psi;leaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol &bull; m-2 &bull; s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- &Delta;&psi;leaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

测量叶片导水率和气孔导度和他们的反应，辐射和脱水蒸发通量法（EFM）

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

使用高通量形状的RNA二级结构预测

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

对于外来体定量和尺寸测量新方法研究

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

在对飞行性能的准确，快速测定方法的改进<em&gt;果蝇</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

组织特异性荧光的斑马鱼胚胎半自动成像

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Relating Stomatal Conductance to Leaf Functional Traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.