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>

<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?...

<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...

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

Research

JoVE Journal

Biology

28.1K 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

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 ...

科研

生物学

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 ...

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.

与气孔导度，以叶功能性状

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis algorithms which can be used effectively to count various biological particles. When counting large numbers of cell samples, the hemocytometer presents a bottleneck with regards to time. Likewise, counting membranes from migration/invasion assays with the ImageJ plugin Cell Counter, although accurate, is exceptionally labor intensive, subjective, and infamous for causing wrist pain. To address this need, we developed two plugins within ImageJ for the sole task of automated hemocytometer (or known volume) and migration/invasion cell counting. Both plugins rely on the ability to acquire high quality micrographs with minimal background. They are easy to use and optimized for quick counting and analysis of large sample sizes with built-in analysis tools to help calibration of counts. By combining the core principles of Cell Counter with an automated counting algorithm and post-counting analysis, this greatly increases the ease with which migration assays can be processed without any loss of accuracy.

This paper describes the quantification of hemocytometer and migration/invasion micrographs through two new open-source ImageJ plugins Cell Concentration Calculator and migration assay Counter. Furthermore, it describes image acquisition and calibration protocols as well as discusses in detail the input requirements of the plugins.

自动定量分析细胞计数程序使用ImageJ插件

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis ...

Clock Scan Protocol for Image Analysis: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na+ or Ca++ within a single cell, as well as to the analysis of spreading activity (e.g., Ca++ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

用于图像分析的时钟扫描协议：ImageJ插件

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...

小鼠白色脂肪组织的半自动分离

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

作者聚焦：使用组织解离剂从小鼠白色脂肪组织中半自动分离基质血管组分  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...