这项工作得到了美国农业部内部资金的支持。

1. 准备
<ol><li>收集设备、材料和软件, 记录和处理工厂冷冻的视频。<ol>
<li>通过将电源开关设置为On来启动可编程冰箱, 并将温度设置为0摄氏度。程序冰箱达到-8 °c 在1摄氏度/小时。</li>
	</ol>
</li>
	<li>将一个6周大的草莓植物和 2-5 花种植在一个1升的容器中, 放入冰箱中。</li>
	<li>使用紧固带和一小块木头设置2台红外摄像机 (例如FLIR T620 相机), 以产生正确的透镜会聚角。 注: 最佳距离空间的镜头中心的2相机通常被认为是相同的距离之间的眼睛1或约7厘米。</li>
	<li>安装两个摄像头到一个 10 x 10 cm 实验室杰克和位置的杰克在冰箱足够接近的植物, 让图像集中。垂直和水平调整相机, 使同一部分的植物是可见的两个摄像头。使用插孔垂直定位两个摄像头, 这样的方式, 2 图像包含整个植物和土壤的一部分。</li>
	<li>使用 usb 连接器将2摄像头连接到计算机上的 usb 插座。</li>
	<li>将2个/C 插座插入两个摄像头, 以允许对植物进行连续的监测。</li>
</ol>2. 记录的计算机和软件设置
<ol><li>双击红外摄像机软件2x 的图标, 打开2个窗口 (每个照相机的1个窗口)。按照 "帮助" 菜单中的说明, 将左侧的照相机与右窗口中的右摄像头连接。 注意: 使用该软件的详细信息可以通过 "帮助" 菜单进行访问。单色调色板最适合此示例, 因为需要使用红色蓝色着色来进行3D 渲染。</li>
	<li>双击该程序的图标, 打开屏幕捕获软件。通过单击并拖动帧来调整捕获帧, 以便同时包括两个摄像头, 同时允许同时对两个照相机进行屏幕捕获。 注意: 从两个摄像头同时捕获视频流是非常关键的, 因为它允许左右视图的完美同步。</li>
	<li>以3小时为增量录制视频, 以便在视频处理软件中进行更简单的处理。 注意: 不可能确切地知道植物何时结冰, 所以在冰冻事件之前记录一段时间是很重要的。在段中记录的选项是该软件的一个功能, 因此建议将其设置为3小时记录。该软件将自动保存3小时的录音, 然后开始一个新的录音。每个3小时记录的文件将自动给出一个数字序列后的名称。每个视频文件将从10到 20 GB, 因此请确保硬盘驱动器上有足够的空间用于此大小的多个文件。</li>
	<li>通过在 "控制器" 菜单中选择 "运行" 并开始屏幕捕获, 启动冰柜程序。然后按窗口上的 "录制" 按钮。确保显示被捕获屏幕区域的轮廓变为红色。</li>
	<li>将草莓植株冷冻至-8 摄氏度, 并将冷冻机的温度保持在1小时。</li>
	<li>将冰箱的温度提高2摄氏度/小时, 直到冷冻机在 +2 摄氏度。停止录音。 注: 总冻结时间为14小时。</li>
	<li>使用文件转换软件将感兴趣的文件从. mp4 格式转换为. mov。 注意: 在这种情况下, 将使用包含1个或更多冻结事件的单个 3 h 文件。</li>
</ol>3. 使用视频影像软件处理视频
注: 视频成像软件将在本例中使用。有关如何使用该软件的教程可在网上获得。本示例将假定软件的基本知识。假设对 "组合"、"层" 和 "渲染队列" 等术语的理解, 以及各种面板以及如何操作它们。
<ol><li>双击 "项目" 面板中的任意位置, 将感兴趣的. mov 文件导入到图像软件中, 并将该文件拖到 "项目" 面板底部的 "组合" 图标中。然后将项目保存到包含原始视频的同一文件夹中。 注意: 录制的视频将在预览窗格中显示。</li>
	<li>单击 "预览" 窗口底部的 "感兴趣区域" 图标, 并使用游标仅勾勒出左侧照相机的录制。</li>
	<li>拖动相同的. mov 视频到组合图标, 创建同一视频的第二个组合。重复步骤 3.3, 但是这次, 使用游标只选择正确的相机。</li>
	<li>选择 "组合" > "裁剪复合" 到左侧视图感兴趣的区域。对正确的视图重复此操作。重命名每个组合, 以指示哪些是左和右。</li>
	<li>通过单击它并在顶部的主菜单中选择 "组合" > "添加到呈现队列" 来突出显示左组合。</li>
	<li>在渲染队列中, 单击输出模块, 并确保视频将呈现为视频 (例如, QuickTime 视频)。单击渲染设置以减少分辨率以允许更快的渲染。单击 "输出", 将视频的名称命名为左侧, 并将其保存到与原始录制和项目相同的文件夹中。单击 "保存", 然后单击呈现面板右上侧的 "呈现" 按钮。</li>
	<li>重复步骤3.6 的草莓正确的成分。</li>
	<li>双击 "项目" 面板, 然后导入刚刚呈现的草莓左侧和草莓的正确视频。</li>
	<li>突出显示两个视频并将其拖动到 "项目" 面板底部的 "组合" 图标中。在要求静止持续时间的弹出式屏幕中, 以5个零为3小时的持续时间输入3。 注意: 两个视频 (精确同步) 将在 "项目" 面板中, 但只有 "组合" 面板中最顶层的视频才可见。</li>
	<li>要查看其他图像, 请单击小眼球关闭图层。按下控制/W以允许在预览面板中使用光标旋转控制图像。使用光标并单击顶层上移和关闭, 调整顶部或底部视图的旋转方面, 以确保两个图像位于同一旋转平面中。然后在3D 眼镜子程序中直接调整 X 和 Y 平面。</li>
	<li>突出显示 "组合" 面板的顶层, 然后从顶部的菜单中选择 "效果" > "透视" > 3D 眼镜。 注: 3D 眼镜效果的参数将在控制面板中弹出。</li>
	<li>在 "控制面板" 中, 单击 "左视图" 右侧的框-如果控制面板未与 "项目" 面板分离, 请单击 "项目" 面板顶部的 "控制面板" 选项卡。在下拉菜单中列出 "组合" 面板中的2视频, 突出显示 "左视图" 列表中的视频。对 "右视图" 重复此步骤。</li>
	<li>在3D 视图右侧的框中, 选择红色蓝色 LR。</li>
	<li>使用红蓝眼镜, 检查项目面板中的视图。如果3D 视图看起来不正确, 请尝试单击 "换行左对"。调整场景收敛和垂直对准, 以消除任何重影和眼张力。</li>
	<li>当视频的3D 方面是可接受的, 通过单击它并选择 "组合" > "添加到呈现队列" (如步骤3.7 中所做的) 来突出显示组合。将视频呈现到项目中其他文件所在的文件夹中。 注意: 此文件将相当大。文件呈现后, 可以使用视频处理软件将其重新呈现为较小的文件大小。</li>
</ol>

<ol>
	<li>Geng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+display+technologies.">Three-dimensional display technologies.</a> Advances in Optics and Photonics. 5, 456-535 (2013).</li><li>Ceccardi, T. L., Heath, R. L., Ting, I. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-temperature+exotherm+measurement+using+infrared+thermography.">Low-temperature exotherm measurement using infrared thermography.</a> HortScience. 30, 140-142 (1995).</li><li>Wimmer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> History of thermal imaging, Security Sales and Integration. , (2011).</li><li>Wisniewski, M., Lindow, S. E., Ashworth, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+of+ice+nucleation+and+propagation+in+plants+using+infrared+video+thermography.">Observations of ice nucleation and propagation in plants using infrared video thermography.</a> Plant Physiology. 113, 327-334 (1997).</li><li>Kuprian, E., Tuong, T., Pfaller, K., Livingston, D. P., Neuner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+supercooling+of+reproductive+shoots+is+enabled+by+structural+ice+barriers+being+active+despite+an+intact+xylem+connection.">Persistent supercooling of reproductive shoots is enabled by structural ice barriers being active despite an intact xylem connection.</a> Public Library of Science ONE. 11, e0163160 (2016).</li><li>Livingston, D. P., Tuong, T. D., Murphy, J. P., Gusta, L., Wisniewski, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-definition+infrared+thermography+of+ice+nucleation+and+propagation+in+wheat+under+natural+frost+conditions+and+controlled+freezing.">High-definition infrared thermography of ice nucleation and propagation in wheat under natural frost conditions and controlled freezing.</a> Planta. 247, 791-806 (2017).</li><li>Boyles, R. P., Raman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+climate+patterns+and+trends+in+North+Carolina+(1949-1998).">Analysis of climate patterns and trends in North Carolina (1949-1998).</a> Environment International. 29 (2-3), 263-275 (2003).</li><li>Poling, E. B., Poling, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Managing+Cold+Events.">Managing Cold Events.</a> A Growers' Guide to Production, Economics and Marketing. , 75-97 (2015).</li><li>Hacker, J., Neuner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice+porpagaion+in+plants+visualized+at+the+tissue+level+by+infrared+differential+thermal+analysis+(IDTA).">Ice porpagaion in plants visualized at the tissue level by infrared differential thermal analysis (IDTA).</a> Tree Physiology. 27, 1661-1670 (2007).</li><li>He, J. Q., Harrison, R. J., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+3D+imaging+system+for+strawberry+phenotyping.">A novel 3D imaging system for strawberry phenotyping.</a> Plant Methods. 13, 93-101 (2017).</li></ol>

作者没有什么可透露的。

这项协议需要两台红外摄像机, 它们必须针对这个主题, 从稍微不同的角度1。这将要求透镜从 5-8 cm 分开, 但两个必须瞄准在将被摄制的主题的同一个地方。把2相机镜头看成是观众眼中的一种替代品。左摄像头类似左眼和右眼相机。后处理软件将色调的左图像的红色和正确的图像, 以蓝色的颜色, 所以通过穿红蓝眼镜, 左眼只能看到左图像和右眼只有正确的图像。这意味着在记录冻...

尽管生活在一个三物理维度的世界, 研究人员通常只限于在2D 中报告视觉观察。虽然2D 图像通常足以传达重要信息, 但这种缺乏深度的信息限制了我们感知和理解真实世界物体的复杂性的能力。1
这一缺乏的深度信息提供了一个动机, 以产生3D 视频主要是在商业电影行业, 因为早期的 1900s1。然而, 在静止图像和视频中生成清晰的3D 信息, 由于生成这些图像所涉及的复杂性而受到阻碍。最简单的生成3D 胶片的方法是基于立体摄影的原理。立体摄影利用两个相同物体的图像, 从略微不同的角度, 在大脑中传递3D 图像。为了使这成为可能, 每只眼睛必须只看它各自的图像 (即左眼在左图和右眼在正确的图像)。因为眼睛不会自然地做这, 立体头饰设计使这成为可能1。在3D 胶片的开发过程中, 采用了几种立体视觉技术, 以及偏振交错、时间复用和头贴显示技术, 但采用红绿 (或青色) 的颜色交错或浮雕方法。眼镜是最简单和最便宜的技术之一。有关3D 成像和所涉及的各种技术的全面审查, 请参阅耿1的评论。
利用红外热像仪对植物中的冰冻进行监测是基于水结冰时, 必须放弃内部能量2的原理。这种能量是以热的形式存在的, 在电磁波谱的 IR 区域是可探测的。自 1929年3以来, 能够记录红外线能量的摄像机已经在使用。首次发表的报告使用红外技术的电影冷冻在植物是从 Cecardi等。2, 但使用的相机的分辨率使得很难准确地确定冻结的组织。维希涅夫斯基等。4用更高分辨率的相机确定了几种植物中冰核的更精确的位置。随着红外热成像技术的改进, 高分辨率图像导致了诸如冻结5的障碍和冰层形成6的精确细胞定位等发现。
红外摄影中的一个难点是由于温差小。这将导致视图字段中的大多数对象都是相似的颜色, 这使得很难精确确定哪些对象是/正在冻结。这在确定特定组织中冷冻的顺序时很重要, 例如小麦的叶子或根部6。如果植物的红外视频可以在3D 中成像, 那么在某一时间点确定该植物的哪一部分是结冰的准确度就能得到改善。
在美国的某些地区, 草莓是一种作物, 在这种情况下, 冷冻温度对种植者相当关注。在某些生长条件下, 草莓花在平均去年春季冷冻前 2-3 周出现。在阿巴拉契亚山脉的某些地区, 冰冻事件可能会发生在7 , 通常会导致花朵死亡。因此, 霜冻保护对草莓种植者在受这些冰冻事件的地区至关重要。例如, 在北卡罗来纳州, 草莓种植者必须在 4-6 霜冻事件发生前和 1-2 在早期绽放期8期间冻结, 以防止霜冻。为了帮助开发更耐冻的草莓基因型, 重要的是要了解冰冻的各个方面, 如冰核的部位和传播到植物的其他部分。红外热成像为解决这些问题提供了有效的手段。
在这里, 我们使用草莓来说明在3D 使用浮雕方法记录冰冻事件的技术。草莓很适合这个例子, 因为树叶和花朵分布在3D 空间, 在2D 红外视频中观看时很难区分。

<table border="0" cellpadding="0" cellspacing="0" width="811">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">T620 Infrared Camera and software</td>
			<td style="width:71px;">FLIR</td>
			<td style="width:119px;">55903-5122</td>
			<td style="width:323px;">2 cameras are needed. Software works only on a Windows-based computer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:299px;">After Effects</td>
			<td style="width:71px;">Adobe</td>
			<td style="width:119px;">15.0.1.73</td>
			<td style="width:323px;">Post-Production Video Editing Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Bandicam</td>
			<td style="width:71px;">Bandisoft</td>
			<td style="width:119px;">4.1.2.1385</td>
			<td>Screen Capture Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Laboratory Scissor Jack&#160;&#160;</td>
			<td style="width:71px;">Eisco</td>
			<td style="width:119px;">CH0642A</td>
			<td>Steel Platform 13X15 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Fastening Strap</td>
			<td style="width:71px;">Velcro</td>
			<td style="width:119px;">90441</td>
			<td>To hold camera on jack.&#160; Should be at least 60cm long by 2cm wide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Media Converter</td>
			<td style="width:71px;">iSkysoft</td>
			<td style="width:119px;">10.0.6</td>
			<td>Software to convert mp4 files to .mov&#160;</td>
		</tr>
	</tbody>
</table>

protocol for producing three-dimensional infrared video of freezing in plants

植物中的冰冻可以用红外线 (IR) 热成像来监测, 因为当水结冰时, 它会发出热量。然而, 色彩对比的问题使得2维 (2D) 红外图像难以解释。在3维度 (3D) 中查看红外图像或植物的视频可以更准确地识别冰核的位置, 以及冻结的进展。在本文中, 我们展示了一个相对简单的方法来生产一个3D 红外视频的草莓植物冷冻。草莓是一种经济上重要的作物, 在世界许多地区遭受意想不到的春季冰冻事件。准确了解草莓冷冻将为育种者和种植者提供更经济的方法, 以防止在冰冻条件下对植物造成任何损害。
该技术涉及两个红外摄像机的定位, 在略微不同的角度, 以拍摄草莓冷冻。两个视频流将精确地同步使用屏幕捕获软件, 同时记录两个摄像头。录音将被导入到成像软件中, 并使用浮雕技术进行处理。使用红蓝色的眼镜, 3D 视频将使其更容易确定的精确地点的冰核在叶片表面。

令人惊讶的是, 草莓植物冷冻的红外视频 (补充视频 1) 表明, 并非所有的叶子/花朵冻结在同一时间。叶子和花朵都在不同的温度下单独冻结, 但树叶比花更早结冰, 温度更高。此外, 冷冻开始于叶子, 但不一定在同一位置上的每一叶。虽然这些结果没有被描述以前在草莓, 相似的结果被发现了在其他植物种类6。一旦树叶结冰, 冰就会沿着叶柄向?...

Watch this Scientific Journal Video about Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants at JoVE.com

Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants

在这里, 我们提出一个协议, 以图像的草莓植物冻结在3维度。两个红外摄像机定位在略微不同的角度被用来产生一个红蓝浮雕视频, 以观察冷冻的植物在3维度。

植物冷冻三维红外视频制作协议

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast ...

protocol-for-producing-three-dimensional-infrared-video-freezing

Research

JoVE Journal

Environment

6.1K 次观看.  United States Department of Agriculture and North Carolina State University. 在这里, 我们提出一个协议, 以图像的草莓植物冻结在3维度。两个红外摄像机定位在略微不同的角度被用来产生一个红蓝浮雕视频, 以观察冷冻的植物在3维度。

视频: Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.

Understanding the role of environmental heterogeneity in species coexistence has typically focused on types of heterogeneity that are extrinsic to the community&#8217;s species composition. We provide novel detailed methods for creating soil heterogeneity treatments using soils subject to plant-soil feedback conditioning, or heterogeneity intrinsic to the community composition.

用于操纵植物诱导土壤异质性的实验协议

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as ...

科研

环境科学

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

重组纤维素酶Cel5A从表达<em&gt;里氏木霉</em&gt;在烟草

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

植物与放射性示踪剂测量矿质营养和毒物的通量

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

高分辨率红外热成像（HRIT）的冰核和冰传播的植物研究中的应用

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

Extracting Metrics for Three-dimensional Root Systems: Volume and Surface Analysis from In-soil X-ray Computed Tomography Data

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to climate change and crop management. Quantitative size information on roots in their native environment is invaluable for studying root growth and environmental processes involving plants. X-ray computed tomography (XCT) has been demonstrated to be an effective tool for in situ root scanning and analysis. We aimed to develop a costless and efficient tool that approximates the surface and volume of the root regardless of its shape from three-dimensional (3D) tomography data. The root structure of a Prairie dropseed (Sporobolus heterolepis) specimen was imaged using XCT. The root was reconstructed, and the primary root structure was extracted from the data using a combination of licensed and open-source software. An isosurface polygonal mesh was then created for ease of analysis. We have developed the standalone application imeshJ, generated in MATLAB1, to calculate root volume and surface area from the mesh. The outputs of imeshJ are surface area (in mm2) and the volume (in mm3). The process, utilizing a unique combination of tools from imaging to quantitative root analysis, is described. A combination of XCT and open-source software proved to be a powerful combination to noninvasively image plant root samples, segment root data, and extract quantitative information from the 3D data. This methodology of processing 3D data should be applicable to other material/sample systems where there is connectivity between components of similar X-ray attenuation and difficulties arise with segmentation.

A methodology for obtaining visual and quantitative root structure information from X-ray computed tomography data acquired in-soil is presented.

从在土X射线计算机断层扫描数据量和表面分析:提取指标三维根系

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to ...

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey bee colony numbers have been declining in the United States and many European countries since 1947. A number of factors play a role in this decline, including the unintentional exposure of honey bees to pesticides. The development of new methods and regulations are warranted to reduce pesticide exposures to these pollinators. One approach is the use of repellent chemistries that deter honey bees from a recently pesticide-treated crop. Here, we describe a protocol to discern the deterrence of honey bees exposed to select repellent chemistries. Honey bee foragers are collected and starved overnight in an incubator 15 h prior to testing. Individual honey bees are placed into Petri dishes that have either a sugar-agarose cube (control treatment) or sugar-agarose-compound cube (repellent treatment) placed into the middle of the dish. The Petri dish serves as the arena that is placed under a camera in a light box to record the honey bee locomotor activities using video tracking software. A total of 8 control and 8 repellent treatments were analyzed for a 10 min period with each treatment was duplicated with new honey bees. Here, we demonstrate that honey bees are deterred from the sugar-agarose cubes with a compound treatment whereas honey bees are attracted to the sugar-agarose cubes without an added compound.

The loss of honey bee colonies presents a challenge to crop pollination services. Current pollinator protection practices warrant an alternative approach to minimize the contact of honey bees to harmful pesticides using repellent chemistries. Here, we provide detailed methods for a visual tracking protocol to screen deterrents for bees.

用于蜂蜜蜜蜂筛选化学成分的视频跟踪协议

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey ...

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.

Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity.

纳米材料在水介质中的分散: 对协议优化的研究

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

利用水下立体视频工具量化鱼类密度的新方法研究进展

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

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 ...

Combining Eye-tracking Data with an Analysis of Video Content from Free-viewing a Video of a Walk in an Urban Park Environment

As individuals increasingly live in cities, methods to study their everyday movements and the data that can be collected becomes important and valuable. Eye-tracking informatics are known to connect to a range of feelings, health conditions, mental states and actions. But because vision is the result of constant eye-movements, teasing out what is important from what is noise is complex and data intensive. Furthermore, a significant challenge is controlling for what people look at compared to what is presented to them.
The following presents a methodology for combining and analyzing eye-tracking on a video of a natural and complex scene with a machine learning technique for analyzing the content of the video. In the protocol we focus on analyzing data from filmed videos, how a video can be best used to record participants&#39; eye-tracking data, and importantly how the content of the video can be analyzed and combined with the eye-tracking data. We present a brief summary of the results and a discussion of the potential of the method for further studies in complex environments.

The objective of the protocol is to detail how to collect video data for use in the laboratory; how to record eye-tracking data of participants looking at the data and how to efficiently analyze the content of the videos that they were looking at using a machine learning technique.

将眼动追踪数据与从自由观看城市公园环境中的步行视频中获得的视频内容进行分析

As individuals increasingly live in cities, methods to study their everyday movements and the data that can be collected becomes important and valuable. ...