Name: protocol for producing three-dimensional infrared video of freezing in plants
Uploaded: 2018-09-12T14:30:00.000+00:00
Duration: 7 min 30 s
Description: Watch this Scientific Journal Video about Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants at JoVE.com

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><tbody><tr><td>T620 Infrared Camera and software</td><td>FLIR</td><td>55903-5122</td><td>2 cameras are needed. Software works only on a Windows-based computer</td></tr><tr><td>After Effects</td><td>Adobe</td><td>15.0.1.73</td><td>Post-Production Video Editing Software</td></tr><tr><td>Bandicam</td><td>Bandisoft</td><td>4.1.2.1385</td><td>Screen Capture Software</td></tr><tr><td>Laboratory Scissor Jack&amp;nbsp;&amp;nbsp;</td><td>Eisco</td><td>CH0642A</td><td>Steel Platform 13X15 cm</td></tr><tr><td>Fastening Strap</td><td>Velcro</td><td>90441</td><td>To hold camera on jack.&amp;nbsp; Should be at least 60cm long by 2cm wide</td></tr><tr><td>Media Converter</td><td>iSkysoft</td><td>10.0.6</td><td>Software to convert mp4 files to .mov&amp;nbsp;</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

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 ...

科研

环境科学

RGB and Spectral Root Imaging for Plant Phenotyping and Physiological Research: Experimental Setup and Imaging Protocols

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop cultivars against environmental stresses. An experimental protocol is presented for RGB and hyperspectral imaging of root systems. The approach uses rhizoboxes where plants grow in natural soil over a longer time to observe fully developed root systems. Experimental settings are exemplified for assessing rhizobox plants under water stress and studying the role of roots. An RGB imaging setup is described for cheap and quick quantification of root development over time. Hyperspectral imaging improves root segmentation from the soil background compared to RGB color based thresholding. The particular strength of hyperspectral imaging is the acquisition of chemometric information on the root-soil system for functional understanding. This is demonstrated with high resolution water content mapping. Spectral imaging however is more complex in image acquisition, processing and analysis compared to the RGB approach. A combination of both methods can optimize a comprehensive assessment of the root system. Application examples integrating root and aboveground traits are given for the context of plant phenotyping and plant physiological research. Further improvement of root imaging can be obtained by optimizing RGB image quality with better illumination using different light sources and by extension of image analysis methods to infer on root zone properties from spectral data.

An experimental protocol is presented for assessment of soil grown plant root systems with RGB and hyperspectral imaging. Combination of RGB image time series with chemometric information from hyperspectral scans optimizes insights into plant root dynamics.

RGB 和光谱根成像植物表型和生理研究: 实验装置和成像协议

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

生物学

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

从长期来看，高解析度激光共聚焦时间流逝成像<em&gt;拟南芥子叶</em&gt;表皮萌发过程中

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

测量时空的Ca<sup&gt; 2 +</sup&gt;在拟南芥信号

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

植物根系的轻质板材荧光显微镜生长在一个凝胶表面

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

一种用于阴南芥叶片安装的通用方法

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

赫连图斯安努斯大型热成像筛选装置新稳压器的鉴定

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Optical Clearing of Plant Tissues for Fluorescence Imaging

It is challenging to directly observe the internal structure of multi-layered and opaque plant specimens, without dissection, under a microscope. In addition, autofluorescence attributed to chlorophyll hampers the observation of fluorescent proteins in plants. For a long time, various clearing reagents have been used to make plants transparent. However, conventional clearing reagents diminish fluorescent signals; therefore, it has not been possible to observe the cellular and intracellular structures with fluorescent proteins. Reagents were developed that can clear plant tissues by removing chlorophyll while maintaining fluorescent protein stability. A detailed protocol is provided here for the optical clearing of plant tissues using clearing reagents, ClearSee (CS) or ClearSeeAlpha (CSA). The preparation of cleared plant tissues involves three steps: fixation, washing, and clearing. Fixation is a crucial step in maintaining the cellular structures and intracellular stability of fluorescent proteins. The incubation time for clearing depends on the tissue type and species. In Arabidopsis thaliana, the time required for clearing with CS was 4 days for leaves and roots, 7 days for seedlings, and 1 month for pistils. CS also required a relatively short time of 4 days to make the gametophytic leaves of Physcomitrium patens transparent. In contrast, pistils in tobacco and torenia produced brown pigment due to oxidation during CS treatment. CSA reduced the brown pigment by preventing oxidation and could make tobacco and torenia pistils transparent, although it took a relatively long time (1 or 2 months). CS and CSA were also compatible with staining using chemical dyes, such as DAPI (4',6-diamidino-2-phenylindole) and Hoechst 33342 for DNA and Calcofluor White, SR2200, and Direct Red 23 for the cell wall. This method can be useful for whole-plant imaging to reveal intact morphology, developmental processes, plant-microbe interactions, and nematode infections.

Here, a method is described for making plant tissues transparent while maintaining the stability of fluorescent proteins. This technique facilitates deep imaging of cleared plant tissues without physical sectioning.

用于荧光成像的植物组织的光学清除

It is challenging to directly observe the internal structure of multi-layered and opaque plant specimens, without dissection, under a microscope. In ...

植物冷冻三维红外视频制作协议

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