We are grateful to B. Stevenson for technical assistance and Dr Marc R. Knight for providing Aequorin transgenic line. This work was funded by the National Institutes of Health Grant R01 GM059138.

1，基于水母发光蛋白钙成像使用FAS系统<ol><li>准备苗木发光成像。消毒拟南芥植物表达水母发光蛋白含0.01％的Triton-100 10％的漂白粉溶液的种子。播下含有全强度MS（Murashige和Skoog基础盐混合物），1％蔗糖和1.2％琼脂的无菌种子在方形板（10×10厘米的正方形培养皿格）。代替板垂直地在分层后培养室中于4℃下进行2天（ 图1A）。 </li><li>转移到苗木电影。在7-10天龄幼苗的生长板的顶部放置一个粘合膜（ 图1B）。轻轻地用手推片，以确保苗附着于膜（ 图1C）。剥离该膜轻轻使得幼苗保持粘附于膜（ 图1D和1E） </li><li>孵育辅苗。将在Adhered幼苗在含正方形板（10×10厘米）将15ml 2微克/毫升H-CTZ（腔肠素）的水。孵育秧苗在室温下搅拌4小时至过夜（ 图1F）。 </li><li>准备发光成像。就拿拍出来的H-CTZ的解决方案，并剪下来的中间，形成两片。将每一块膜用苗面对两种不同的板块。留在暗处将板5分钟。 </li><li>获得发光图像。在黑暗中，将两个板彼此相邻的发光成像系统（ 图1G）的阶段。后，立即同时加入20ml的刺激溶液于板获得图像。 </li><li>分析发光图像。选择相同的显示范围为所有发光图像。作物感兴趣区域（ROI）的区域，并生成图像，JPEG文件（ 图2A）。另外，导出图像作为固相萃取格式的文件，并将其导入到ImageJ的图像分析软件。设置测量投资回报率的平均灰度值的计算。选择ROI区域的大小相同，并测量平均灰度和本数据作为柱状图（ 图2B）。 </li></ol> 2，活细胞共聚焦钙成像<ol><li>准备苗木共聚焦成像。消毒拟南芥植物表达case12含0.01％的Triton 10％的漂白粉溶液的种子。母猪在含有全量MS盐类，1％蔗糖，1.2％琼脂平板无菌种子。代替板垂直地在分层后培养室中于4℃下进行2天。 </li><li>安装成像钱伯斯<ol><li>装配使用幻灯片和盖玻片（室）的成像室。将一块浸透水的脱脂棉上滑动的中间。然后从板传送一个或两个5天龄幼苗到水浸泡药棉的顶部。坚持小块粘土的盖玻片，在每一个角落d将盖玻片上的幼苗创造盖玻片和载玻片之间的间隙（室）（ 图3A，左图）的顶部。聚乙烯管（0.58mL毫米直径）的一端连接至1ml注射器和放置管紧邻所述腔室的另一端。用胶带（ 图3A，右图）握住管到位。 </li><li>装配用有机玻璃室（B室）成像室。 <ol><li>散布一层薄薄的硅脂围绕两个聚乙烯管（0.58mL毫米直径），然后按涂覆管放置在有机玻璃室的每一侧的信道（ 图3B左面板）。散布一层薄薄的硅脂到包含所述管槽的腔室的表面上，按盖玻片入润滑脂，以密封腔室开口（切出）的一侧。 </li><li>从板块转移日龄苗成室，并放置了一块脱脂棉用开水浸泡在顶部ØF表示苗子。传播硅脂到所述腔室的另一面，然后按上顶端以密封另一盖玻片。其中一个管的一端连接到1毫升注射器。离开其他管开放（ 图3B右面板）的端部。 </li></ol></li><li>采用腔盖玻璃（C室）准备成像室。消毒腔盖玻璃用70％乙醇和离开它引擎盖上直到干燥。添加0.6毫升或0.2含1％蔗糖和0.5％植物凝胶成2孔腔的每个孔或8孔腔室（ 图3C右侧面板），分别为全强度MS培养基中。种子消毒，直接在含有一层薄薄的透明凝胶的墓室盖玻璃播下的种子，让他们上下5天（ 图3C）成长。 </li></ol></li><li>掌握用共聚焦显微镜图像。施加压力刺激，如盐，寒冷或过氧化物，慢慢注入大约200微升的150 mM氯化钠，冰栏ð水或1毫米的H 2 O 2获取图像，或轻轻地加刺激液500微升或100微升至2或8孔室的很好，分别前夕溶液进入室（厅A或B） 。拍摄的图像将立即使用倒置尼康A1R共聚焦激光扫描显微镜20倍水浸泡镜片（数值孔径0.75）刺激后的解决方案。收集在4秒的间隔与488 nm和500的激发和发射波长的时间序列图像 - 550纳米，分别，并且在512×512像素的分辨率。 </li><li>图像分析 使用尼康元素，投资回报是吸引每一个感兴趣的单元格（或区域）周围。每个感兴趣区域内的总强度是使用时间测量对话框（ImageJ的可以用来代替）随时间测量。总强度测量输出和数据图进行处理。 钙尖峰幅度定义为峰值强度减去休息的强度，持续时间为次起始和尖峰完成后，并作为两个尖峰相邻尖峰峰值之间的时间间隔周期E之间的时间。 吨 -test用于比较的装置。 </li></ol>

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The authors have nothing to disclose.

我们已经展示了FAS系统记录拟南芥幼苗的时空钙离子的响应。这FAS 钙记录系统提供了一个可能被适用于测量通过钙除了在此处提出的非生物胁迫的刺激各种刺激引起离子动力简便，灵敏，强健的方法。使用这个系统，我们可以很容易地在整株水平比较组织或刺激特定的时空钙离子动态。 FAS的高灵敏度允许使用低强度刺激的用于检查的 Ca 2 +的反应?...

植物细胞响应该坐标细胞动作经由信令环境。早期的细胞在应对环境刺激信号事件是一个短暂的钙增加。的图案，或一过性升高签名中游离的 Ca 2 +浓度的特征在于，其振幅，频率和持续时间。不同的时空钙签名规范不同的细胞活动1。特定刺激，如热，冷，盐，干旱，光，或植物激素，可以微调膜本地化的 Ca 2 +通道和/或转运的时空活性，导致具体的 Ca 2 +的签名。虽然钙离子转运都得到很好的特点，鲜为人知的是， 钙离子通道在植物1分子的身份和功能。对于突变体遗传筛选具有改变的 Ca 2 +响应强调刺激可能是一种有效APPRoach用于识别构成的 Ca 2 +的签名的组件。最近基于几个水母发光蛋白的 Ca 2 +检测系统已被开发，以促进遗传筛选的钙响应于病原体攻击和非生物胁迫2-4信令组件。 水母发光蛋白最早是用于检测钙在植物体内离子信号在90年代初5。从那时起，水母发光蛋白已经被定位到不同的细胞区室，如细 ​​胞质5，核6，叶绿体7，液泡膜8，线粒体9和基质10，以及不同的细胞类型中的根来监视特定的Ca 2细胞+信号11。基于水母发光蛋白钙离子的测量揭示细胞应力刺激人口的空间和时间的 Ca 2 +的响应。然而，在大多数情况下，单个细胞的钙离子的反应是unsynchroni捷思在响应组织4。因此，水母发光蛋白的 Ca 2 +的记录不一定报告在个体细胞中的钙信号。近年来，基因编码荧光蛋白（FP）为基础的 Ca 2 +的指标，如黄色卡默莱昂（YCS）12和CASEs12 13已被用于研究的 Ca 2 +具有高亚细胞分辨率的信号。 YCS是荧光共振能量转移（FRET）为基础的 Ca 2 +的指标，含有CFP和YFP的变体由钙挂钩2 +结合蛋白钙调蛋白和钙调蛋白结合肽M13。钙调蛋白发生构象变化，因为它结合的 Ca 2 +，由此带来的CFP和YFP更加靠近，从而增加了能量转移（FRET增强）。随着时间的荧光共振能量转移的水平，大约为YFP对CFP信号强度的比值计算，反映了细胞内钙离子动态。几个YC版本已被用于在植物中。 YC3.6为吨argeted到细胞质14,15，核16，线粒体17，和质膜18，和YC4.6和D4ER被定位到ER 15,19，和D3cpv被靶向过氧化物酶体20。转基因植物表达YCS允许不同类型的细胞不同的细胞内钙离子动态的活细胞成像。例（大概钙 lcium 本身 nsor）是单圆置换荧光蛋白（cpFPs）窝藏钙调蛋白和钙调蛋白结合肽M13。结合后的 Ca 2 +，情况发生构象变化，从而导致增加的荧光强度。该案例的荧光响应和Ca 2 +浓度之间的相关性使得细胞内钙离子动力学来进行定量测定。在钙 -饱和脂肪形式Case12变种12倍的增加荧光。N。 benthiminana植物瞬时EXPRessing Case12或拟南芥稳定表达病例12为材料，研究钙信号在国防和非生物胁迫4,21。异步时空钙离子振荡，细胞应对病原体的攻击，或脱水的压力已经显露了Case12基于钙离子成像。 在这里，我们提出的组织-和刺激基于水母发光蛋白发光成像特定的CA在拟南芥幼苗离子动力，并为细胞内的共焦成像与核钙的详细说明，在拟南芥根细胞离子的动态表达的FAS案例12发光成像可适于分析在完整植物或此处未描述的组织应力诱导的 Ca 2 +的动态，或筛选诱变的拟南芥属植物种群，对具有改变的应力诱导的 Ca 2 +信号的突变体。活细胞的 Ca 2 +成像设置可适于分析钙使用其他钙指标内的不同亚细胞区室或在不同类型的细胞离子动力。

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			<td>Nikon Instruments Inc.</td>
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measuring spatial and temporal ca2+ signals in arabidopsis plants

发展与环境线索诱发的 Ca 2 +的波动在植物细胞中。刺激特定的空间-时间的 Ca 2 +的图案是由发起的 Ca 2 +信号传导级联的细胞的 Ca 2 +结合蛋白的检测。但是，我们仍然知之甚少如何刺激产生特定的钙信号。 à 钙离子信号的特殊性可能是由于钙离子通道和/或转运活动的复杂调控响应给定的刺激。识别这些细胞成分和理解它们的功能，关键的是要使用的系统，允许的 Ca 2 +信号的灵敏和鲁棒记录同时在组织和细胞水平。基因编码的定位到不同的细胞钙指标都规定了细胞钙信号活细胞共聚焦成像的平台。在这里，我们描述的指令S为使用两钙离子检测系统：基于水母发光蛋白FAS（薄膜粘合苗）发光钙成像和case12基于活细胞共聚焦荧光钙离子成像。使用FAS系统发光成像提供了一种简单，可靠和灵敏的检测的空间和时间的 Ca 2 +信号，在组织水平的，同时采用Case12活细胞的共聚焦成像提供同时检测胞质和核的 Ca 2 +信号中的较高的分辨率。

甘露糖醇，氯化钠和H 2 O 2被用作代理进行脱水，盐和氧化应激刺激，分别。检查，如果重金属离子铜离子协同作用与任何这三个应力刺激，我们比较了的 Ca 2 +响应于在铜离子的存在或不存在各刺激。如示于图2中 ，FAS发光成像显示，Arabidoposis苗反应不同脱水，盐和氧化应激。为刺激我们研究在本研究中的浓度，75mM氯化钠诱导的第40秒的曝光（...

Watch this Scientific Journal Video about Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants at JoVE.com

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

的 Ca 2 +信号调控植物中不同的生物过程。在这里，我们提出的方法用于监测非生物胁迫诱导的时空钙在拟南芥的细胞，并使用基因编码的钙指标水母发光蛋白或Case12组织2 +信号。

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+ ...

measuring-spatial-and-temporal-ca2-signals-in-arabidopsis-plants

Research

JoVE Journal

Biology

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

12.0K 次观看.  Purdue University.  的 Ca 2 +信号调控植物中不同的生物过程。在这里，我们提出的方法用于监测非生物胁迫诱导的时空钙在拟南芥的细胞，并使用基因编码的钙指标水母发光蛋白或Case12组织2 +信号。 

视频: Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

建模生物膜与电路板，并在轴突测量电气信号：学生实验室练习

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

科研

生物学

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

冰帽：为发展方法拟南芥和番茄植株在96孔板高通量的基因分型

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

测量细胞内Ca<sup&gt; +</sup在人类精子的变化采用4项技术：传统的荧光，停流荧光，流式细胞术及单细胞成像

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

动态钙的自动分析<sup&gt; 2 +</sup&gt;在图像序列信号

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

实时在体记录了拟南芥钙信号在使用荧光传感器的昆虫饲喂时

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

时空映射和粒子分析技术在细胞内 ca2 +原位信令定量中的应用

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

激光捕获微分块RNA-测序空间和时间组织特异性基因表达分析

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. Upon wounding of a leaf, an increase in cytosolic calcium ion concentration (Ca2+ signal) occurs at the wound site. This signal is rapidly transmitted to undamaged leaves, where defense responses are activated. Our recent research revealed that glutamate leaking from the wounded cells of the leaf into the apoplast around them serves as a wound signal. This glutamate activates glutamate receptor-like Ca2+ permeable channels, which then leads to long-distance Ca2+ signal propagation throughout the plant. The spatial and temporal characteristics of these events can be captured with real-time imaging of living plants expressing genetically encoded fluorescent biosensors. Here we introduce a plant-wide, real-time imaging method to monitor the dynamics of both the Ca2+ signals and changes in apoplastic glutamate that occur in response to wounding. This approach uses a wide-field fluorescence microscope and transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-based Ca2+ and glutamate biosensors. In addition, we present methodology to easily elicit wound-induced, glutamate-triggered rapid and long-distance Ca2+ signal propagation. This protocol can also be applied to studies on other plant stresses to help investigate how plant systemic signaling might be involved in their signaling and response networks.

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

阿拉伯多普西斯局部和系统伤口信号的广域实时成像

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

一种简单灵活的接种方法，用于准确评估拟南芥和其他植物的白粉病感染表型

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...