这项工作是由国家机构支持部分卫生赠款HL-085887，HL-092992，S10RR027535和MOP-93676。

1，血管夹层和成像<ol><li>从国内少年猪一样的马腾斯等 27所描述的收获组织。将收获的猪右心室成聚二甲基硅氧烷（PDMS）含有HEPES缓冲生理盐水溶液（PSS）的底部夹层菜。 <ol><li>用立体显微镜的帮助下，解剖，取出左前从使用镊子和剪刀弹簧通过从周围的心脏组织的层去除血管区段周围组织降支冠状动脉（约8毫米长，0.5毫米直径）的片段。注意：小心不要刺破血管壁。 </li><li>放置一个PDMS块（1×0.5×0.5公分）放入解剖盘，并用针将其脚的底部。削减约40微米直径的钨丝成十二个〜0.3厘米长段，形成m​​icropins，并把这些在盘子里。使用镊子，固定血管段的一端与一个micropi块Ñ​​。 </li><li>小心地插入小弹簧剪刀成血管腔，并纵向切开向下容器的一侧，以完全打开。定位打开的血管段与内皮起来。 </li><li>使用剩余的micropins以固定打开血管区段的边界方框，使得该容器形成一个扁平的矩形。注意：Micropin面应被弯曲到90°和插入，直到与块表面齐平。应小心，以拉伸容器制备而不过度拉伸;最终宽度应〜1.5倍的首发未拉伸宽度。 </li><li>准备一个小体积的通过在HEPES混合的Fluo-4 AM（10μM）溶解在DMSO中与聚醚（0.03％）的Fluo-4AM加载溶液（〜1毫升）中缓冲的PSS（含有以mM计：134 NaCl的，​​6氯化钾，1氯化镁，10 HEPES，10葡萄糖），然后将整个块成在室温下在黑暗中约40分钟的加载溶液。 </li><li>加载后，在HEPES缓冲洗块PSS 5-10分钟。 </li><li>安装块到50〜100微米厚的垫片中含有HEPES缓冲PSS一个玻璃罩底部室。注：金属针可以用作间隔物​​，并确保血管区段朝下和不接触垫片。 </li><li>放置在一个倒置显微镜配备的共焦成像的舞台腔，专注于内皮细胞层上。 </li><li>为〜3分钟，在20倍的放大倍率，并使用共聚焦图像序列AQUISITION软件每秒〜8帧的帧速率基底相对荧光的采集时间间隔的图像序列。 </li><li>后记录基础荧光〜3分钟，取代的HEPES缓冲液的PSS与P物质（PM 100）相同体积（约1毫升）溶解在HEPES缓冲的PSS，并记录为一个addtional 3分钟。 </li></ol></li></ol> 2，自动分析<ol><li>呈现荧光活性钙的共聚焦采集软件为8位，G图像序列（S）rayscale&#39;。TIF“格式的文件，没有规模的信息。 </li><li>打开ImageJ的，单击文件菜单中的“打开”，然后选择在浏览器窗口中相应的图像序列中的ImageJ查看图像序列（S）。 </li><li>通过使用矩形ROI工具来估计活动的图像序列（次）内的空间传播的上限和下限确定一个适当的ROI的直径。注：平均长方形的直径为直径的投资回报率一个合适的选择。 </li><li>创建计算机硬盘驱动器上的一个新文件夹，并添加图像序列（S）到该文件夹​​目录。 </li><li>在ImageJ的，点击“插件”窗口，然后单击“LC_Pro”开始分析。 </li><li>进入投资回报直径值，然后选择过滤器的阈值（无论是0.05或0.01）。 </li><li>点击“药物治疗”复选框，并立即将药物加入前，后（以秒为单位）输入时间点的值。 </li><li>点击“确定”，并进入图像序列目录到文件浏览器窗口。 </li></ol> 3，图形输出<ol><li>从下载R版本3.0.2 <a href="http://www.r-project.org/">http://www.r-project.org/</a> 。 </li><li>打开32位版本的河</li><li>点击“文件”，然后“打开脚本”，然后选择“traceplot.R&#39;R脚本生成图形化的实验报告。 </li><li>通过选择“套餐”安装校准，并gplots包，“安装软件包”，并选择appropirate包。 </li><li>点击“文件”，然后“更改目录”命令，并使用资源管理器窗口中，选择对应于LC_Pro分析输出的目录。 </li><li>单击脚本窗口，然后在“运行所有&#39;命令来运行该脚本。 </li></ol>

<ol>
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作者什么都没有透露。

在细胞和多细胞水平进行译码复杂的Ca 2 +信号，将需要严格的实验和分析方法。这里，这种方法描述了其中时间分辨共焦图象的Ca 2 +依赖的荧光的序列进行识别及量化统计学相关的Ca 2 +内完好蜂窝字段在特定情况下，信号表示的自动分析，动脉段是孤立从猪的心脏，寄托打开，露出内皮，装入的Ca 2 +指示剂fluo-4 AM，受到共聚焦成像，并使用自定义算法LC_Pro评估。?...

钙离子是一种普遍存在的第二信使的信号分子和细胞内Ca 2 +的水平受到高度监管。细胞内Ca 2 +信号是复杂的，包括分离的瞬变，振荡，并传播波是1 - 4。的Ca 2 +的空间和时间的控制被认为是背后的生理信号的特殊性，因此对Ca 2 +的信号模式的分析是相当大的兴趣在多个领域的5调查。 的Ca 2 +指示剂染料如荧光4和Fura-2的通常用于测量细胞内Ca 2 +信号用荧光显微镜5-12。典型地，时间的Ca 2 +信号被评价为用户定义的区域内的平均荧光随时间变化，或感兴趣区域（ROI）的5,6,13 - 16。目前，人工ROI分析是既费时又劳力由于病程中它要求用户识别许多的ROI，并执行重复计算17 - 19。这些技术也可能会受到相当大的用户错误，包括引进人工信号模式和排除低振幅或弥漫性信号18,20。 自动ROI检测算法先前已采用多种统计方法，以确定最佳的ROI的位置实现的，但他们一般都限于线扫描或伪线扫描图像，这限制了分析，以一个单一的空间维度中时间17分析， 19 - 22。此外，许多现有的算法都不足以涵盖钙，范围从周期性的，本地化的瞬态传播的波23,24 2 +释放事件的多样性。生理的Ca 2 +信号的综合评价通常进一步由显著图像ARTIF存在复杂行动是混淆信号，在许多实验系统噪音的歧视。 此前，自动ROI检测算法解决的Ca 2 +信号瞬态检测，实现为插件NIH ImageJ的软件（卫生，马里兰州贝塞斯达的美国国家研究院），是开发和验证25,26。这种算法，称为LC_Pro，目的是确定和分析的ROI无所不包的Ca 2 +信号瞬态二维时间推移图像序列。在这里，一个实用的实验方案和算法猪冠状动脉内皮细胞的应用有代表性的示范提供，使用开源统计处理软件研发，生成可用的图形输出额外的后处理。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissection dish</td>
			<td style="width:71px;">Fisher Sci</td>
			<td style="width:119px;">#08-772-70</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polydimethylsiloxane (PDMS)</td>
			<td style="width:71px;">Fisher Sci</td>
			<td style="width:119px;">#NC9644388</td>
			<td>elastomer kit, must be molded into dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES-buffered PSS</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">#H3375-250G</td>
			<td>HEPES acid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">stereomicroscope</td>
			<td style="width:71px;">Nikon Inst.</td>
			<td style="width:119px;">#MNA42000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">forceps</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">#11223-20</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">spring scissors</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">15003-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">tungsten wire</td>
			<td style="width:71px;">Scientific Inst Svcs</td>
			<td style="width:119px;">#406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluo-4 AM</td>
			<td style="width:71px;">Life Tech.</td>
			<td style="width:119px;">#F-14201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pluronic F-127</td>
			<td style="width:71px;">Life Tech.</td>
			<td style="width:119px;">#P3000MP</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">metal pins</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">#26002-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cover-glass bottom chamber</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Custom designed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">spinning disc confocal microscope</td>
			<td style="width:71px;">Perkin Elmer</td>
			<td style="width:119px;">RS-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">imageJ software</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://rsbweb.nih.gov/ij/download.html">download at: http://rsbweb.nih.gov/ij/download.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LC_Pro plugin for imageJ</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://rsbweb.nih.gov/ij/plugins/lc-pro/index.html">download at: http://rsbweb.nih.gov/ij/plugins/lc-pro/index.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">R software</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://www.r-project.org/">download at: http://www.r-project.org/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">R traceplot script</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
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automated analysis of dynamic ca2+ signals in image sequences

细胞内Ca 2 +信号通常研究了用荧光钙离子指 ​​示剂染料和显微镜技术。然而，Ca 2 +的成像数据的定量分析是费时并受偏压。基于感兴趣区域（ROI）的检测区域自动信号分析算法已实施了一维的线扫描测量，但没有目前的算法集成在二维图像序列优化的识别和投资回报的分析。这里的算法进行快速采集和投资回报分析图像序列进行描述。它利用椭圆拟合到噪声滤波的信号，以确定最佳的ROI的位置，并且计算的Ca 2 +信号的幅度，持续时间和空间扩散参数。该算法被实现为一个可自由查看的插件ImageJ的（NIH）的软件。连同开源的统计处理软件研发书面分析脚本，这种方法提供了一种高容量的管道，用于执行实验的输出的快速统计分析。作者认为，使用这种分析的协议会导致生理的Ca 2 +信号的更全面和公正的表征。

自定义算法，LC_Pro，开发并为了实现来执行CA的自动分析的共聚焦图像序列2 +动态。正如如图1所示，该算法采用顺序处理模块，A）检测和动态的Ca轨道站点​​2 +更改上述统计（P &lt;0.01），噪音，B），自动在活动现场中心定义的感兴趣区（ROI）区域， C）计算平均荧光强度的投资回报，以确定具体的事件参数。该算法的图形概述使用已知的强度和位置的计算机生成的...

Watch this Scientific Journal Video about Automated Analysis of Dynamic Ca2+ Signals in Image Sequences at JoVE.com

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

在这里，一种新型的基于排序的二维时间推移图像序列中分配给积极信号的地区最适合椭圆利益分析协议的区域是证明。该算法可以使研究人员综合分析生理的Ca 2 +信号以最小的用户输入和偏见。

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+ ...

automated-analysis-of-dynamic-ca2-signals-in-image-sequences

Research

JoVE Journal

Biology

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

17.0K 次观看.  University of South Alabama. 在这里，一种新型的基于排序的二维时间推移图像序列中分配给积极信号的地区最适合椭圆利益分析协议的区域是证明。该算法可以使研究人员综合分析生理的Ca 2 +信号以最小的用户输入和偏见。 

视频: Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

光固化牙科粘合树脂安装电极或微透析探针在慢性实验中的应用

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

科研

生物学

Semi-automated Optical Heartbeat Analysis of Small Hearts

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

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

半自动小红桃光学心跳分析

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

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

马赛克斑马鱼转基因增强子序列，以评估

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

机器人技术和植物组织的基因表达研究的动态图像分析

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

技术成像钙 2 +信号在人类精子

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent 
proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest1, recent 
developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association 2, 3. One small 
tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH 
(green fluorescent), with high specificity even in live cells 2. The TC/biarsenical dye system offers far less steric constraints to the host protein than 
fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions 4-7. We recently developed 
a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington 
disease 7. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only 
binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer 
content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) 
with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.

ReAsH / FLASH标签和图像分析Tetracysteine​​传感器蛋白在细胞中

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

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+ ...

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, ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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 ...