这项工作是由Hukriede，NCI / NIH（P01 CA78039）沃格特，NHLBI /美国国立卫生研究院（1R01HL088016）曾NICHD的/美国国立卫生研究院（1R01HD053287）。

1）设置斑马鱼交配和BCI治疗。 <ol><li>前两天的治疗，查明纯合子男性TG（dusp6：d2eGFP）PT6斑马鱼和地点单独进入交配坦克1。添加分隔符和野生型AB *女鱼交配笼留下过夜。 </li><li>第二天早晨去掉分隔，并收集1小时后的胚胎。胚胎转移至100mm菜E3（5毫米氯化钠，0.17 mM的氯化钾，氯化钙，0.33毫米0.33 mM的硫酸镁），解决方案，并在6小时28 ° C孵育。去除死皮和未受精的胚胎，补充新鲜的E3解决方案，并孵育过夜。 </li><li>排序类似的阶段（24hpf）和统一的绿色荧光蛋白表达的转基因胚胎。阵列胚胎分别为每孔96孔板。 </li><li>除去多余E3的解决方案与微。添加到每口井的多通道移液器200μL的E3的解决方案。 </li><li>添加列出下列化合物： <table border="1"><tbody><tr><td></td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td><td> 8 </td><td> 9 </td><td> 10 </td><td> 11 </td><td> 12 </td></tr><tr ><td> 一 </td><td> 2μL 二甲基亚砜</td><td> 2μL BCL （0.1MM） </td><td> 2μL BCL （0.5MM） </td><td> 2μL BCL （1MM） </td><td> 2μL BCL （2MM） </td><td> 2μL BCL （2.5） </td><td> 2μL BCL （0.1MM） </td><td> 2μL BCL （0.5MM） </td><td> 2μL BCL （1MM） </td><td> 2μL BCL （2MM） </td><td> 2μL BCL （2.5） </td><td> 2μL 二甲基亚砜</td></tr><tr ><td> 乙 </td><td> 2μL 二甲基亚砜</td><td> 2μL BCL （0.1MM） </td><td> 2μL BCL （0.5MM） </td><td> 2μL BCL （1MM） </td><td> 2μL BCL （2MM） </td><td> 2μL BCL （2.5） </td><td> 2μL BCL （0.1MM） </td><td> 2μL BCL （0.5MM） </td><td> 2μL BCL （1MM） </td><td> 2μL BCL （2MM） </td><td> 2μL BCL （2.5） </td><td> 2μL 二甲基亚砜</td></tr><tr ><td> 彗星 </td><td> 2μL 二甲基亚砜</td><td> 2μL BCL （0.1MM） </td><td> 2μL BCL （0.5MM） </td><td> 2μL BCL （1MM） </td><td> 2μL BCL （2MM） </td><td> 2μL BCL （2.5） </td><td> 2μL BCL （0.1MM） </td><td> 2μL BCL （0.5MM） </td><td> 2μL BCL （1MM） </td><td> 2μL BCL （2MM） </td><td> 2μL BCL （2.5） </td><td> 2μL 二甲基亚砜</td></tr><tr ><td> ð </td><td> 2μL 二甲基亚砜</td><td> 2μL BCL （0.1MM） </td><td> 2μL BCL （0.5MM） </td><td> 2μL BCL （1MM） </td><td> 2μL BCL （2MM） </td><td> 2μL BCL （2.5） </td><td> 2μL BCL （0.1MM） </td><td> 2μL BCL （0.5MM） </td><td> 2μL BCL （1MM） </td><td> 2μL BCL （2MM） </td><td> 2μL BCL （2.5） </td><td> 2μL 二甲基亚砜</td></tr><tr ><td> Ë </td><td> 2μL 二甲基亚砜</td><td> 2μL BCL （0.1MM） </td><td> 2μL BCL （0.5MM） </td><td> 2μL BCL （1MM） </td><td> 2μL BCL （2MM） </td><td> 2μL BCL （2.5） </td><td> 2μL BCL （0.1MM） </td><td> 2μL BCL （0.5MM） </td><td> 2μL BCL （1MM） </td><td> 2μL BCL （2MM） </td><td> 2μL BCL （2.5） </td><td> 2μL 二甲基亚砜</td></tr><tr ><td> F </td><td> 2μL 二甲基亚砜</td><td> 2μL BCL （0.1MM） </td><td> 2μL BCL （0.5MM） </td><td> 2μL BCL （1MM） </td><td> 2μL BCL （2MM） </td><td> 2μL BCL （2.5） </td><td> 2μL BCL （0.1MM） </td><td> 2μL BCL （0.5MM） </td><td> 2μL BCL （1MM） </td><td> 2μL BCL （2MM） </td><td> 2μL BCL （2.5） </td><td> 2μL 二甲基亚砜</td></tr><tr ><td> 摹 </td><td> 2μL 二甲基亚砜</td><td> 2μL BCL （0.1MM） </td><td> 2μL BCL （0.5MM） </td><td> 2μL BCL （1MM） </td><td> 2μL BCL （2MM） </td><td> 2μL BCL （2.5） </td><td> 2μL BCL （0.1MM） </td><td> 2μL BCL （0.5MM） </td><td> 2μL BCL （1MM） </td><td> 2μL BCL （2MM） </td><td> 2μL BCL （2.5米M） </td><td> 2μL 二甲基亚砜</td></tr><tr ><td> H </td><td> 2μL 二甲基亚砜</td><td> 2μL BCL （0.1MM） </td><td> 2μL BCL （0.5MM） </td><td> 2μL BCL （1MM） </td><td> 2μL BCL （2MM） </td><td> 2μL BCL （2.5） </td><td> 2μL BCL （0.1MM） </td><td> 2μL BCL （0.5MM） </td><td> 2μL BCL （1MM） </td><td> 2μL BCL （2MM） </td><td> 2μL BCL （2.5） </td><td> 2μL 二甲基亚砜</td></tr></tbody></table>终浓度1μM，5μM，10μM，20μM，25μm的景气指数在1％DMSO。覆盖铝箔板和孵育6小时，在28 ° C。 </li></ol> 2）自动成像96孔板和分析。 <ol><li>添加tricaine（0.61mM）加入10μl，每孔麻醉30hpf胚胎。 </li><li>装入Imagexpress超分子器件板块。 </li><li>打开Metaxpress软件。选择一个具有4倍的目标和最大的针孔直径的配置，配置激光自动对焦检测板的底部，并确定最佳的Z平面检测感兴趣区域。收购五百二十五分之四百八十八纳米（GFP）的激发/发射波长的图像。设置扫描面积为2000x2000像素（4 x 4毫米），没有分级的单一站点。配置的激光功率和增益等，阳性对照图像中最亮的地区不饱和。在一些正面和负面的控制井确认正确的图像采集。 </li><li>启动盘扫描。该仪器将自动获得所有的水井和归档他们在SQL Server数据库中的图像。 </li><li>上传到Definiens的开发，使用的Cellenger应用模块和进口过滤器，承认Imagexpress超分子器件平台产生的图像格式和文件结构的图像。 </li><li>定制设计的认知网络技术算法（也称为规则集）的过程映像。设计的算法，我们定制开发的基于最近公布的规则集2，自动识别胚，蛋黄，和头部，并量化表达GFP的头结构的亮度和面积。在其中没有发现胚胎或头部不能被明确指定（例如，具有高毒性化合物或出重点图像）井是从分析中排除。配置的算法，使用一些负面控制图像（车辆处理）和阳性对照图像（BCI的治疗），正确地确定两个头和蛋黄等。 “头结构”平均蛋黄强度的倍数检测阈值设置，直到明亮的头部结构的检测与目视观察。定义参数的出口。在这个例子中，最翔实的参数是头以下称为“总头结构的亮度”（图1），GFP阳性区域内的总集成的亮度。 <img src="/files/ftp_upload/1900/1900fig2.jpg" alt="图1" /> 图1 图depicitng的BCI的剂量依赖效应，对绿色荧光蛋白在转基因胚胎的表达。 </li><li>运行在所有板块的图像，使用集成的作业调度算法。 Definiens的软件使用多个处理器的分布式计算（“引擎”），使多个图像的同时分析。该算法计算，这是处理后的图像的副本保存在数据库中的图像分析应用与分析，结果为基础的数值措施。 </li></ol>代表性的成果： 所有的图像进行分析后，数据可以被可视化内Cellenger或出口到第三方软件（EXCEL，GraphPad棱镜）作进一步的分析和图形表示。图2显示从处理过的车辆存档的扫描图像和一个BCI的无CNT的分析应用，良好的待遇。图1文件GFP报告基因的表达在TG（dusp6：d2EGFP）的BCI的剂量依赖效应PT6胚胎。引出一个半最大反应（EC50）所需的浓度约为12微米。 <img src="/files/ftp_upload/1900/1900fig1.jpg" alt="图2" /> 图2 自动图像采集和分析，转基因斑马鱼的胚胎。 （一）二甲基亚砜车辆处理TG（dusp6：d2eGFP）30hpf PT6胚胎。 （二）与20μMBCI显示处理过的转基因胚胎中GFP的表达增加。 （三）从胚胎图像（a）在运行规则集Definiens的头部找到GFP表达。橙色点表示该软件的措施GFP的强度。 （四）从胚胎图像（b）在运行规则集Definiens公司在头部发现GFP的表达。橙色点表示该软件的措施GFP的强度。请注意，规则集能够找到处理后的胚胎中GFP表达的扩大面积。

<ol>
	<li>Molina, G. A., Watkins, S. C., Tsang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+FGF+reporter+transgenic+zebrafish+and+their+utility+in+chemical+screens.">Generation of FGF reporter transgenic zebrafish and their utility in chemical screens.</a> BMC Dev Biol. 7, 62-62 (2007).</li><li>Vogt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+image-based+phenotypic+analysis+in+zebrafish+embryos.">Automated image-based phenotypic analysis in zebrafish embryos.</a> Dev Dyn. 238, 656-663 (2009).</li><li>Molina, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+chemical+screening+reveals+an+inhibitor+of+Dusp6+that+expands+cardiac+cell+lineages.">Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages.</a> Nat Chem Biol. 5, 680-687 (2009).</li></ol>

限制的斑马鱼化工屏幕的吞吐量的一个主要因素是缺乏系统的成像和分析过程的96孔板的方法。由于是复杂的多细胞生物体的图像，低对比度和性质异构，现有的图像分析算法故障检测和量化机体内部的具体结构。因此，大多数公布的斑马鱼化工屏幕涉及视觉由专门人员检查整个过程中个别井。这通常可以防止的数值读数生成和屏幕图像和数据的完整归档。此外，评价手册消除图像为基础的筛查，即能力，画面表现进行回顾性分析，研究表型的变化，没有甄别运动（如毒性或发育缺陷）的主要重点和跨的几个关键优势参考过去或将来的屏幕上使用相同的化合物库的筛选数据。 在这份报告中，我们强调和斑马鱼的胚胎在多孔板自动成像分析的方法，无需用户干预。 PT6胚胎在96孔板，并开发了一种图像分析算法基于Definiens公司的认知网络技术，量化的GFP：我们ImageXpress超激光扫描共聚焦阅读器（分子器件，位于美国加州Sunnyvale）的形象TG（d2EGFP dusp6）自动图像捕捉表达的转基因胚胎的头。该方法提供了分级响应和FGF信号的一种小分子活化剂在体内的活动量化。与epifluorescence基于ArrayScan II（Cellomics公司，匹兹堡PA）获得了类似的结果证明它是可以实现的各种市售板读者2斑马鱼胚胎的自动图像捕获。方法论的发展，自动分析多细胞生物体图像消除由人类观察者视觉得分的需要，并启用了回顾性分析，并与未来的屏幕比较的数值数据和图像的生成和归档。

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development of automated imaging and analysis for zebrafish chemical screens.

我们演示了基于图像的高内涵筛选（HCS）的方法来识别，可以调节斑马鱼的胚胎中的FGF / RAS / MAPK通路的小分子的应用程序。斑马鱼的胚胎是在体内高含量的化学屏幕的理想系统。 1日龄胚胎直径约1mm，可以很容易地排列到96孔板，为高通量筛选的标准格式。在发展的第一天，胚胎透明，大部分目前的主要器官，从而使组织形成的胚胎发育过程中的可视化。然而，斑马鱼化工屏幕的完整的自动化仍然是一个挑战，特别是在自动化图像采集和分析的发展。我们先前生成的转基因绿色荧光蛋白（GFP）的FGF活动的控制下，记者一行表示，并展示了在化学屏幕它们的效用<sup&gt; 1</sup&gt;。要建立高吞吐量的整个机体屏幕的方法，我们开发了96孔板自动成像系统和分析斑马鱼的胚胎在受精后24-48小时（HPF）<sup&gt; 2</sup&gt;。在这个视频中，我们突出24hpf转基因胚胎阵列多孔板的程序，除了一个小分子（BCI）hyperactivates FGF信号<sup&gt; 3</sup&gt;。板孵育6小时tricaine此外，麻醉一个分子器件ImageXpress超激光扫描共聚焦的HCS读者幼虫前自动成像。图像处理Definiens的认知网络技术的算法，我们开发的检测和定量表达绿色荧光蛋白转基因胚胎的头的软件开发人员使用。在这个例子中，我们突出了该算法的能力来衡量处理的胚胎中GFP报告基因表达的BCI剂量依赖效应。

Watch this Scientific Journal Video about Development of automated imaging and analysis for zebrafish chemical screens. at JoVE.com

Development of automated imaging and analysis for zebrafish chemical screens.

我们报告的自动成像系统和分析在多孔板的转基因斑马鱼的胚胎的发展。这表明成纤维细胞生长因子记者的基因表达的能力来衡量的一个小分子，BCI，​​剂量依赖效应，并提供技术建立高通量的斑马鱼化工屏幕。

斑马鱼化工屏幕自动成像和分析的发展。

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

development-automated-imaging-analysis-for-zebrafish-chemical

Research

JoVE Journal

Biology

11.3K 次观看.  University of Pittsburgh Drug Discovery Institute. 我们报告的自动成像系统和分析在多孔板的转基因斑马鱼的胚胎的发展。这表明成纤维细胞生长因子记者的基因表达的能力来衡量的一个小分子，BCI，​​剂量依赖效应，并提供技术建立高通量的斑马鱼化工屏幕。

视频: Development of automated imaging and analysis for zebrafish chemical screens.

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

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 ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

在斑马鱼胚胎的代谢标记和Bioorthogonal点击化学成像聚糖

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

定量和自动化高通量的全基因组RNAi屏幕 C。线虫</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

在斑马鱼发育的细胞追踪使用Photoconvertible蛋白

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

组织特异性荧光的斑马鱼胚胎半自动成像

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

开源高内涵分析利用自动荧光寿命成像显微镜

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

斑马鱼胰岛单细胞分辨率钙成像对β细胞功能的分析

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Automated Detection and Analysis of Exocytosis

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic events in cell culture. To perform an unbiased, efficient identification and analysis of such events, a computer-vision based approach was developed and implemented in MATLAB. The analysis pipeline consists of a cell segmentation and exocytic-event identification algorithm. The computer-vision approach includes tools for investigating multiple parameters of single events, including the half-life of fluorescence decay and peak &#916;F/F, as well as whole-cell analysis of the frequency of exocytosis. These and other parameters of fusion are used in a classification approach to distinguish distinct fusion modes. Here a newly built GUI performs the analysis pipeline from start to finish. Further adaptation of Ripley&#39;s K function in R Studio is used to distinguish between clustered, dispersed, or random occurrence of fusion events in both space and time.

We developed automated computer vision software to detect exocytic events marked by pH-sensitive fluorescent probes. Here, we demonstrate the use of a graphical user interface and RStudio to detect fusion events, analyze and display spatiotemporal parameters of fusion, and classify events into distinct fusion modes.

外分泌物的自动检测和分析

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic ...