We thank O. Subach for providing the original H2B-PSmOrange plasmid and our fish facility team for fish care. We are grateful to the Nikon Imaging Center at the University of Heidelberg for access to microscopy equipment and analysis software. We acknowledge the support of the Core Facility Live Cell Imaging Mannheim at the CBTM (DFG INST 91027/10-1 FUGG). This work was supported by the Excellenzcluster CellNetworks, EcTop Spatio-temporal coordination of signaling processes (EcTop 2), University of Heidelberg to C.A.B. and the Medical Faculty Mannheim of the University Heidelberg and the DFG (FOR 1036/2, 298/3-1 and 298/6-1) to M.C.

1. H2B-PSmOrange基因体外转录 mRNA纯化<ol><li>线性化使用根据制造商的说明的NotI限制性内切酶含有PCS2 +质粒H2B-PSmOrange。 注意:请使用适当的保护措施，如手套和白大褂，以防止污染的mRNA降解，并在接下来的步骤。 </li><li>纯化使用根据制造商的协议的PCR纯化试剂盒或苯酚 - 氯仿基础的方法的线性化的DNA。 </li><li>根据制造商的说明使用线性模板DNA 1微克了SP6-mRNA的转录。 </li><li>通过在37℃下加入1.0单位RNAse游离DNA酶I 10-20分钟除去的DNA。 </li><li>使用的RNA，根据制造商的协议清理试剂盒清理的mRNA。 </li><li>以增加mRNA的纯度和浓度，通过加入6微升乙酸钠（3.0M，pH5.2）和150微升96％乙醇沉淀出的mRNA。孵育并购在-20℃ixed溶液至少30分钟，长达24小时。 </li><li>通过在4℃下纺丝在18.407 XG的溶液45分钟收集的mRNA。弃去液体，并在150微升70％乙醇重悬的mRNA。混合和离心的溶液在4℃额外的15分钟。弃去液体，干燥在冰上5分钟，将沉淀，并在20微升超纯无RNase水重悬的mRNA。 </li><li> 100的mRNA稀释液（在99微升超纯无RNase水1微升mRNA）的:由1的光度测量评估mRNA的浓度和纯度。 </li><li>对于短期存储，保持在-20℃的mRNA的溶液。对于长期存储，保持的mRNA在-80℃。 </li></ol> 2. H2B-PSmOrange基因微量注射的斑马鱼胚胎<ol><li>设置使用TG注射前对交配夜（FOXD3:GFP）; TG（FLH:GFP）的双转基因鱼（或任何其他的GFP转基因鱼）。离开鱼放置一个隔板隔开在它们之间，以控制交配的时间。 </li><li>除去在注射的早晨隔板和让鱼交配20分钟。 </li><li>在交配时，淡化H2B-PSmOrange的mRNA 130皮克的最终浓度/ NL（在无RNA酶的水），并转让6微升注射溶液成使用微加载尖端的显微注射毛细管。检查注射量与校准的载玻片。调节注射压力注射2 NL的mRNA溶液（260皮克）。 </li><li>收集鸡蛋放入含有1x胚胎（E3）中无菌塑料培养皿。 </li><li>转移20到30的胚胎，使用一个塑料巴斯德吸管的喷射盘。 </li><li>注入含有溶液到细胞或正下方的细胞进入蛋黄在单细胞阶段11 2 NL的mRNA。 </li><li>转印注射胚胎成含有1x E3介质的培养皿，除去未受精或不能正常发育中的胚胎，并在28℃下生长它们。 注:轻保障oF中的胚胎，不需要在整个实验。 </li><li>提高在1×E3介质斑马鱼的胚胎和原肠胚形成后添加0.2mM的PTU（1-苯基-2-硫脲），以抑制色素沉着。改变介质每天两次，以减少细菌污染的危险。 </li></ol> 3.胚胎嵌入<ol><li>选择的GFP（绿色）和PSmOrange使用装有荧光灯和适当的发射滤光片双目显微镜（橙色/红色）共表达的胚胎。转移的积极胚胎成含有1x E3介质的无菌培养皿。 </li><li> Dechorionate胚胎使用镊子12在立体显微镜下。 </li><li>在含有1.0毫升1.5毫升管传送一个胚胎预热用的切口尖端吸管（P200）在超纯水中制备1.0％低熔点琼脂糖（LMA）。注:在预热至LMA 80℃和嵌入胚胎前冷却在室温下3-5分钟。 </li><li>转移胚胎在150微升LMA成腔玻璃罩和调节胚胎的取向，例如背侧中为倒共聚焦激光扫描显微镜A1R +（或任何其它合适的显微镜）中描述的实验下来，用细塑料小费。当向LMA是聚合的，用含有0.2mM的PTU和0.02％3-氨基苯甲酸乙酯甲磺酸盐（三卡因）麻醉鱼水填充腔室。 </li><li>使样品成像之前，等待15分钟，以确定三卡因的效果。麻醉防止胚胎运动，可以使用装备有明照明立体显微镜进行监测。 注意:室可两次重复使用。 </li></ol> 4. PSmOrange光转化<ol><li>放置共焦显微镜下的样品和扫描试样，以确定该地区使用488纳米和561纳米激光器分别成像GFP和PSmOrange到photoconvert。 注意:使用倒置激光共聚焦显微镜A1R +上的倒站GE TiEcontrolled用显微镜成像软件，并配备了488纳米，561纳米和640纳米激光器光转化和成像。然而，应用程序当然没有限制，只要转换和成像激光线路可用限于此显微镜。 <ol><li>把20X空中目标到位（NA:0.75; WD 1.0毫米; FOV:0.64点¯x0.64毫米）。 </li><li>使用下面的显微镜设置光转化之前检测PSmOrange蛋白质:561纳米的激光，在聚焦平面上的目标上述测定0.74毫瓦。 注意:这对应于40％的在该系统上（在聚焦平面的测量是确定有效功率的唯一方式）。根据实验的需要为H2B-PSmOrange注射的效率可以变化调整激光功率。 </li><li>添加缩放因子，突出感兴趣的区域。放大倍率越高，降低图像采集时间，因此光毒性。 </li></ol></li><li>收购Z堆栈覆盖结构的S使用488纳米和在顺序模式（线模式1-&gt; 4）561纳米激光器的兴趣。修正1.0和2.0微米之间的z步骤。线平均和扫描频率可用于优化图像质量。 </li><li>扫描样本并选择感兴趣（ROI）工具，该地区要突出以photoconvert的区域。修复投资回报率的刺激区域。选择照片激活/漂白模块，通过检查各个框激活488纳米激光器和488nm的激光设定为80％（在目标测量1毫瓦的激光功率）。设置扫描速度0.5秒/帧。 </li><li>打开光转化工具（ND刺激），并指定光转换设置。 <ol><li>点击"添加"命令指定的光转化协议。 </li><li>在"采集/刺激"菜单"获取"设置"相位"1和表示在"循环"菜单光转换之前获得的图像的数量。 </li><li>设置"相位"2"圣要执行imulation"和输入的刺激的事件的数量。 </li><li>调整"相位"3作为"相位"1可选描述，插入一个等待"相位"通过输入时间上轮图像采集前要等待"相位"后2。 </li><li>一旦所有的参数都设置，应用刺激设置和运行该光转化。 注意:激光功率，扫描和迭代次数为每一轮光转化可以变化的频率和从胚胎不同胚胎。的设置来启动与表1中示出。 </li></ol></li><li>收购使用488纳米，561纳米和640纳米的激光应用设置如上一个最终的Z堆栈。设置640纳米的激光来可视化使用高的激光功率的转换PSmOrange（高达4.5毫瓦）。 </li></ol> 5. LMA胚胎下马<ol><li>除去从显微镜容纳室胚胎。小心地从琼脂糖ü取出胚胎唱镊子。 </li><li>转移胚胎成新灭菌塑料培养皿或进入无菌含有1x E3和0.2mM的PTU 6-孔板。孵育在28℃的胚胎直至发展所需的阶段。 </li></ol> 6.分析光转换PSmOrange蛋白表达细胞的命运<ol><li>下点3.3和3.4中所述重新嵌入胚胎。 </li><li>放置共聚焦显微镜下的样品和扫描样本，以确定的利息（488纳米）的GFP转基因结构光转换细胞（640纳米）。 </li><li>收购Z堆栈覆盖感兴趣的结构采用488纳米，561纳米和连续模式（行模式1-&gt; 4）640 nm激光。修正1.0和2.0微米之间的z步骤。线平均和扫描频率可用于优化图像质量。 </li><li>使用下列方法之一来识别细胞，其共表达GFP和所述光转换蛋白。 <ol><li>定制自动斐济图片Ĵ宏3 <ol><li>卷积使用"高斯模糊"插件原来栈（→处理→过滤器→高斯模糊），并减去从原来的使用"图像计算器"插件（→处理→图像计算器）顺利堆栈。使用此步骤可视化感兴趣的结构，并减少计算时间用于分析。 </li><li>适用于绿色和远红外通道特定的阈值，以突出的光转换单元（→图像→调整→阈值）。使用"分析粒子"工具，使用合适的设置来检测阈值的区域（→分析→分析颗粒）。 </li><li>打开"图像计算器"插件和黄（→处理→图像计算器）显示在绿色和远红外通道重叠的投资回报。 </li></ol></li><li>显微镜图像处理软件的3D数据评估<ol><li>显示在堆栈3D使用show量视图选项（→三维可视化菜单→显示卷视图）。使用图形界面来选择绿色，红色和远红色频道，调节亮度，对比度和作物的三维堆叠以突出显示光转换区域（ 图1）。 注意:类似的方法来分析在大多数用于图像分析的通用软件的数据是可用的。 </li></ol></li></ol></li></ol>

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The authors have nothing to disclose.

携带荧光记者转基因胚胎有助于从根本上了解胚胎发育。然而，仍然有启动子的基本需要，以促进特定结构的特定的可视化。在他们的缺席，研究人员依靠技术，如荧光蛋白的光转化，了解他们的利益结构的起源和发展。这又是鉴定参与在其发展的分子机制的关键先决条件。在斑马鱼领域的技术进步，现在允许更换FP在方法14,15使用CRISPR-Cas9介导的敲除转基因系与，例如，photoconvertible蛋白?...

Exponentially improving imaging techniques allow following developmental processes over time periods of up to about four consecutive days3. In zebrafish and many other animal model systems, specific cells, tissues, axonal or vascular structures are marked by transgenic green or sometimes red or yellow fluorescent proteins to facilitate visualization. However, in most transgenic lines the transgene is not specifically expressed in the cells of interest but also additional structures, which hinders the precise tracking of for instance single cells or groups of cells.
Fluorescent photoconvertible proteins are well suited for cell tracking during embryonic development. The prerequisites for the application of such proteins are a long-lived nature, a well-separated emission range upon conversion and bright fluorescence. Available photoconvertible proteins comprise those that change their emission range upon conversion such as Kaede4, KiGR5, mEos26, PS-CFP2 or Dendra27 and others which are only fluorescent when photoactivated (PAmCherry8, PAGFP9 or PATagRFP10). Their applications to track cells in existing FP-transgenic animals are however limited as they often involve a green fluorescent state or do not fulfill all of the above criteria. Only recently, Subach and colleagues reported the PSmOrange protein, which changes its emission from orange/red to far red upon photoconversion and was successfully applied in cells in culture and cultured cells injected into mice2.
To investigate the protein's suitability for cell tracking in a living embryo, we generated an expression construct for the microinjection of nuclear-tagged H2B-PSmOrange into zebrafish embryos. We find that the protein fulfills all prerequisites for successful cell tracking in GFP transgenic backgrounds during the first 4 (and possibly more) days of zebrafish embryonic development. During this time, most of the cell migratory events are completed in fish making the PSmOrange system an excellent addition to the zebrafish toolkit.

<table><tbody><tr><td>PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>mMESSAGE mMACHINE SP6 Transcription kit</td><td>Ambion</td><td>AM1340</td><td></td></tr><tr><td>RNeasy MiniElute Cleanup kit</td><td>Qiagen</td><td>74204</td><td></td></tr><tr><td>Plastic Pasteur</td><td>alpha laboratories</td><td>LW4000</td><td></td></tr><tr><td>Original H2B-PSmOrange Plasmid</td><td>Addgene</td><td>31920</td><td>The plasmid described in the paper is available in the Carl lab</td></tr><tr><td>FemtoJet Microinjector</td><td>Eppendorf</td><td>5247 000.013</td><td></td></tr><tr><td>Forceps (5 Inox)</td><td>NeoLab</td><td>2-1633</td><td></td></tr><tr><td>Lab-Tek II Chambered #1.5 German Coverglass System&amp;nbsp;</td><td>Nunc</td><td>155382</td><td></td></tr><tr><td>Nikon A1R+</td><td>Nikon GmbH Germany</td><td>No Number</td><td></td></tr><tr><td>Nikon PLAN Apo &amp;lambda; 20X air objective&amp;nbsp;</td><td>Nikon GmbH Germany</td><td>No Number</td><td></td></tr><tr><td>NIS Elements AR Software (v. 4.30.02)</td><td>Nikon GmbH Germany/Laboratory Imaging</td><td>No Number</td><td></td></tr></tbody></table>

tracking cells in gfp-transgenic zebrafish using the photoconvertible psmorange system

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

图1示出了PSmOrange光转换系统的一个例子。松果体复杂的是在脊椎动物背间脑的保守结构。像在许多其他脊椎动物中，此复合体由在间脑和左双面parapineal细胞的中心松果体器官。优雅但费时解笼锁的实验表明，parapineal细胞松果体器官13的前部产生。在TG（FOXD3:GFP）; TG（FLH:GFP）转基因胚胎，无论松果体和parapineal细胞发育过程中的标签。为了评估PSmOrange?...

Watch this Scientific Journal Video about Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System at JoVE.com

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

在GFP-转基因斑马鱼使用Photoconvertible PSmOrange系统追踪单元

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

tracking-cells-gfp-transgenic-zebrafish-using-photoconvertible

Research

JoVE Journal

Developmental Biology

8.6K 次观看.  Heidelberg University. We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

视频: Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

科研

发育生物学

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

在斑马鱼胚胎中通过细胞 Photoconversion 的心内膜组织运动后

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

生物学

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles.
Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion.
This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.

在活细胞荧光蛋白的双探头光学突出纯净的光转化的

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

多色成像定时转基因斑马鱼：可视化由针对性的神经细胞消融激活视网膜干细胞

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

单细胞在斑马鱼的命运映射

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

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 ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

的分散Presomitic中胚层细胞培养的斑马鱼分割时钟在单细胞成像代

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

ROS Live Cell Imaging During Neuronal Development

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H2O2) is best characterized with respect to roles in cellular signaling. H2O2 has been implicated during the development in several species. For example, a transient increase in H2O2 has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H2O2 source, NADPH oxidase (NOX), impairs nervous system development such as the differentiation, axonal growth, and guidance of retinal ganglion cells (RGCs) both in&#160;vivo and in vitro. Here, we describe a method for imaging intracellular H2O2 levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H2O2-specific biosensor, roGFP2-Orp1. This probe can be transiently or stably expressed in zebrafish larvae. Furthermore, the ratiometric readout diminishes the probability of detecting artifacts due to differential gene expression or volume effects. First, we demonstrate how to isolate and culture RGCs derived from zebrafish embryos that transiently express roGFP2-Orp1. Then, we use whole larvae to monitor H2O2 levels at the tissue level. The sensor has been validated by the addition of H2O2. Additionally, this methodology could be used to measure H2O2 levels in specific cell types and tissues by generating transgenic animals with tissue-specific biosensor expression. As zebrafish facilitate genetic and developmental manipulations, the approach demonstrated here could serve as a pipeline to test the role of H2O2 during neuronal and general embryonic development in vertebrates.

This protocol describes the use of a genetically encoded hydrogen peroxide (H2O2)-biosensor in cultured zebrafish neurons and larvae for assessing the physiological signaling roles of H2O2 during nervous system development. It can be applied to different cell types and modified with experimental treatments to study reactive oxygen species (ROS) in general development.

神经元发育过程中的 ROS 活细胞成像

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the ...

神经科学

Protein Phase Separation Assay: An Optogenetic Method for Mutant RNA-Binding Protein Phase Separation in Spinal Motor Neurons of Zebrafish Larvae

Source: Asakawa, K. et al. <a href="https://www.jove.com/v/62932">Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae.</a> J. Vis. Exp. (2022)
In this video, we describe a phase separation assay, wherein intracellular proteins comprising intrinsically disordered regions fused to a photosensitive oligomerization domain are induced via blue-light exposure to self-associate into membrane-less liquid-like condensates.

蛋白相分离试验:斑马鱼幼虫脊髓运动神经元突变 RNA 结合蛋白相分离的光遗传学方法

Source: Asakawa, K. et al. Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae. J. Vis. Exp. (2022)
In this video, we ...

在GFP-转基因斑马鱼使用Photoconvertible PSmOrange系统追踪单元

摘要

探索更多视频

活完好斑马鱼的单细胞 Photoconversion

在斑马鱼胚胎中通过细胞 Photoconversion 的心内膜组织运动后

在现场的斑马鱼胚胎的基于双光子Photoactivation

在活细胞荧光蛋白的双探头光学突出纯净的光转化的

多色成像定时转基因斑马鱼：可视化由针对性的神经细胞消融激活视网膜干细胞

单细胞在斑马鱼的命运映射

在斑马鱼发育的细胞追踪使用Photoconvertible蛋白

的分散Presomitic中胚层细胞培养的斑马鱼分割时钟在单细胞成像代

神经元发育过程中的 ROS 活细胞成像

蛋白相分离试验:斑马鱼幼虫脊髓运动神经元突变 RNA 结合蛋白相分离的光遗传学方法

在GFP-转基因斑马鱼使用Photoconvertible PSmOrange系统追踪单元

摘要

探索更多视频

活完好斑马鱼的单细胞 Photoconversion

在斑马鱼胚胎中通过细胞 Photoconversion 的心内膜组织运动后

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

在活细胞荧光蛋白的双探头光学突出纯净的光转化的

多色成像定时转基因斑马鱼：可视化由针对性的神经细胞消融激活视网膜干细胞

单细胞在斑马鱼的命运映射

在斑马鱼发育的细胞追踪使用Photoconvertible蛋白

的分散Presomitic中胚层细胞培养的斑马鱼分割时钟在单细胞成像代

神经元发育过程中的 ROS 活细胞成像

蛋白相分离试验:斑马鱼幼虫脊髓运动神经元突变 RNA 结合蛋白相分离的光遗传学方法

在现场的斑马鱼胚胎的基于双光子Photoactivation