我们感谢伯纳德 Kulemaka 和史密斯实验室的成员为他们提供有用的评论和试剂指导, Sam 康奈尔和布伦特. 雷德福3i 的成像问题和黛博拉砰, 凯伦注意和凯斯图尔特为斑马鱼护理。这项工作得到了圣母大学、伊丽莎白和迈克尔？加拉格尔家族的支持, 在巴黎圣母院, 圣母大学斑马鱼研究中心, 圣母大学的干细胞和再生医学中心。圣母.所有动物研究都是按照圣母大学 IACUC 博士的要求进行的。

所有动物研究都是由圣母大学机构动物保育和使用委员会批准的。
1. 斑马鱼标本的制备
<ol><li>将一个成年男性和一个成年女性 Tg (可转换蛋白) 放入每个标准过程的交配室13。在这篇手稿中, 使用 Tg (sox10: eos) 鱼9由于访问, 但其他转基因线与 photoconvertible 蛋白可以同样使用。设置一个以上的房间, 以防鱼不下蛋。允许鱼一夜之间留在房间里。</li>
	<li>第二天早上, 在100毫米培养皿中收集鸡蛋。在筛查前允许卵成熟到24小时后受精 (hpf)。</li>
	<li>如果上面的动物是杂合子的转基因, 屏幕 24 hpf 菜肴为 Tg (sox10: eos) + 胚胎与 488 nm 光源和 GFP 过滤器集在解剖显微镜。分离 Tg (sox10: eos) + 胚胎, 并允许成熟到 48 hpf。</li>
	<li>用针或镊子手动 Dechorionate 胚胎。</li>
	<li>准备和微波5毫升的0.8% 低熔点琼脂糖溶液。<ol>
<li>一旦琼脂糖是凉爽的触摸, 地方3-4 麻醉 Tg (sox10: eos) + 48 hpf 鱼的中心10毫米玻璃盖玻片底部培养皿。</li>
		<li>添加足够的琼脂糖, 以覆盖盖玻片的表面, 大约1毫升。用探针针在它们的两侧排列鱼。允许琼脂糖固化以确保动物的安装。可能需要不断重新安排斑马鱼, 直到琼脂固化14。凝固需要大约2分钟。</li>
	</ol>
</li>
	<li>琼脂糖凝固2分钟后, 慢慢加入含有0.02% 苯甲酸酸酯 (Tricaine) 的胚培养基, 直到琼脂和盘子的底面被淹没。这应该是大约3mL。</li>
</ol>2. 显微镜安装和预转换成像
<ol><li>打开共焦软件并选择捕获和焦点窗口 [图 1]。在 "捕获" 窗口下, 选择 "捕获设置" 下拉选项卡下的实验室特定的转换成像设置 [图 1]。在这里, 实验室特定的设置称为 "鱼映像"。</li>
	<li>将标本放在共聚焦范围内, 使用过程和微调旋钮将试样聚焦。</li>
	<li>打开焦点窗口, 找到所需的感兴趣区域 (即背根神经节)。</li>
	<li>在 "过滤器集" 菜单下选择 c488 激光器。将曝光率设置为300毫秒, 激光功率为 5, 并增强为 75 [图 2]。</li>
	<li>选中捕获类型部分下的3D 框。在3D 捕获部分中, 选择使用当前位置并在当前周围检查范围。在同一节中, 将范围设置为 35, 将平面数设为 36, 并将步长调整为1。范围可以增加或减少, 以适应成像区域的深度。对于脊髓, 35-40 栈之间的范围值通常是足够的 [图 5]。</li>
	<li>选择当前位置 [图 5]。</li>
	<li>单击 "捕获" 窗口底部的 "开始" 以获取图像。</li>
</ol>3. 单细胞 Photoconversion
<ol><li>打开共焦软件并选择捕获和焦点窗口 [图 1]。在 "捕获" 窗口下, 选择 "捕获设置" 下拉选项卡下的实验室特定的转换成像设置 [图 1]。在这里, 实验室特定的设置称为 "鱼消融全芯片。</li>
	<li>在 "过滤器集" 菜单下选择 c488 和 c541 激光器。如果不使用相同的显微镜软件, 找到菜单选择不同的激光器, 并选择 488 nm 和 541 nm 激光器。将曝光设置为300毫秒, 激光功率为 5, 并增强为 75 [图 2]。这些激光设置是根据产生足够的荧光信号而不引起漂白或毒性而选择的。如果毒性或漂白可视化, 减少激光功率或曝光。</li>
	<li>打开焦点窗口, 然后单击 "焦点" 窗口中的 "photomanipulation" 选项卡。相应地调整激光参数。将激光堆栈电源更改为 2, 然后单击转到。将光栅块大小更改为 1, 然后单击设置。请将双击大小更改为4。将激光线更改为 v405 [图 3]。</li>
	<li>在 "捕获" 窗口中打开高级捕获设置。选择 "photomanipulation" 选项卡, 然后将双击重复次数更改为2。单击 "确定" (图 4)。</li>
	<li>在焦点窗口中选择 XY 选项卡。双击 photomanipulation 选项卡中的步骤2中的激光参数 [图 3,图 4]。设置激光设置, 以 photoconvert 细胞的兴趣, 没有 photoconversion 的周围细胞。<ol>
<li>如果相邻细胞 photoconversion 存在, 则减少激光功率。如果 photoconversion 细胞不发生, 激光功率可以增加。优选地, 设置激光功率 photoconvert 仅感兴趣的区域和不附近区域。</li>
	</ol>
</li>
	<li>选中捕获类型下的 timelapse 框。然后, 单击开始[图 6A]。</li>
	<li>打开 "实时 timelapse" 窗口后, 选择顶部工具栏上的 "圆圈" 工具 [图 7]</li>
	<li>在单元格的靠近中心区域中绘制一个圆。右键单击绘制的圆圈, 选择酶区域, 然后等待3秒。选定区域应变暗 [图 8]。单击停止捕获。</li>
	<li>如果成像多个动物, 请返回焦点菜单中的 XY 选项卡, 然后选择 "位置 2"。然后, 重复步骤 4.1-4.6。</li>
	<li>要转换单元格的数量, 请按照步骤 4.1-4.4 中的协议和激光参数进行操作, 但不要绘制一个圆来酶感兴趣的区域, 请使用 "线条" 工具。在感兴趣的区域上绘制一条线, 并使用上面列出的相同参数酶区域。</li>
</ol>4. photoconversion 后成像
<ol><li>一旦所有的点都 photoconverted。选择 precoversion 映像步骤3中描述的实验室特定的标准图像堆栈设置。这是在捕获窗口的 "捕获设置" 下拉选项卡下找到的 [图 5]。</li>
	<li>选择 c488 激光器并将曝光设置为 300ms, 激光功率为 5, 并加强到 75 [图 2]。</li>
	<li>选择与 c488 激光器在同一菜单下发现的 c541 激光器。将曝光率设置为500毫秒, 激光功率为 10, 并增强为 75 [图 9]。</li>
	<li>选中捕获类型部分下的3D 框。在3D 捕获部分中, 选择使用当前位置并检查当前范围。在同一节中, 将范围设置为 35, 将平面数设为 36, 并将步长调整为1。范围数可能会增加或减小以适应所需的图像深度 [图 5]。</li>
	<li>如果 "XY 焦点" 选项卡中有多个点, 请在 "捕获" 窗口中选择 "多位列表" 选项。如果没有, 请选择 "当前位置"。</li>
	<li>单击 "捕获" 窗口底部的 "开始" 以获取图像。</li>
</ol>

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作者没有什么可透露的。

在复杂组织中, 不同的细胞类型组织成特定的领域。最近的技术已经被用来标记这些大型组织结构中的单个细胞1,2,3。在这里, 我们演示了两种技术, 同样可以用来可视化的单细胞相互作用和细胞群的相互作用, 在复杂的组织。photoconversion 技术的优势在于, 科学家必须对细胞进行清晰的标记。借助显微镜, 它能够在较大的成像窗口...

多个不同的细胞相互作用, 建立和维持复杂的动物和植物组织。这些细胞往往是夹层和难以区分与邻居在一个单一的细胞水平没有高分辨率显微镜, 需要固定的组织。然而, 要了解这些组织是如何形成的, 如何保持, 并成为病态, 就必须研究组织内的单个细胞在一段时间内是如何相互作用的。理想情况下, 这些实验要求在不需要固定的情况下, 用非侵入性的方式对组织内的单个细胞进行标记。科学家们现在已经开发了许多技术来完成这个任务1,2,3,4。
水母绿色荧光蛋白 (GFP) 的发现和实施是一种令人兴奋的方法, 允许在组织环境1中标记不同的细胞。使用特定于单元格的启动器, 可以从遗传学上选择标记为1的单元格子集。或者, 病毒诱导的 gfp 表达可以用于 gfp3,4的用户选择的表达式。虽然很有用, 基因介导的 GFP 表达不允许用户选择的表达在组织中的细胞子集;和病毒表达的 GFP, 虽然有利, 可侵入。随着 GFP 衍生物的出现和像 Brainbow 这样的巧妙的技术, 在组织中表达出不同的荧光蛋白更稀少, 它已经成为可能的可视化单个细胞和它们之间的相互作用在复杂的组织2,5. 但是, 这些方法以随机方式标记单元格。如果所需的实验需要对实验者定义的单个细胞或细胞的数量进行可视化, 那么它们是有限的。通过这样的实验 , 有一个基因表达的荧光蛋白 , 可以纵 , 以区别 , 在一个单一的细胞的方式 , 它从其他荧光和非荧光细胞 , 这将是有利的。
为了实现这一目标, 并可视化一个复杂的活体组织内单细胞生物学, 科学界使用的单一细胞 photoconversion 的独特的荧光蛋白6,7,8。使用 photoconvertible 蛋白的基因控制表达 (即, eos, 枫等), 当暴露于紫外线 (488 nm) 光时, 从绿色到红色荧光状态过渡, 我们可以区分单个细胞与它的荧光标记邻居6,7,8。这种方法利用连接到我们共焦显微镜的仪器, 它可以将激光栈中的光定向到感兴趣的衍射有限区域。使用此技术, 我们可以用用户定义的方式 (9、10、11) 来标记单个单元格或更大的填充。这项技术是微创与单细胞注射的病毒 GFP。作为概念的证明, 我们表明, 我们可以 photoconvert 在神经节内的单个细胞在周围神经系统和 photoconvert 更大的人群, 如位于脊髓腹侧的细胞9,10, 11,12。然后, 我们可以想象这些 photoconverted 细胞群24小时后, 以了解他们的运动和分化过程中的发展。

<table><tbody><tr><td>&lt;font class=&quot;font5&quot;&gt;Tg(&lt;em&gt;sox10:eos&lt;/em&gt;)&lt;/font&gt;&lt;font class=&quot;font0&quot;&gt; zebrafish animals&lt;/font&gt;</td><td></td><td></td><td>Fish were obtained and crossed from the fish facility at the University of Notre Dame</td></tr><tr><td>100 X 15 mm petri dish</td><td>VWR</td><td>25384-302</td><td></td></tr><tr><td>Embryo medium</td><td></td><td></td><td>Embryo medium is made weekly and provided by the fish facility at the University of Notre Dame containing 5L RO water, 30uL methylene blue, and 200mL salt stock.</td></tr><tr><td>0.8% Low Melting Point Agarose</td><td>dot scientific inc</td><td>9012-36-6</td><td></td></tr><tr><td>35 X 10 mm glass-coverslip bottom petri dish</td><td>Ted Pella Inc.</td><td>14021-20</td><td></td></tr><tr><td>Needle dissecting probe</td><td></td><td></td><td></td></tr><tr><td>0.002% 3-aminobenzoic acid ester (Tricaine)</td><td>Fluka analytical</td><td>A5040-250G</td><td></td></tr><tr><td>Fluorescent Dissecting Microscope with GFP filters</td><td>Zeiss Axiozoom</td><td></td><td></td></tr><tr><td>Confocal microscope with lasers to excite GFP and RFP filter sets</td><td>3i spinning disk confocal</td><td></td><td></td></tr><tr><td>UV light source (laser) for photoconversion</td><td>405 nm laser</td><td></td><td></td></tr><tr><td>Slidebook software</td><td>3i</td><td></td><td></td></tr><tr><td>Methylene blue</td><td>Kordon</td><td></td><td></td></tr></tbody></table>

single-cell photoconversion in living intact zebrafish

动物和植物组织由细胞的不同的人口组成。这些细胞随着时间的推移相互作用, 以建立和维持组织, 并可能导致疾病时中断。科学家们通过在细胞子集中表达荧光蛋白, 开发出巧妙的技术来研究这些细胞在完整组织中的特性和自然动力学。然而, 有时, 实验需要更多的细胞在组织内的可视化, 有时候是在单细胞或细胞群的方式。为了实现这一目标, 并在细胞中显示单个细胞, 科学家们利用了荧光蛋白的单细胞 photoconversion。为了演示这项技术, 我们在这里展示了如何将紫外线照射到一个在完整的、活的斑马鱼身上表达出感兴趣的 Eos 细胞。然后, 我们在24小时后对那些 photoconverted Eos+单元格进行映像, 以确定它们在组织中的变化。我们描述了两种技术: 单细胞 photoconversion 和 photoconversions 的细胞群。这些技术可用于可视化细胞相互作用, 细胞命运和分化, 细胞迁移, 使其成为一种技术, 适用于许多生物学问题。

Photoconversion 荧光蛋白可用于标记组织中的不同细胞6。为了证明这一点, Tg (sox10: eos)鱼9被用来表达 photoconvertible 蛋白 eos 下的调节序列sox10。Tg (sox10: eos)动物在 48 hpf 第一次安装, 然后成像, 以检测任何可能发生的非特定 photoconversion。然后, 将 eos 未转换的荧光信号与小 Eos photoconverted 荧光信号进行可视化 (<strong class="xfig...

Watch this Scientific Journal Video about Single-cell Photoconversion in Living Intact Zebrafish at JoVE.com

Single-cell Photoconversion in Living Intact Zebrafish

在这里, 我们提出了一个协议, 以显示如何实现细胞 photoconversion 是通过紫外线照射到特定区域表达荧光蛋白, Eos, 在活体动物。

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 ...

single-cell-photoconversion-in-living-intact-zebrafish

Research

JoVE Journal

Developmental Biology

6.0K 次观看.  University of Notre Dame. 在这里, 我们提出了一个协议, 以显示如何实现细胞 photoconversion 是通过紫外线照射到特定区域表达荧光蛋白, Eos, 在活体动物。

视频: Single-cell Photoconversion in Living Intact Zebrafish

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

科研

发育生物学

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.

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 ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

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 ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

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 ...

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 ...

In Vivo Confocal Fluorescence Imaging of Neural Activity Induced by Sensory Stimulation in Partially Restrained Larval Zebrafish

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural circuitry facilitate the use of optogenetic techniques in cellular analyses of behavior. Fluorescent indicators of in vivo neural activity, such as GCaMP6s, have been widely used to study the neural activity associated with simple behaviors in larval zebrafish. Here, we present a protocol for detecting sensory-induced activity in semi-restrained zebrafish larvae using the transgenic line Tg(elav3:GCaMP6s). In particular, we use the chemical agent allyl isothiocyanate to induce a robust, reproducible fluorescent response in a brain region at the border of the hindbrain and spinal cord. We discuss the potential uses of GCaMP6s for optical monitoring of neural activity during a range of behavioral paradigms and the limitations of this technique. Our protocol outlines an accessible approach for monitoring dynamic, behavior-related in vivo neural activity in the larval zebrafish brain.

Here, we present a detailed protocol to examine neural activity in brain regions of transgenic zebrafish that express GCaMP calcium indicators using confocal microscopy.

体内研究 部分束缚斑马鱼幼鱼感觉刺激诱导的神经活动的共聚焦荧光成像

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural ...

活完好斑马鱼的单细胞 Photoconversion

摘要

探索更多视频

在活斑马鱼胚胎成像的亚细胞结构

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

现场共聚焦显微镜对斑马鱼的胚胎脑成像

在斑马鱼胚胎中通过细胞 Photoconversion 的心内膜组织运动后

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

在现场的斑马鱼胚胎的基于双光子Photoactivation

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

体内研究部分束缚斑马鱼幼鱼感觉刺激诱导的神经活动的共聚焦荧光成像

活完好斑马鱼的单细胞 Photoconversion

摘要

探索更多视频

在活斑马鱼胚胎成像的亚细胞结构

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

现场共聚焦显微镜对斑马鱼的胚胎脑成像

在斑马鱼胚胎中通过细胞 Photoconversion 的心内膜组织运动后

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

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

体内研究 部分束缚斑马鱼幼鱼感觉刺激诱导的神经活动的共聚焦荧光成像

在现场的斑马鱼胚胎的基于双光子Photoactivation

体内研究部分束缚斑马鱼幼鱼感觉刺激诱导的神经活动的共聚焦荧光成像