Name: single-cell photoconversion in living intact zebrafish
Uploaded: 2018-03-19T14:00:00
Description: Watch this Scientific Journal Video about Single-cell Photoconversion in Living Intact Zebrafish at JoVE.com

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。...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="1846">
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt;width:320pt" width="427">Tg(sox10:eos) zebrafish animals</td>
			<td style="width:125pt" width="167"></td>
			<td style="width:85pt" width="113"></td>
			<td style="width:854pt" width="1139">Fish were obtained and crossed from the fish facility at the University of Notre Dame</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">100 X 15 mm petri dish</td>
			<td>VWR</td>
			<td>25384-302</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">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 height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">0.8% Low Melting Point Agarose</td>
			<td>dot scientific inc</td>
			<td>9012-36-6</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">35 X 10 mm glass-coverslip bottom petri dish</td>
			<td>Ted Pella Inc.</td>
			<td>14021-20</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Needle dissecting probe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">0.002% 3-aminobenzoic acid ester (Tricaine)</td>
			<td>Fluka analytical</td>
			<td>A5040-250G</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Fluorescent Dissecting Microscope with GFP filters</td>
			<td>Zeiss Axiozoom</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Confocal microscope with lasers to excite GFP and RFP filter sets</td>
			<td>3i spinning disk confocal</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">UV light source (laser) for photoconversion</td>
			<td>405 nm laser</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Slidebook software</td>
			<td>3i</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">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 ...

Research

JoVE Journal

Developmental Biology

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

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 ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

成像清除胚胎和出生后心脏在单细胞分辨率

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

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 ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

小鼠胰腺内分泌细胞的单细胞 Transcriptomic 分析

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

将DNA微注射到异种胚胎的眼蕾中，以及GFP表达光学轴状轴向轴向的成像，活的Xenopus大白鼠

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

使用"彩虹"和"延时共聚焦成像"可视化活斑马鱼中发育中的脑

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...