Name: confocal imaging of single mitochondrial superoxide flashes in intact heart or in vivo
Uploaded: 2013-11-05T19:00:00
Description: Watch this Scientific Journal Video about Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo at JoVE.com

作者要感谢博士。郑和平，惠良张和斯蒂芬Kolwicz的宝贵意见，并在开发这种方法的技术支持。这项研究是由美国国立卫生研究院资助和科学家发展资助从美国心脏协会WW支持。

1。实验准备<ol><li>制备含有等渗平衡盐溶液（50毫升）：140 mM氯化钠，5mM的氯化钾，2.5mM的氯化钙 ，2毫摩尔MgCl 2和10mM HEPES（pH 7.2）中， 在体内骨骼肌成像。 </li><li>制备1升含的Krebs-Henseleit缓冲液（KHB）：118 mM氯化钠，5.3 mM的氯化钾，1.2mM的MgSO 4干燥，0.5mM的EDTA和25mM的NaHCO 3。 </li><li>气泡的KHB与含氧气体95％O 2/5％CO 2 10-15分钟之前，加入2mM的CaCl ...

<ol>
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成像在活的动物或器官灌注线粒体单事件具有比传统方法的线粒体功能评价17,19,21,22,24,25显著优势。这里描述的技术可以实现实时的在一个真实的生理状态在亚细胞分辨率线粒体功能的原位测定。这是特别有用的，当与其它测量值相结合，系统地研究线粒体在体内特定器官或细胞类型的正常功能的作用。这是一个复杂的技术，它结合了共焦显微术和复杂的灌注系统的各...

线粒体在细胞生物能量核心作用，自由基信号，氧化还原平衡，离子调控和细胞命运决定1,2。线粒体功能障碍常伴和的基础疾病3-6的发病机制。尤其是在肌肉系统，如心脏和骨骼肌线粒体呼吸提供了广大的ATP以支持及时调控细胞内钙离子和强大的开发力量7,8。这些肌肉具有大量线粒体经常占据高达总细胞体积的20-40％，并在肌丝2之间被“固定”的。 尽管大量的研究，我们对线粒体功能的调节，特别是在体内和在生理学相关条件的理解，是有限的。其中一个原因是，多数的评价线粒体功能开发的方法依赖于在VITRo或体外的方法，如监测分离线粒体辅以人工基质的耗氧量，并通过形态学（ 如电子显微镜），酶的活性（ 如乌头酸酶活性）的间接测定线粒体功能，或细胞内ATP水平9-11 。 最近，小分子荧光指示剂与线粒体相对富集已应用于提供一瞥线粒体信号，包括膜电位，钙和活性氧簇（ROS），在完整细胞中11-13。此外，一些绿色荧光蛋白（GFP）的氧化还原和ROS指标，已经开发了实现了条块细胞内的氧化还原或ROS信号14-16的更具体的评价。这中间，我们开发了一种基因编码的超指标，圆形排列的黄色荧光蛋白，并targeteD它进入线粒体（MT-cpYFP）17。 MT-cpYFP可以在405或488 nm的激发与在515 nm的两个发射峰。在488nm激发的发光是专门响应于在体外和体内的校准17,18所示由以前超氧。在405nm处激发的发光被用作内部控制（请参考图1号17的对MT-cpYFP的发射光谱和激发光谱的详细信息在各种条件下）。随着时间推移共焦成像，这一指标检测爆棚超生产活动，名为超氧化物闪烁，在单个线粒体完整细胞。超闪作为线粒体呼吸，伴随的瞬态线粒体膜去极化和活性氧的产生17-20的复合函数。最近，我们已经生成使用PUC-CAGGS-MT-cpYFP矢量17,19上C57/BL6背景的泛组织MT-cpYFP转基因小鼠和验证了强烈的表达锡永这一指标在心脏，骨骼肌等组织中（ 图2）。根据要求和MTA批准由华盛顿大学的转基因小鼠将供感兴趣的学术研究。 在这项研究中，我们描述了在超闪烁的Langendorff灌流心脏原位成像以及体内成像的麻醉MT-cpYFP转基因小鼠17,19骨骼肌Flash事件。此技术允许在生理相关的条件下或在体内21,22单个线粒体ROS产生事件的实时监控。它也是可行使用该系统来监测其它单线粒体参数如膜电位和钙与适当的荧光指示剂。与细胞内事件（ 如钙瞬变）或心脏功能（线粒体功能进一步，同步或并行的评价如。射血分数）就可以实现。病理扰动，如局部缺血和再灌注，可以被应用到灌注心脏评估应力对未受影响的心肌单线粒体功能的影响。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Blebbistatin</td>
			<td>Toronto Research Chemicals</td>
			<td>B592500</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Acros Organics</td>
			<td>AC34961-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP120-500</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-1</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H7006-500</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541-1</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#8226;6H2O</td>
			<td>Fisher Scientific</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#8226;7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M1880-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>S8282-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S6014-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P2256-25</td>
			<td></td>
		</tr>
		<tr>
			<td>TMRM</td>
			<td>Invitrogen</td>
			<td>T-668</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Confocal Line Scanning Microscope (LSM 510 Meta, Zeiss), software version 4.2 SP1 including &#34;Physiological Analysis&#34; module.</td>
		</tr>
	</tbody>
</table>

confocal imaging of single mitochondrial superoxide flashes in intact heart or in vivo

线粒体是负责能源生产和细胞内信号在真核系统中的关键细胞内的细胞器。线粒体功能障碍常伴随并有助于人类疾病。已开发，以评估线粒体功能和功能障碍的方法的大多数都是在体外或离体测量的基础上。这些实验的结果具有有限的在确定体内的线粒体功能的能力。在这里，我们描述了一种新的方法，利用激光共聚焦扫描显微镜对完整的组织的活缩醛胺的成像，其允许单个线粒体功能的评价在一个实时的方式在体内 。首先，我们生成表达线粒体超氧针对性的指标，循环置换黄色荧光蛋白（MT-cpYFP）转基因小鼠。麻醉MT-cpYFP鼠标固定在一个特制的舞台适配器和时间推移图像拍摄fROM中的后肢暴露的骨骼肌。鼠标随后被处死，心脏被设置为Langendorff灌流与生理溶液在37°C灌注心脏被定位在一个特殊的腔上的共聚焦显微镜阶段，温柔的压力施加到固定的心脏，抑制心脏的跳动引起的运动伪影。超氧化物闪烁是由实时二维共焦成像，每秒1帧的频率检测到。该灌注溶液可修改成包含不同呼吸底物或其它荧光指示剂。灌注也可以进行调整，以产生疾病模型，如缺血再灌注损伤。这种技术是用于确定单个线粒体在完整的组织和体内的功能的独特的方法。

原位成像灌注心脏（ 图1），根据该协议，单个线粒体事件的体内成像可以在麻醉小鼠的骨骼肌完成之后。的成像条件的最佳设置将确保清晰的图像的完整肌肉组织中，并与单一的线粒体的分辨率（ 图2）。 TMRM经常被用来验证的mt-cpYFP的位置和应显示一个完整的重叠图案与MT-cpYFP信号（ 图2）。 TMRM对于在完整细胞中23线粒体膜电位的测定...

Watch this Scientific Journal Video about Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo at JoVE.com

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

共聚焦扫描显微镜是适用于在成像灌注心脏或骨骼肌线粒体单事件活的动物。单个线粒体过程，如超氧化物闪烁和膜电位的波动进行实时监控，使线粒体功能的评价在生理相关的背景和病理过程中的扰动。

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

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Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Single Cell Electroporation in vivo within the Intact Developing Brain

在体内的单细胞电穿孔内完整的大脑发育

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

科研

生物学

Single-cell Suction Recordings from Mouse Cone Photoreceptors

单从小鼠锥感光细胞吸录音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

钙火花的评估在正常肌肉纤维

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

一氧化氮和超氧化物歧化酶恩的人脸检测在完整的动脉内皮细胞层

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

高分辨率呼​​吸测量，以评估线粒体功能的通透和完整细胞

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

果蝇的制备及心功能体内使用光学相干显微镜成像纵向（OCM）

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

神经肽 Y-pHluorin 的共焦成像: 一种完整的小鼠和人胰岛胰岛素颗粒胞的可视化技术

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

单个细胞分辨率三维成像的有机体

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Quantification of Mitochondrial Membrane Potential and Superoxide Levels

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells (Video) | JoVE 

使用HeLa细胞中的实时成像对线粒体膜电位和超氧化物水平进行基于荧光的定量

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, ...