Name: in vivo two-photon microscopy of single nerve endings in skin
Uploaded: 2014-08-24T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin at JoVE.com

作者要感谢Neurotar有限公司提供技术援助，CIMO基金会和FGSN提供财政支持。

涉及动物主题手续已批准的全国动物实验局，芬兰。 1，动物准备成像<ol><li>由intraperitional（IP）注射氯胺酮（每体重0.08毫克）和甲苯噻嗪（每体重0.01毫克），麻醉鼠标。检查与后脚趾捏反射麻醉。 </li><li>沉浸动物的眼睛滴眼液（，涉及的），以保护眼睛免受脱水。 </li><li>把鼠标放在一个加热垫（仕霸）在37℃，以防止体温过低。 </li><li>清洁指定用于成像，用70％乙醇的足垫。 </li><li>添?...

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在这个视频协议，我们证明了该方法的单神经末梢的非侵入性的纵向双光子成像。 皮肤神经支配的动力学在这些疾病如牛皮癣和外周神经病变2，并且在创伤性损伤9受到影响。双光子成像允许神经纤维结构中的胶原基质的详细分析。利用转基因小鼠的报道中，有助于避免与神经纤维的染色的问题。 THY1-YFP-H菌株似乎是形态学数据分析10足够坚固，而大?...

皮肤的神经末梢发生在不同的病理生理状态的动态变化。神经纤维可以通过变性和再生或重组过程中这种疾病的过程中如周围神经病变2莫顿神经瘤3。跌打损伤后，神经末梢动态皮肤的一个重要组成部分是受损区域的神经再支配。然而，对于神经末梢调查的通常的方法是在体外组织学切片，缺乏对正在进行的流程4的实时信息。用遗传编码的荧光标记物，所以能够掌握其神经末梢活体动物的皮肤，从而获得关于结构变化丰富和显著更多相关信息。皮神经末梢的调查能够使用常规荧光显微镜，然而，皮肤组织的强烈的光散射强烈破坏了质量数据的获取5。多光子显微镜能够取得在强烈散射组织中的高清晰度图像，由于只从物镜的焦点导致荧光发射的激发光的光子能量的非线性总和。这个效果导致了强劲增长的穿透深度和改善信号对噪声比的测量在皮肤组织6。使用相同的激光作为成像，所以能够产生神经纤维7的选择性清扫。在下面的协议，我们将展示在体内的皮肤神经末梢纵向成像的记者转基因小鼠结合使用市售的多光子显微镜系统选择性激光损伤的方法。

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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Round cover glass&#160;</td>
			<td style="width:209px;">Electron Microscopy Science</td>
			<td style="width:119px;">72296-05</td>
			<td style="width:687px;">5 mm diameter #1.5 thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eye drops Viscotears</td>
			<td style="width:209px;">Novartis</td>
			<td style="width:119px;"></td>
			<td>2mg/g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Superglue Loctite 401</td>
			<td style="width:209px;">Henkel</td>
			<td>135429</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketaminol</td>
			<td style="width:209px;">Intervet</td>
			<td style="width:119px;"></td>
			<td>Ketamine 50 mg/ml, working solution 10 mg/ml (use it 80 &#181;g per gramm of animal weight)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rompun</td>
			<td style="width:209px;">Bayer Healthcare</td>
			<td style="width:119px;"></td>
			<td>Xylazine 20 mg/ml, working solution 1.25 mg/ml (use it 10 &#181;g per gramm of animal weight)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td style="width:687px;">Working solution is a mixture of Ketamine 10 mg/ml and Xylazine 1.25 mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol 70%</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Distilled water or Milli-Q water</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringes 1ml&#160;</td>
			<td style="width:209px;">BD</td>
			<td style="width:119px;">300013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">30G 1/2&#34; needles</td>
			<td style="width:209px;">BD</td>
			<td style="width:119px;">304000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic packaging material</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td>Could be purchaized from general hardware store</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FV-1000MPE microscope</td>
			<td style="width:209px;">Olympus</td>
			<td style="width:119px;">FV-1000MPE</td>
			<td>Microscope with motorized stage and rigid metal bar for fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25X XLPlan objective</td>
			<td style="width:209px;">Olympus</td>
			<td style="width:119px;">XLPLN 25XWMP</td>
			<td>Water imersion objective optimized for multiphoton imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mai-Tai DeepSee laser (2W)</td>
			<td style="width:209px;">SpectraPhysics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heating pad</td>
			<td style="width:209px;">Supertech</td>
			<td style="width:119px;">TMP-5b</td>
			<td>Heating pad with a temperature controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metal ring fixator</td>
			<td style="width:209px;">Neurotar Ltd.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td style="width:209px;">NIH</td>
			<td style="width:119px;"></td>
			<td>Open source software for image processing and analysis, http://rsbweb.nih.gov/ij/&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thy1-YFPH mice strain</td>
			<td style="width:209px;">JaxLab</td>
			<td style="width:119px;">003782</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo two-photon microscopy of single nerve endings in skin

在皮肤神经末梢参与生理过程，如感测1以及在病理过程，如神经性疼痛2。他们贴近地表的定位有利于皮肤神经末梢的生活完整的动物显微成像。使用多光子显微术，它能够获得精细图像克服的皮肤组织的强烈的光散射问题。表达的THY-1启动子在神经元（包括周感觉神经元）的控制下EYFP记者转基因小鼠是适合于个别的神经末梢在延长的时间周期的纵向研究长达数月甚至终生。此外，使用相同的飞秒激光作为用于成像，所以能够产生神经纤维的高选择性病变的神经纤维重组的研究。在这里，我们提出了一个简单而可靠的协议进行纵向多光子活体成像和基于激光显微对小鼠皮肤的神经末梢。

使用所描述的技术也能够掌握其损伤后在同一光纤，并研究了受损的神经末梢（ 图1）的降解。采集的堆栈120-150微米的厚度通常是正确的重复成像期间数天，以保持在整个纤维中的视场。 病灶通常可以简明地产生时的塑料包装材料被调整到弄平皮肤，使胶原蛋白层出现在图像上均匀的强度（ 图2）。神经末梢的胶原层之上是，以产生精确的病变最合?...

Watch this Scientific Journal Video about In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin at JoVE.com

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

在皮肤中的单神经末梢体内双光子显微镜

该协议的动态纵向成像和神经末梢记者在转基因小鼠中的选择性激光损伤呈现。

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Research

JoVE Journal

Neuroscience

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

在体内脊髓的鼠标线使用双光子显微镜成像

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

科研

神经科学

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

同时双光子<em&gt;在体内</em&gt;突触输入的影像和突触后目标中的鼠标Retrosplenial的Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

利用双光子显微镜的艾滋病毒引起的神经炎症的小鼠模型脑血管结构的量化

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

在毛囊披针形结局生活神经末端标记的光学监测<em&gt;离体</em&gt;鼠耳皮肤

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...