Name: harmonic nanoparticles for regenerative research
Uploaded: 2014-05-01T13:45:00
Description: Watch this Scientific Journal Video about Harmonic Nanoparticles for Regenerative Research at JoVE.com

作者要感谢来自欧洲FP7研究项目NAMDIATREAM（NMP4-LA-2010-246479，http://www.namdiatream.eu）和INTERREG IV法国 - 瑞士NAOMI项目的部分资金。

1，文化与扩展人类的ESC <ol><li>制备含有基因敲除DMEM细胞繁殖培养基（称为PM）的补充有20％敲除血清，1％MEM非必需氨基酸，1％L-谷氨酰胺的200mM，1％青霉素 - 链霉素，和3.5微升β-巯基乙醇。 </li><li>在8ml下午中等解冻人类胚胎（胚胎干细胞）和他们离心5分钟，在115 XG放弃DMSO-辅以冷冻介质。 </li><li>加入1 ml PM培养基含有10mM摇滚抑制剂以阻止细胞凋亡和促进细胞存活时解冻（仅24小时培养）。 </li>...

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纳米技术的应用，干细胞研究是一个相对较新的，但迅速扩大的领域。正如关于这一主题的各种评论文章，利用纳米颗粒的可应用来完成不同的任务的研究，从细胞追踪（ 在体外和在体内 ），向细胞内递送的蛋白质和基因，建立至少不仿生蜂窝环境中的特定分化优惠政策刺激/抑制通路19,20。在本协议中所述的方法是有限的，以光学跟踪，虽然采用红外线激发的可能性，导致...

非线性显微镜系统中，由于其固有的三维切片能力，已越来越多地用于触发光稳定的荧光团与双光子吸收带在近红外的需求。仅在过去的几年中，以补充基于荧光的标签（染料，量子点，上转换纳米颗粒），不同的成像方法已被开发利用了一种新的非线性固有的纳米粒子的家庭作为标签， 即发展谐波纳米粒子（的HNP）已对多光子显微镜是专门开发的。这些标签的基础上，无机非中心对称晶体，施加的光学对比度产生激励频率的SH：例如通过转换的近红外脉冲激发光的一小部分（λ= 800纳米）转换成可见光的蓝光（λ/ 2 = 400nm）的。几位作者在最近的过去已经测试了不同的材料，包括铁碘酸铁（IO <suB&gt; 3）3 1，铌酸钾（的KNbO 3）2，铌酸锂（LiNbO 3晶体）3，钛酸钡（BaTiO 3的）4,5，磷酸氧钛钾（KTiOPO4，KTP）6-8，和氧化锌（ZnO ）5,9,10。相比于荧光探针的HNP具有一系列有吸引力的性质，如完全没有漂白和闪烁，窄发射带，激发波长可调谐性（从紫外到红外），取向检索能力和相干光响应的。这些独特的性能已在两次全面审查文件11,12最近解释。在红外光谱区域，它通过最小化散射和吸收增加成像深度的工作，也有可能大大限制了样品光降解13,14。此外，通过的HNP保证了无限的光稳定的信号，使它们成为理想的探针进行长期跟踪的细胞，这是我更是尤为吸引人再生医学应用15。 在这个可视化实验，提供了协议的细节进行体外标记的人类胚胎干细胞的（胚胎干细胞）与未官能化的HNP。胶体悬浮液的合成与制备详见以前的出版物和参考文献16和超出了工作范围。方法通过对多光子显微镜和其分化成心肌集群（如保持长期气 - 液培养）人类胚胎干细胞研究被提出。人类ESC可以让两种不同的方法，或者通过EB形成集落的碎片在悬浮液，或者，迫使单细胞进入胚体使用Aggrewell板聚集，如示于图1A中所谓的类胚体（EB）内，以区分。培养簇跳动心肌细胞对聚四氟乙烯（PTFE）多孔过滤因子ilitates他们的长期维护进行进一步的研究（例如电动作电位测量）。 扫描显微镜的激发源应当能够提供超短脉冲（与脉冲持续时间超过300飞秒较小的样本），以达到执行的HNP的二次谐波成像所需要的峰值功率。例如，用于成像的最常见FSEC源是可调谐的Ti：蓝宝石激光器。可替换地，其他超快激光器可以采用，例如铒离子17，铬镁橄榄石18或Ti：蓝宝石泵浦红外光参量振荡器。显微镜可以配备一个客观与优选相当高的数值孔径。非常重要的是，之前的测量值，并且目标是，每次更换，这是强制性的，通过优化激光脉冲预压缩机的设置在工作，以减少存在于该集合式（镜片）的分散波长选择。此过程中，在协议中详细描述的，确保了激光脉冲是尽可能接近到变换有限的持续时间（ 即最短可能的话）在焦平面和最大限度地提高了样品的非线性响应。 在协议结束时描述的图像分析的目标是识别并跟踪在跳动的心脏簇的节律性收缩相关联的三维运动的HNP。在图像平面中的跟踪纳米粒子被简单地通过确定它们在连续的动画帧的位置来实现。以提取的轴向运动信息，将非线性响应强度作为轴向位移的一个函数的现有校准是必须的。请注意，对于长期测量，样品的轴向位置的有源干涉控制是必需的，以保持校准曲线的有效性在热和/或机械漂移的存在。 这里使用的TRAC的的HNP聚集在ê跳动电池是基于铌酸钾氧化（的KNbO 3），但其他可用的非线性纳米材料中详细Staedler 等[16]的工作进行检讨。 大部分至今调查纳米材料的非线性光学效率是非常类似的。为的KNbO 3的选择基本上是由胶体溶液的稳定性好，并具有良好的生物相容性，对几种人类细胞系，即使在相当高的浓度和较长的论述16次测试的动机。 鉴于用于这项工作的纳米材料的新颖性，的HNP相比荧光灯/荧光生物标记物的主要特征都显示在很短的原始计算机影视动画由作者来实现。

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			<td height="22" style="height:22px;width:231px;">Microscope Incubator</td>
			<td style="width:231px;">OKO LAB</td>
			<td style="width:205px;">UNO package (top stage)</td>
			<td style="width:343px;">&#160;37&#176;C, 5% CO2, moisturized</td>
		</tr>
		<tr height="21">
			<td height="64" rowspan="2" style="height:64px;width:231px;">Multiphoton microscope</td>
			<td rowspan="2" style="width:231px;">Nikon</td>
			<td rowspan="2" style="width:205px;">AR1-MP</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:343px;">Fast scanning, four non photomultiplier descanned detectors</td>
		</tr>
		<tr height="21">
			<td height="64" rowspan="3" style="height:64px;width:231px;">Filters SHG and autofluorescence</td>
			<td rowspan="3" style="width:231px;">Semrock</td>
			<td style="width:205px;">FF01-360/12-25</td>
			<td rowspan="3" style="width:343px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">FF01-395/11-25&#160;</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:205px;">FF02-485/20-25</td>
		</tr>
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			<td height="64" rowspan="3" style="height:64px;width:231px;">Microscope objectives</td>
			<td rowspan="3" style="width:231px;">Nikon</td>
			<td style="width:205px;">CFI Plan Fluor 10x</td>
			<td style="width:343px;">NA 0.30, WD 16 mm</td>
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			<td height="21" style="height:21px;width:205px;">CFI Plan Apo 20x</td>
			<td style="width:343px;">NA 0.75, WD 1.0 mm, VC</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:205px;">CFI Apo 40x</td>
			<td style="width:343px;">NA 1.25, WD 0.18mm &#955;S, Nano-Crystal Coat</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:231px;">Rhock inhibitor&#160;</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:205px;">Y-27632</td>
			<td style="width:343px;"></td>
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			<td height="43" style="height:43px;width:231px;">Knockout DMEM</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:205px;">10829</td>
			<td style="width:343px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Knockout Serum&#160;</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:205px;">10828</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">MEM Non-Essential Amino Acids&#160;</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:205px;">1140</td>
			<td style="width:343px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:231px;">L-glutamine 200mM&#160;</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:205px;">25030</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Penicillin-streptomycin</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:205px;">15140</td>
			<td style="width:343px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:231px;">&#946;-mercaptoethanol&#160;</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:205px;">M7522</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Collagenase IV&#160;</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:205px;">17104-019</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Roller scraper tool&#160;</td>
			<td style="width:231px;">StemPro EZPassage, Invitrogen</td>
			<td style="width:205px;">23181-010</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">StemPro Accutase&#160;</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:205px;">S11105-01</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Aggrewell system&#160;</td>
			<td style="width:231px;">StemCell Technologies</td>
			<td style="width:205px;">27845</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Hyclone serum&#160;</td>
			<td style="width:231px;">Thermo Scientific</td>
			<td style="width:205px;">SH30070.03</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Gelatin</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:205px;">G9391</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">6-well plates&#160;</td>
			<td style="width:231px;">Falcon</td>
			<td style="width:205px;">353046</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">24-well plates&#160;</td>
			<td style="width:231px;">Nunclon</td>
			<td style="width:205px;">142475</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:231px;">Polytetrafluoroethylene (PTFE) filters</td>
			<td style="width:231px;">Millipore&#160;</td>
			<td style="width:205px;">NA76/25</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Inserts</td>
			<td style="width:231px;">Millipore</td>
			<td style="width:205px;">PICM03050</td>
			<td style="width:343px;"></td>
		</tr>
	</tbody>
</table>

harmonic nanoparticles for regenerative research

在这个可视化实验，提供了协议的细节进行体外标记的人类胚胎干细胞的（胚胎干细胞）与二次谐波产生的纳米颗粒（的HNP）。后者是最近推出的用于标记的生物样品的多光子成像探测的一个新的家庭。的HNP是能够通过产生二次谐波的非线性光学过程与激发波长没有限制激发光的频率加倍。 多光子为基础的方法进行人类胚胎干细胞分化成心肌集群（如保持长期的气 - 液培养）将详细介绍。尤其是，显示在如何在三维监测跳动心脏组织中的3D最大化激烈二次谐波（SH）分离的HNP排放的证据。所得到的图像的分析来检索三维位移模式也详细描述。

之前评估由共焦成像的打浆作用，进行了仔细表征的PTFE过滤器的非线性光学响应的​​，单独的或的HNP的存在下，在高浓度（1毫克/毫升）。它确保：i）该裸基材双光子激发荧光很弱，不能防止测量相关的生物样本，以及ii）从孤立的HNP了SH排放可以通过外延检测模式的基板很容易获得通过成像（ 图2）。其目的是有参考控件，用于三维位移的量化。 在图3?...

Watch this Scientific Journal Video about Harmonic Nanoparticles for Regenerative Research at JoVE.com

Harmonic Nanoparticles for Regenerative Research

纳米谐波再生研究

提供用于体外标记的人类胚胎干细胞的二次谐波产生的纳米颗粒协议的细节。方法通过对多光子显微镜和其分化成心肌人类胚胎干细胞群的调查也被提出。

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Research

JoVE Journal

Bioengineering

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

构建交付的干细胞壳聚糖微球的胶原凝胶

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

科研

生物工程

Viral Nanoparticles for In vivo Tumor Imaging

Viral Nanoparticles for In vivo Tumor Imaging

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Biofunctionalized普鲁士蓝纳米粒子多式联运分子成像应用

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

新生儿心脏支架：再生研究新矩阵

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

液体中脉冲激光烧蚀制备金属纳米粒子：研究纳米粒子抗菌性能的工具

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

20 mJ, 1 ps Yb:YAG Thin-disk Regenerative Amplifier

20 mJ，1 ps Yb：YAG薄盘再生放大器

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with ...