Name: highly resolved intravital striped-illumination microscopy of germinal centers
Uploaded: 2014-04-09T21:20:00
Description: Watch this Scientific Journal Video about Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers at JoVE.com

我们感谢R. HEINTZMANN的富有成果的讨论的条带照明的方法，K. Rajewsky和A.哈伯曼的发展过程中提供的C57BL / 6 B1-8 GFP转基因小鼠。我们承认在授予NI1167/3-1德意志研究联合会（到RN），HA5354/4-1和SFB633，项目A15（至AEH），下批拉结 - 赫希奖学金的查理特（到RN）的资金支持。我们特别感谢网络JIMI进行富有成效的讨论及基础设施支援

多波束设置的条纹照明TPLSM 这里所使用的安装程序是一个专门的多波束双光子激光扫描显微镜，如先前所描述的6,20。该系统示于图1，本方法可以应用到其它的双光子激光扫描显微镜，其能够与本galvoscanner反射镜的运动同步相机采集，即使它们只能够执行单光束扫描。在这种情况下，缺点将是较低的采集速度，但是类似于标准的PMT基于TPLSM采集速度。为了达...

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活体光学成像的目的是动态地和功能上可视化细胞运动和相互作用，以了解组织和在健康和疾病5器官功能。最有力的技术来实现这一点，多光子激光扫描显微术，仍然要克服与波前畸变，散射，缓慢采集，光漂白和光损伤，这限制了它的空间分辨率的限制，对比度以及其时间分辨率。 有两种成功的解决方案，以实现更好的激发（和发射）光子管理在深层组织导致更?...

双光子激光扫描显微镜（TPLSM），其优点为深层组织成像与红外超短脉冲激发1，已通过揭示各种运动和交互模式彻底改变了我们的看法在单细胞水平的生命过程细胞亚群在活体动物2-5。然而，目前的技术仍不足以澄清器官的功能和功能障碍的机制，作为开发新的治疗策略的先决条件，因为它使得大约只有在健康和疾病组织中的细胞反应的分子基础稀疏信息。当前技术使由于散射几百微米只有一个空间窗口和波上的空间分辨率和信号噪声比6，这是特别明显的成年动物的高度紧凑的组织面前的失真效应。这些散射和波前畸变的影响是由于高度异质的的折射率在组织三维各向异性分布，导致在第一步骤中，以三维空间分辨率的深度依赖性恶化，最后到信号从弹道激发光子， 即损失的信号-噪声比始发的总损耗。在生物科学和生物医学深层组织的应用程序而言，这意味着目前的技术是无法明确地揭示蜂窝通信，因为分辨率差，会导致假阳性的相互作用，而信号噪声比的减少将导致系统忽略了一些暗淡的结构之间的相互作用。 为了清楚地检测以动态方式的细胞相互作用，一个高度改善的空间分辨率，需要深的组织内。基于特殊的数值算法， 如结构照明方式，对第一激发态损耗， 如目前推出了功能强大的纳米显微技术</em&gt;受激发射损耗，RESOLFT，或在分子的定位， 例如 dSTORM，PALM，已经发现许多应用中固定的细胞，以及在活细胞培养7。然而，为了这些应用程序扩展到组织切片，活组织和有机体，我们仍然需要克服严重的技术困难。双光子激发的受激发射损耗具有不同波长，以及与单个波长（sw2PE-STED），用于激励和受激发射已被应用，以提高横向分辨率在大脑切片8或人工基质与包埋细胞9，分别在同一轴向分辨率为标准TPLSM。利用单光子受激发射损耗，树突棘的动态可以在大脑皮质（长达10-15微米的深度）的表面活Thy1系EGFP小鼠在67纳米10的分辨率成像。一个多功能的工具，用于发育生物学是由多灶性结构照明显微镜，它提供了两方面的改进二维提供分辨率。然而，这种技术可用于仅在与光散射如斑马鱼胚胎11倾向低的生物。不过，没有这些技术可以在成年动物的高散射组织被应用在数百微米，这是至关重要的模型与发病的疾病在出生后的生物医学和临床研究。 独立用于计算衍射限制的波前的形状， 即该点扩散函数（PSF）的近似值，聚焦通过透镜后，沿着光轴（轴向分辨率）的PSF的宽度大于至少3倍PSF的宽度在垂直于光轴（横向分辨率）12。波不同的订单面前的扭曲泽尔尼克的量化系数大大修改深层组织成像聚焦电磁波导致更大的PSF，特别是沿光纤的波前形状人轴13-15。因此，这两个衍射法和波前畸变效应指向沿着光轴在深层组织成像的限制因素的分辨率。而纳米显微技术专注于抵消衍射的极限只有一个技术，提高了轴向分辨率和通过抵消两个衍射和波前畸变效应的对比度是需要高分辨率的活体成像。理想情况下，这种技术应该也足够快，以便监测细胞动力学的。 PSF畸变和使用TPLSM自适应光学对比度损失的实时校正已被广泛研究，并在过去十年13,14,16-18改善，因此它是目前市面上最好的选择，从而更好地管理弹道导弹激励14的光子。不过，由于大部分波前校正方法在自适应光学系统中使用的其实是迭代和他们公顷已经被重复为小区域（数10×10微米2）由于在组织中的折射率的高异质性，采集速度比所需的成像细胞运动和通信显著更低。此外，在自适应光学的物理极限提高TPLSM仍然由衍射确定。 照明（SPIN）和检测侧（铲）时间调制的空间调制已经从理论上提出了要施加到激光扫描显微镜来提高分辨率。在活体成像的实际应用还有待证明19。 综上所述，对于技术，有效地提高了分辨率为深层组织成像在活成年动物发展的高需求。在这项工作中，我们实现了激励模式的，通过控制在多波束条带照明的多光子激光 - S的扫描过程空间调制罐头镜（MB-SI-MPLSM）20。相反，以结构化的照明方法，其中，所述激发光束的横截面进行空间调制，我们只使用扫描过程来实现激发的空间调制。通过扩大激发到更长的波长，我们是独立的光能够提高在高散射组织（ 如淋巴结，自由散射路径47微米6）深层组织双方的空间分辨率和信号噪声比，我们检测到的非线性信号， 例如荧光，产生二次谐波或其它混频现象。使用这种方法在激发波长为900毫微米，我们能够动态图像的细胞的蛋白质结构上的几个分子在小鼠淋巴结生发中心的规模。因此，我们可以更好地可视化在探测反过程滤泡树突细胞和B细胞的表面上的抗原携带单元之间的相互作用期间在次级淋巴器官的免疫应答根。

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">ATTO590 &#8211; NSH for coupling</td>
			<td style="width:99px;">ATTO Tec, Germany</td>
			<td style="width:119px;">AD-590-35</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">CD21/35 &#8211; Fab fragment &#8211; ATTO590</td>
			<td style="width:99px;">DRFZ, Berlin</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">The coupling reaction was performed in the central lab of our institution.</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">B1-8 Jk-/- EGFP+</td>
			<td style="width:99px;">Prof. Anne Habermann, Yale Univ., CA, US</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">Can be also found at Jackson Laboratories (JAX).</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">EasySep Negative Selection Mouse B Cell Enrichment&#160;</td>
			<td style="width:99px;">StemmCell Technologies, Germany</td>
			<td style="width:119px;">19854</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Polystyren beads (605), 0.2 &#181;m</td>
			<td style="width:99px;">Life Technologies, Germany</td>
			<td style="width:119px;">F8803</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Name of Equipment</td>
			<td style="width:99px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">TriMScope I</td>
			<td style="width:99px;">LaVision Biotec, Bielefeld, Germany</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">OPO (manual version)</td>
			<td style="width:99px;">APE, Berlin, Germany</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Ti:Sa Laser Chameleon Ultra II</td>
			<td style="width:99px;">Coherent, Duisburg, Germany</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">water-immersion objective lens, 20x, NA 0.95, IR, WD 2 mm</td>
			<td style="width:99px;">Olympus Germany, Hamburg, Germany</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
	</tbody>
</table>

highly resolved intravital striped-illumination microscopy of germinal centers

监测蜂窝通信由活体深层组织的多光子显微镜的关键是理解厚组织标本和器官在健康和疾病中的免疫细胞的命运。通过控制多光子显微镜扫描模式和运用恰当的数值算法，我们开发了一个条纹的照明方式，使我们能够实现3倍更好的轴向分辨率和改进的信号噪声比， 即相反，在超过100μm的组织深度内高散射淋巴器官组织的标准相比，多光子显微镜。采集速度以及光漂白和光损伤作用类似于标准的光电倍增管为基础的技术，而成像深度是由于使用领域探测器略低。通过使用条带照明的方法，我们能够观察到的二次滤泡树突状细胞的免疫复合物沉积动力学̵1;上的几个蛋白质分子在生发中心的水平。

在淋巴结空间分辨率实际点扩展函数（EPSF）的尺寸对应于显微镜12的空间分辨率。我们通过我们的MB-SI-TPLSM获取在淋巴结胶原纤维的二次谐波生成的信号相比，建立了双光子激光扫描显微技术， 即场检测TPLSM（由CCD相机装置）测定该三维函数和点检测TPLSM（由光电倍增管的方式）。 从这些测量结果可以很明显地，在试件的表面，无论是横向和?...

Watch this Scientific Journal Video about Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers at JoVE.com

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

高分辨率的活体成像具有增强对比度高达120微米的深度在成年小鼠的淋巴结被空间调制多焦点双光子显微镜的激励模式来实现。在100微米的深度，我们测量了487 nm的分辨率（横向）和551纳米（轴向），从而绕过散射和衍射极限。

生发中心的高度已解决活体条纹照明显微镜

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

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Immunology and Infection

Intravital Microscopy of the Inguinal Lymph Node

腹股沟淋巴结的活体显微镜

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

科研

免疫与感染

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

追踪中性粒细胞腔内的爬行，活体视频显微镜Transendothelial组织中的迁移和趋化

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

肝脏活体如下显微成像<em&gt;利什曼原虫</em&gt;感染：肝脏血流动力学的评估

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

在Mesenterial静脉的活体显微镜白细胞，内皮细胞和血小板白细胞相互作用小鼠

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

直立活体显微术制备小鼠颌下腺涎腺

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...