Name: 3d orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles
Uploaded: 2014-10-01T13:00:00
Description: Watch this Scientific Journal Video about 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles at JoVE.com

This project was supported by grants NIH NIGMS 8P41 GM103540-28 and P50-GM076516

1，样品制备<ol><li>保持在使用补充有10％胎牛血清和100 IU / ml青霉素链霉素50微克/毫升的组织培养瓶中的CHOK1细胞。培养细胞在5％二氧化碳培养箱在37℃。 </li><li>嘉实然后板CHOK1细胞在14毫米直径的微孔与0.16毫米的表面厚度。种子细胞用于成像的最佳密度，周围60-70％汇合。 </li><li>过夜孵育细胞，在37℃，5％CO 2。在HBSS（汉克缓冲盐溶液）中含有50nM的Lysotracker DND26绿色和150纳米的?...

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	<li>So, P. T., Dong, C. Y., Masters, B. R., Berland, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+photon+excitation+fluorescence+microscopy.">Two photon excitation fluorescence microscopy.</a> Annu Rev Biomed Eng. 2, 399-429 (2000).</li><li>Dupont, A., Lamb, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+three+dimensional+single+particle+tracking.">Nanoscale three dimensional single particle tracking.</a> Nanoscale. 3, 4532-4541 (2011).</li><li>Estrada, L. 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S., Lakadamyali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+live+cell+and+superresolution+microscopy+reveals+cargo+transport+dynamics+at+microtubule+intersections.">Correlative live cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 3375-3380 (2013).</li><li>Huotari, J., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosome+maturation.">Endosome maturation.</a> Embo Journal. 30, 3481-3500 (2011).</li><li>Ragan, T., So, P. T., Kwon, H. 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尽管荧光显微技术及仪器，在过去几年取得的荧光粒子在三维空间运动轨迹具有高时间分辨率的巨大进步一直保持在该领域的挑战。如果高时间分辨率已经在两个维度上被实现跟踪颗粒，延伸到轴向方向通常带来大幅减少了系统10的频率响应。 在这个视频协议，我们重点跟踪单个粒子在三维空间中活的细胞中利用双光子显微镜的高时间分辨率（32毫秒在这个实验中）?...

大量方法已被开发出迄今为止为在使用显微镜的三维跟踪荧光发色粒子。大多数方法依赖于使用的快速照相机，非常适合以跟踪在两个维度上，典型地结合了镜的出射光学系统的定制修改，以实现跟踪在轴向方向上。激光扫描显微镜（或者共聚焦或两个光子）通常可以追踪的荧光粒子通过进行的Z-堆栈的时刻的顺序，尽管这种过程通常是耗时的，并且会产生合理的时间分辨率（10赫兹）只有当被跟踪的粒子是由有源反馈机制2保持在一个小的光栅成像区域的中心。 锁入到该颗粒的想法是在轨道的跟踪方法的基础。取而代之的是光栅扫描的圆形轨道围绕的荧光粒子进行。荧光沿强度轨道精确本地化粒子的位置4。 该颗粒的局部位置然后可用于致动显微镜扫描器和重新中心在粒子位置的轨道。显微镜的检流计扫描仪是由一个模拟电压驱动。这个电压的AC分量允许用聚焦激光束进行的轨道， 即正弦波和施加到X和Y扫描反射镜将允许执行一个圆形轨道上的余弦波。直流信号的偏移有助于改变轨道中心的位置。一旦一个轨道期间被确定并且对于交流信号的波形构成，所述反馈系统需要更新信号的唯一的直流分量。 软件能够读取从检测器收集到的荧光信号是必需的，以控制扫描仪和检测器，来计算粒子的位置和更新的直流断设定。对于一个成功的反馈成像仔细选择的轨道参数（大小和定时）的需要和这些参数具有基于所述荧光体颗粒，有必要进行跟踪的特性来进行调整。 该技术的物理和数学基础，在过去4,5 2D和3D应用程序进行了描述。在这个协议中，我们简要地描述了安装的主要硬件部件，并且更详细的参数的选择和样品制备所需的典型的实验中，使我们以下溶酶体中的活细胞内的高时间分辨率的位移。

<table border="0" cellpadding="0" cellspacing="0" width="525">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:197px;">Name</td>
			<td style="width:160px;">Company</td>
			<td style="width:168px;">Model</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lysotracker DND 26</td>
			<td>Life technologies</td>
			<td>L-7526</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tubuline tracker Green</td>
			<td>Life technologies</td>
			<td>T34075</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mitotracker RED</td>
			<td>Life technologies</td>
			<td>M7512</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coherent Chamelon- ultra II TI</td>
			<td>Coherent</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcite polarize</td>
			<td>Melles Griot Glan&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Galvanometer-motor mirror</td>
			<td>Cambridge technologies</td>
			<td>M 6350</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dichroic mirror</td>
			<td>Chroma technologies</td>
			<td>700 DCSPXR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Motorized stage</td>
			<td>ASI</td>
			<td>MS2000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Piezo&#160;</td>
			<td>PI</td>
			<td>P721-LLQ</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photomultiplier tube</td>
			<td>Hamamatsu</td>
			<td>H7422P-40</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Data acquisition card</td>
			<td>IO tech</td>
			<td>PCI 1128-4000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaging software</td>
			<td>LFD</td>
			<td>Global for images-SimFCS</td>
		</tr>
	</tbody>
</table>

3d orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles

这个视频协议的目的是讨论如何使用修改后的双光子显微镜1执行和分析三维荧光轨道粒子跟踪实验。相对于传统的方法（光栅扫描或基于一个堆栈帧的宽域），三维轨道跟踪允许定位和跟踪具有高空间（10 nm的精度）和时间分辨率（50赫兹的频率响应）的三维位移移动荧光粒子的长度尺度数百微米2的。该方法是基于一种反馈算法来控制，以围绕该对象执行的圆形轨道上的双光子激光扫描显微镜的硬件要跟踪：反馈机制将保持在中心的荧光物，通过控制的位移扫描光束3-5。为了证明这项技术的优势，我们跟着快速移动的细胞器，溶酶体，内人艾尔文细胞6,7。细胞根据标准方法镀，并用市售溶酶体染料染色。我们简要讨论的硬件结构和详细的控制软件，以执行在活细胞内的3d轨道的跟踪实验。我们详细讨论，以控制所述扫描镜和使该光束的动作在颗粒周围的封闭轨道所需要的参数。我们得出结论：通过展示如何此方法可有效地用于活细胞内跟踪沿在三维微管标记的溶酶体的快速运动。溶酶体可以用移动的速度在0.4-0.5微米/秒的范围内，通常显示沿微管网8定向运动。

根据这一协议，快速的3D单粒子跟踪可以在里面用改进的双光子显微镜来跟踪荧光标记的溶酶体的位移活细胞中进行。进行实验包括跟踪一个孤立的溶酶体内体成熟过程后，9移动单元格中的。溶酶体用荧光绿色染料染色，激发在930 nm的利用双光子激发。我们的数据表明，有可能获得的x，y和z的位移轨迹具有32毫秒（ 图1a）的时间分辨率。地毯分析显示沿各轨道上的时间?...

Watch this Scientific Journal Video about 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles at JoVE.com

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

3d轨道跟踪的改进双光子显微镜之应用：细胞内的囊泡的跟踪

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Research

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Bioengineering

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

在封装的细胞聚集跟踪缺氧信号

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

科研

生物工程

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

细胞外基质刚性三维肿瘤模型的纵向测量用粒子跟踪Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

在生物膜的力学性能原位制图粒子追踪Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

微孔阵列平台中微生物群落发展的组装与跟踪

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

A Protocol for Real-time 3D Single Particle Tracking

一种实时3D 单粒子跟踪协议

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

o 型血管哺乳动物细胞产生均匀集料的轨道晃动培养

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

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Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

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Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...