Name: a protocol for real-time 3d single particle tracking
Uploaded: 2018-01-03T13:00:00
Description: Watch this Scientific Journal Video about A Protocol for Real-time 3D Single Particle Tracking at JoVE.com

这项工作得到了国家卫生研究院的国家综合医学研究所的支持, R35GM124868 和杜克大学颁发的奖项。

1. 设置布局和对齐
<ol>
	<li>跟踪励磁激光器的安装与准直<ol>
		<li>使用国产安装将激光器粘贴到光学工作台上。该安装是一个简单的铝板与安装孔的激光头和光学表。激光应牢固地附着在金属支架上, 以稳定散热。对于这项工作, 使用 488 nm 固体激光器照明 (图 1), 虽然波长可以选择适合特定的荧光或实验。一个关键的因素是, 激光波长适合 2 d-爆炸物的工作范围, 这是由两个器...

<ol>
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尽管近年来出现了许多3D 单粒子跟踪方法, 但在低光子计数率下, 以简单的设置进行的高速3D 扩散的鲁棒 real-time 跟踪仍然是一个挑战, 这限制了它在重要的生物问题.本协议中描述的3维 DyPLoT 方法以多种方式解决了这些难题。首先, 将激励和检测路径与其他实现简单而健壮的方法相比, 大大简化了。其次, 移动激光光斑和位置估计算法为反馈回路提供精确的位置估计, 使反馈更加稳定。第三, 动态移动...

先进的成像技术的出现, 打开了一个窗口, 看到更详细的细胞现象的结构, 一路下降到分子水平。方法如随机光学重建显微镜 (风暴)1,2,3, 照片激活的本地化显微镜 (PALM)4,5,6,7, 结构化照明显微镜 (SIM)8,9,10,11, 和受激辐射损耗显微镜 (STED)12,13, 14已经远远超出了衍射极限, 从而将空前的细节提供给了活细胞的结构和功能。然而, 充分了解这些系统的行为需要动态信息和结构信息。上面列出的超分辨率方法涉及空间分辨率和时间分辨率之间的权衡, 从而限制了动态过程的探测时间精度。提供高空间精度和时间分辨率的方法是 RT-3D-SPT15、16、17、18、19、20 21,22,23,2425262728在这里, 我们将传统的3维的30和 RT-3D-SPT 区分开来. 传统的3维单标需要一个时间序列的三维图像数据 (可以用共焦显微镜或荧光显微镜获取)给定正确的配置)。在传统的3维标度中, 通过在每个图像堆栈中定位粒子并在连续的卷中连接位置来创建轨迹, 来确定粒子的坐标。对于这些方法, 最终的时间分辨率取决于容积成像率。对于共焦显微镜, 这是很容易在几秒到几十秒的规模。对于荧光的方法 , 其中的光学路径纵 , 使轴向位置信息可以提取 , 时间分辨率是有限的相机曝光或读取时间。这些 epifluorescent 的方法是有限的范围内轴向信息可以收集, 虽然最近的进展, 在傅里叶平面相掩模设计和自适应光学是将这些范围扩展到10µm 或更多31,32,33,34。
相比之下, RT-3D-SPT 不依赖于获取3D 图像堆栈并在事实之后找到粒子。相反, real-time 的位置信息是通过单点检测器提取的, 通过使用高速压电阶段, 反馈被应用到有效地 "锁定" 物镜焦距中的粒子。这就允许连续测量粒子的位置限制, 只有多少光子可以收集。此外, 这种方法能够在粒子在长距离移动时对其进行光谱审讯。RT-3D-SPT 的效果类似于一个无力的纳米物体的光阱, 其中粒子是连续探测和测量在 real-time 不需要大的激光功率或光学力量。鉴于 RT-3D-SPT 提供了一种连续审讯快速扩散对象的方法 (最多20µm2/秒)25,29在低光子计数率三,20, 35, 它应提供一个窗口, 进入快速或瞬态的生物过程, 如细胞内货物运输, 配体受体结合, 和细胞外动力学的单一病毒。然而, 在这一点上, RT-3D-SPT 的应用仅限于少数几个致力于推动这一技术的团体。
一个障碍是 RT-3D-SPT 方法所要求的光学布局的复杂性, 这是多种多样的。对于大多数方法, 光反馈是由压电工作台提供的。当粒子在 X、Y 或 Z 中产生小的运动时, 从单点探测器的读数转换为错误函数, 并以高速向压电 nanopositioner, 这反过来又移动样品来抵消粒子的运动, 有效地将其锁定在相对于物镜的位置。要测量 X、Y 和 Z 中的小位置移动, 可以使用多个检测器 (4 或5取决于实现)15、18、21或多个励磁点 (2-4), 如果锁定放大器用于使用旋转激光光斑提取 X 和 Y 位置)25,28被应用。这些多个检测和发射点的重叠使得系统难以对齐和维护。
在这里, 我们提出了一个高速目标锁定 3 d-标的方法与简化光学设计称为 3 d-DyPLoT29。3 d-DyPLoT 使用 2 d-爆炸物和标记透镜36,37,38 , 以高速率 (50 赫 XY, 70 赫 Z) 动态移动聚焦激光光斑。结合激光聚焦位置和光子到达时间, 使粒子的3D 位置可以迅速获得, 即使在低光子计数率。2 d-爆炸物排爆行动驱动激光焦点在骑士的游览样式39以正方形大小 1 x 1 µm 在 x Y 平面和标记透镜移动激光焦点在轴向范围 2-4 µm。3D 粒子位置得到优化的位置估计算法29,40在3D。在现场可编程门阵列 (FPGA) 上对3D 动态移动光斑、雪崩光电二极管 (APD) 的光子计数、real-time 粒子位置计算、压电级反馈和数据记录进行了控制。在本协议中, 我们描述了如何建立一个3维 DyPLoT 显微镜 step-by 步, 包括光学对准, 校准与固定粒子, 最后自由粒子跟踪。作为一个示范, 110 纳米荧光珠连续跟踪在水中的时间分钟。
本文所描述的方法是一个理想的选择, 在任何应用, 它是希望连续监测快速移动荧光探针在低光水平, 包括病毒, 纳米颗粒, 和小泡, 如体。与以往的方法相比, 只有一个单一的励磁和单一的检测路径, 使对准和维护一目了然。此外, 这个大的探测区域使这个显微镜能够很容易地快速地拾取粒子, 而在低信号水平 (下至10赫) 跟踪的能力使得这种方法适合微光应用程序29。

<table border="0" cellpadding="0" cellspacing="0" width="1075">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">2D Electro-optic Deflector&#160;</td>
			<td style="width:145px;">ConOptics</td>
			<td style="width:160px;">M310A</td>
			<td style="width:541px;">2 required</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Power supply for EOD</td>
			<td style="width:145px;">ConOptics</td>
			<td style="width:160px;">412</td>
			<td>Converts FPGA ouput to high voltage for EOD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">TAG&#160; Lens</td>
			<td style="width:145px;">TAG Optics</td>
			<td style="width:160px;">TAG 2.0</td>
			<td>Used to deflect laser along axial direction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">XY piezoelectric nanopositioner</td>
			<td style="width:145px;">MadCity Labs</td>
			<td style="width:160px;">Nano-PDQ275HS</td>
			<td>Used for moving the sample to lock the particle in the objective focal volume in&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Z piezoelectric nanoposiitoner</td>
			<td style="width:145px;">MadCity Labs</td>
			<td style="width:160px;">Nano-OP65HS</td>
			<td>Used to move the objective lens to follow the diffusing particle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Micropositioner</td>
			<td style="width:145px;">MadCity Labs</td>
			<td style="width:160px;">MicroDrive</td>
			<td>Used to coarsely position sample and evaluate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Objective Lens</td>
			<td style="width:145px;">Zeiss</td>
			<td style="width:160px;">PlanApo</td>
			<td>High numerical aperture required for best sensitivity. 100X, 1.49 NA, M27, Zeiss</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">sCMOS camera</td>
			<td style="width:145px;">PCO</td>
			<td style="width:160px;">pco.edge 4.2</td>
			<td>Used to monitor the particle&#39;s position</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">APD</td>
			<td style="width:145px;">Excelitas</td>
			<td style="width:160px;">SPCM-ARQH-15</td>
			<td>Lower dark counts beneficial</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Field programmable gate array</td>
			<td style="width:145px;">National Instruments</td>
			<td style="width:160px;">NI-7852r</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Software</td>
			<td style="width:145px;">National Instruments</td>
			<td style="width:160px;">LabVIEW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Tracking excitation laser</td>
			<td style="width:145px;">JDSU</td>
			<td style="width:160px;">FCD488-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-150-A-ML</td>
			<td>L1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-200-A-ML</td>
			<td>L2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Pinhole</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">P75S</td>
			<td>PH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Glan-Thompson Polarizer</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">GTH5-A</td>
			<td>GT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Half-wave plate</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">WPH05M-488</td>
			<td>WP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-75-A-ML</td>
			<td>L3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-250-A-ML</td>
			<td>L4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-200-A-ML</td>
			<td>L5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-200-A-ML</td>
			<td>L6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Dichroic Mirror</td>
			<td style="width:145px;">Chroma</td>
			<td style="width:160px;">ZT405/488/561/640rpc</td>
			<td>DC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Fluorescence Emission Filter</td>
			<td style="width:145px;">Chroma</td>
			<td style="width:160px;">D535/40m</td>
			<td>F</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">10/90 beamsplitter</td>
			<td style="width:145px;">Chroma</td>
			<td style="width:160px;">21012</td>
			<td>BS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">190 nm fluorescent nanoparticles</td>
			<td>Bangs laboratories</td>
			<td>FC02F/9942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">110 nm fluorescent nanoparticles</td>
			<td>Bangs laboratories</td>
			<td>FC02F/10617</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-545A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Powermeter</td>
			<td style="width:145px;">Thorlabs</td>
			<td style="width:160px;">PM100D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">CMOS</td>
			<td style="width:145px;">Thorlabs</td>
			<td style="width:160px;">DCC1545M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Iris</td>
			<td style="width:145px;">Thorlabs</td>
			<td style="width:160px;">SM1D12D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td style="width:145px;">Mad City Labs</td>
			<td style="width:160px;">RM21</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for real-time 3d single particle tracking

实时三维单粒子跟踪 (RT-3D-SPT) 有可能在蜂窝系统中对快速的3D 过程产生影响。尽管近年来提出了各种 RT-3D-SPT 方法, 但在低光子计数速率下跟踪高速3D 扩散粒子仍然是一个挑战。此外, RT-3D-SPT 设置通常是复杂和难以实施, 限制其广泛应用于生物问题。该协议提出了一个 RT-3D-SPT 系统, 名为3D 动态光子定位跟踪 (3 维 DyPLoT), 它可以跟踪高扩散速度 (高达20µm2/秒) 在低光子计数率 (下至10赫) 的粒子。3 d-DyPLoT 采用2D 电光偏转器 (2 d-爆炸物) 和可调谐的声学梯度 (标签) 镜头, 以驱动一个单一的聚焦激光点动态在3D。结合一种优化的位置估计算法, 3 维 DyPLoT 可以锁定在高跟踪速度和高定位精度的单粒子上。由于单一的励磁和单一的检测路径布局, 3 维 DyPLoT 是健壮的, 易于设置。本协议讨论如何逐步构建3维 DyPLoT。首先, 介绍了光学布局。然后, 通过光栅扫描190纳米荧光珠与压电 nanopositioner 对系统进行了标定和优化。最后, 为了证明 real-time 3D 跟踪能力, 110 nm 荧光珠在水中跟踪。

固定粒子扫描 (图 4) 和自由扩散 110 nm 荧光粒子跟踪 (图 5) 是按照上述协议执行的。粒子扫描是通过移动压电 nanopositioner 和 bin 光子, 同时计算在扫描的每个点的粒子的估计位置。扫描图像显示一个均匀强度的平方 (图 4a), 估计的位置显示一个与粒子的实际位置的线性关系, 在 x、y 和 z 方向上的 1 x 1 x 2 µm ?...

Watch this Scientific Journal Video about A Protocol for Real-time 3D Single Particle Tracking at JoVE.com

A Protocol for Real-time 3D Single Particle Tracking

本协议详细介绍了一种 real-time 3D 单粒子跟踪显微镜的构造和操作, 能够跟踪高扩散速度和低光子计数率的纳米荧光探针。

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 ...

Research

JoVE Journal

Bioengineering

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

一个简单的悬滴细胞生成三维球体文化协议

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

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 ...

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

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

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

订货单细胞和单胚胎的三维约束：高内涵筛选新设备

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

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 ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

在原位生物过程单细胞形态实时测定的显微镜

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved ...

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

在微生物发酵过程中制造一种纳菲安涂层、还原石墨烯 Oxide/Polyaniline 化学电阻传感器, 以实时监测 ph 值

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

人骨髓间充质干细胞吸收含有法卡林多醇的新型脂质包覆纳米颗粒

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...