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 (图 1: W2) 之间的波板主要确定。488 nm 激光可以有效激发绿色或黄色荧光蛋白 (GFP/YFP), 这是常见的荧光标签在活细胞实验。</li>
		<li>使用一对介质镜像 (图 1: M) 调整激光束的高度和方向. 确保激光光束平行于光学工作台, 并将其调整到合适的高度。 注: 应根据显微镜平台选择高度。对于本协议中使用的所有后续镜像, 都应保留此高度, 但不会明确声明。</li>
		<li>准用一对透镜 (图 1: L1 和 L2) 对光束进行了分析。焦距的镜头, 应谨慎选择, 以避免激光斑点被修剪的光圈 2 d-爆炸物。通过 2 d-爆炸物排爆后, 激光束的形状变化明显。在这里的准直的质量是非常重要的, 因为像差将被随后的透镜放大, 任何分歧将恶化的性能 2 d-爆炸物的偏转。理想情况下, 通过将光束聚焦到至少20米以外的点来准光束。</li>
	</ol>
	</li>
	<li>将一个针孔 (图 1: PH) 放置在第一个准直透镜 (图 1: L1) 的焦点上。<ol>
		<li>选择合适尺寸的针孔。可根据以下计算选择针孔尺寸: 
		<img alt="Equation 1" src="/files/ftp_upload/56711/56711eq1.jpg"> 其中, λ是激光的波长, f是第一个透镜的焦距, D是输入光束的直径。</li>
		<li>在3D 平移阶段安装针孔, 使其能够精确放置在第一准直透镜的焦点上。</li>
		<li>调整针孔的位置。将激光功率计放在针孔后, 调整 XYZ 的针孔位置, 使电能表读数最大化。如果观察到一个衍射环, 用虹膜放置在 2 d-排爆前。</li>
	</ol>
	</li>
	<li>安装格兰-汤普森偏光片<ol>
		<li>将格兰-汤普森偏光片 (图 1: GP) 放在第二个准直透镜 (图 1: L2) 之后, 以清除激光束的偏振。旋转偏光片, 以找到最大的传输功率计。</li>
	</ol>
	</li>
	<li>安装 EODs<ol>
		<li>使用 2 EODs (图 1: EOD1 和 #38; EOD2) 将激光偏转到 X 和 Y 方向。通过调整每个爆炸物的偏航、俯仰和高度来最大化激光传输。</li>
		<li>使用制造商在每个偏转器一侧提供的校准标记对齐两个 EODs。</li>
		<li>将测试模式应用于2维爆炸物的控制器, 以检查爆炸物的输出。这可以是骑士的巡回 (图 2) 通过下面所描述的 FPGA 或由函数发生器提供的一个简单的正弦波。为了得到最佳性能, 每个偏转器的偏转应与压电 nanopositioner 的 X 或 Y 方向平行。这个方向是由器关于传输轴的角方向决定的。在不移动2维爆炸物的情况下旋转偏转方向, 在2维爆炸物排爆后放置一只鸽子棱镜, 使偏转轴与压电 nanopositioner 轴对准。</li>
	</ol>
	</li>
	<li>波板安装<ol>
		<li>将波板 (图 1: W1) 放在格兰-汤普森偏光片 (图 1: GP) 和 2 d-爆炸物排爆器之间, 使入射激光束极化与 2 d-爆炸处理的偏转轴线对准。放置一个长焦距镜头 (300 毫米) 后, 2 d 的爆炸物, 并放置一个 sCMOS 相机在激光焦点。接下来, 打开 2 d-爆炸物, 以生成下面描述的骑士之旅 (图 2)。旋转波板, 直到在相机上观察到均匀的方形激光分布。</li>
	</ol>
	</li>
	<li>光束扩展透镜对和继电器透镜副的安装<ol>
		<li>放置一对镜片 (图 1: L3 和 L4) 在 2 d-排爆后扩展光束以填充物镜的后孔 (图 1: OL) 和另一对镜片 (图 1: L5 和 L6) 以将焦平面中继到显微镜物镜。确保在这两对镜片之间留有足够的空间, 因为在后面的步骤中, 它们之间会安装标签镜头。</li>
	</ol>
	</li>
	<li>安装分色滤光片, 10/90 分束器, 聚焦透镜, APD, 物镜, 荧光发射过滤器, 完成检测通路。<ol>
		<li>安装分色滤镜 (图 1: DC) 以反射激光朝向物镜。将激光对准物镜的中心, 使用长透镜管的顶部和底部有两个虹膜。</li>
		<li>安装10/90 分束器 (图 1: BS), 其中10% 的光被反射到 sCMOS 相机 (如下所述) 和90% 的通过到 APD 进行跟踪。</li>
		<li>对准 APD将激光聚焦到物镜上的片上。使用片的反射来对齐所有下游元素, 通过在目标焦点上放置一个片并检查 APD 读数的强度。在分束器后, 在3D 平移阶段安装 apd 聚焦透镜 (图 1: L7), 然后是 apd。定位透镜, 使激光反射从物镜进入透镜中心。</li></ol></li></ol>在片上, 通过调整3D 位置, 使激光反射的强度最大化, 可以优化 APD 的位置。当探测器位置在任意方向移动时, APD 位置处于最佳位置, 从而降低了强度。
		<li>安装一个 longpass 荧光发射过滤器 (图 1: F), 然后将光束分配器移除反射和散射光。</li>
	
	
	<li>标记透镜的安装<ol>
		<li>将标记镜头放在两对镜片之间 (图 1: L4 和 L5之间), 如前所述, 并显示在图 1中。调整偏航, 俯仰, 高度和横向位置, 使光束通过垂直通过标记透镜的中心。</li>
	</ol>
	</li>

2. 样品制备。
<ol>
	<li>固定粒子制备<ol>
		<li>在 PBS 中稀释 190 nm 荧光珠到 ~ 5 x 108珠/毫升。添加400µL 珠解决方案上的片和安装在压电舞台的样品持有人 (珠的体积将取决于样品持有人; 这里使用的样品盒的直径是18毫米)。在这里使用的珠子, PBS 导致微粒存放在片。</li>
	</ol>
	</li>
	<li>自由运动微粒准备<ol>
		<li>稀释 110 nm 荧光珠到〜 5 x 108珠/毫升与 DI 水。添加400µL 珠解决方案到一个片和安装到样品持有人的压电阶段。</li>
	</ol>
	</li>
</ol>
3. 优化跟踪参数
<ol>
	<li>光栅扫描固定粒子<ol>
		<li>在显微镜上放一个固定的微粒样品。</li>
		<li>打开激光, 微控制器, 压电 nanopositioner 控制器, 标签镜头控制器, 和爆炸物控制器。请注意, 该顺序并不重要。在闭合回路中运行压电 nanopositioner。</li>
		<li>光栅扫描 XYZ 中的示例使用自定义扫描软件程序 (图 S3, 根据作者的请求提供的软件), 它在骑士之旅 (图 2) 中驱动 2 d-排爆, 从 APD 收集计数, 驱动压电nanopositioner, 并执行位置计算 (图 S2)。步长为 40 nm, 扫描范围为2µm。<ol>
			<li>将片放在目标的焦点上, 去掉荧光发射滤波器, 利用激光从片的反射来最大化信号强度作为微 Z 位置的函数。在将样品放置在焦平面上后, 将荧光发射过滤器放回。</li>
			<li>打开扫描程序。通过在 "开始"、"完成" 和 "步骤" 中键入数字来设置扫描范围和步骤大小。首先设置一个大的扫描范围和步长, 以找到一个粒子 (例如, 10 x 10 µm 范围与 200 nm 步大小)。找到微粒后, 缩小扫描范围并减小步长 (例如, 2 x 2 µm 范围, 100 nm 步长)。单击 "扫描" 按钮执行3D 光栅扫描以查找荧光焦点。</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>标记镜头设置 (另请参见图 3)<ol>
		<li>点击标签镜头控制软件。依次单击 "连接"、"通电"。</li>
		<li>为了改变触发信号的输出相位, 必须改变输出触发模式。首先将模式从 "RGB" 更改为 "多", 然后将其更改回。现在可以更改该阶段 (图 3b)。</li>
		<li>将输出阶段设置为0°、90°和270°。虽然可以使用覆盖相空间的任何三阶段, 但发现这三的工作经验很好 (图 3c)。</li>
		<li>选择68500赫兹频率设置。</li>
		<li>为了找到最佳频率, 经常需要改变频率搜索范围。单击 "高级"、"设置"。更改 "最大值"。频率 (hz) 的0列从7万赫兹到 71500 hz。这可以调整到任何频率范围是适当的。单击 "保存校准"、"退出校准" (图 3d)。</li>
		<li>为了使新的校准生效, 请将共振切换到另一个频率 (例如, 189150 hz), 然后切换回68500赫兹频率设置 (图 3e)。</li>
		<li>将振幅逐渐改变为35%。单击 "锁定共振"。锁定频率后, 单击 "解锁共振" (图 3e)。标记透镜现在可以使用了。</li>
		<li>要校准, 更改估计中使用的参数, 使估计位置等于实际位置, 如图 4e所示, f. 输入 x、y 和 z 的扫描范围, 然后单击 "扫描" 按钮以在 XYZ 中扫描示例, 以移动粒子通过移动激光光斑。粒子的位置, 因为它是通过激光聚焦扫描应该同意的估计粒子位置确定的位置估计回路 (图 S1)。估计位置和实际位置 (图 4d) 之间的关系可以从估计位置图像 (图 4f) 中提取。如果位置不一致, 请调整位置估计循环中使用的激光位置 (ck) 的值 (图 S1)。</li>
		<li>对齐标签镜头<ol>
			<li>当标签镜头控制器关闭, 光栅扫描粒子图像 (如下所述) 在安装标记镜头和安装标记透镜后, 应该看起来相同。接下来, 通过移动目标与 z nanopositioner 进行荧光粒子 z 堆栈扫描, 以调整标记透镜的位置和角度 (图 4)。调整标记透镜的位置, 直到沿 z 方向的 xy 平面中的粒子图像没有漂移, 这是当粒子的 xy 位置没有变化 (图 4d) 的函数时实现的。跟踪系统可以在标签镜头对准后使用。</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>微粒监测用 sCMOS 相机的安装<ol>
		<li>安装一个 sCMOS 相机, 以可视化粒子, 而他们被跟踪。仅在安装并优化了跟踪系统的所有组件后才安装 sCMOS。</li></ol></li></ol>在显微镜上加载一个固定的荧光粒子样本, 并运行跟踪程序, 锁定一个粒子在目标的焦点体积。然后, 安装 100 mm 焦距透镜 (图 1: L8) 和 sCMOS。调整 sCMOS 位置, 使粒子的图像聚焦到最小和最亮的点。
	
	

4. 自由扩散纳米粒子的实时3D 跟踪
<ol>
	<li>将 110 nm 的自由运动微粒样品放在显微镜上。</li>
	<li>打开激光, 微定位控制器, 压电 nanopositioner 控制器, 标签镜头控制器, 和爆炸物控制器。在开环中运行压电 nanopositioner。根据步骤3.2 设置标签镜头软件。</li>
	<li>打开并运行跟踪软件 (可根据作者的要求提供)。将位置估计参数设置为其优化值, 在3节中找到。</li>
	<li>将片放在目标的焦点上, 去掉荧光发射滤波器, 利用激光从片的反射来最大化信号强度作为微 Z 位置的函数。在找到片后, 增加焦点位置15µm 将激光从片聚焦到解决方案中。放回荧光发射过滤器。</li>
	<li>设置整型控制常数。积分控制常数可以设置为低值开始, 并慢慢增加, 直到振荡可以看到在粒子的位置读数。一旦观测到振荡, 将积分控制常数设置为引起振荡的值的80%。典型值将是约0.012 的 XY ' 积分增益 ' 和0.004 的 Z ' 积分增益 '。</li>
	<li>在 "跟踪阈值" 和 "最小跟踪" 中设置跟踪阈值, 并通过单击 "搜索" 和 "自动跟踪" 启动跟踪实验。终止跟踪的阈值设置略高于后台级别, 触发跟踪的阈值大约比背景高两倍。在这里, 触发跟踪的阈值是3赫, 终止轨迹的阈值是1.5 赫。</li>
</ol>

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</ol>

作者声明没有竞争的金融利益。

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><tbody><tr><td>2D Electro-optic Deflector&amp;nbsp;</td><td>ConOptics</td><td>M310A</td><td>2 required</td></tr><tr><td>Power supply for EOD</td><td>ConOptics</td><td>412</td><td>Converts FPGA ouput to high voltage for EOD</td></tr><tr><td>TAG&amp;nbsp; Lens</td><td>TAG Optics</td><td>TAG 2.0</td><td>Used to deflect laser along axial direction</td></tr><tr><td>XY piezoelectric nanopositioner</td><td>MadCity Labs</td><td>Nano-PDQ275HS</td><td>Used for moving the sample to lock the particle in the objective focal volume in&amp;nbsp;</td></tr><tr><td>Z piezoelectric nanoposiitoner</td><td>MadCity Labs</td><td>Nano-OP65HS</td><td>Used to move the objective lens to follow the diffusing particle</td></tr><tr><td>Micropositioner</td><td>MadCity Labs</td><td>MicroDrive</td><td>Used to coarsely position sample and evaluate</td></tr><tr><td>Objective Lens</td><td>Zeiss</td><td>PlanApo</td><td>High numerical aperture required for best sensitivity. 100X, 1.49 NA, M27, Zeiss</td></tr><tr><td>sCMOS camera</td><td>PCO</td><td>pco.edge 4.2</td><td>Used to monitor the particle&#039;s position</td></tr><tr><td>APD</td><td>Excelitas</td><td>SPCM-ARQH-15</td><td>Lower dark counts beneficial</td></tr><tr><td>Field programmable gate array</td><td>National Instruments</td><td>NI-7852r</td><td></td></tr><tr><td>Software</td><td>National Instruments</td><td>LabVIEW</td><td></td></tr><tr><td>Tracking excitation laser</td><td>JDSU</td><td>FCD488-30</td><td></td></tr><tr><td>Lens</td><td>ThorLabs</td><td>AC254-150-A-ML</td><td>L1</td></tr><tr><td>Lens</td><td>ThorLabs</td><td>AC254-200-A-ML</td><td>L2</td></tr><tr><td>Pinhole</td><td>ThorLabs</td><td>P75S</td><td>PH</td></tr><tr><td>Glan-Thompson Polarizer</td><td>ThorLabs</td><td>GTH5-A</td><td>GT</td></tr><tr><td>Half-wave plate</td><td>ThorLabs</td><td>WPH05M-488</td><td>WP</td></tr><tr><td>Lens</td><td>ThorLabs</td><td>AC254-75-A-ML</td><td>L3</td></tr><tr><td>Lens</td><td>ThorLabs</td><td>AC254-250-A-ML</td><td>L4</td></tr><tr><td>Lens</td><td>ThorLabs</td><td>AC254-200-A-ML</td><td>L5</td></tr><tr><td>Lens</td><td>ThorLabs</td><td>AC254-200-A-ML</td><td>L6</td></tr><tr><td>Dichroic Mirror</td><td>Chroma</td><td>ZT405/488/561/640rpc</td><td>DC</td></tr><tr><td>Fluorescence Emission Filter</td><td>Chroma</td><td>D535/40m</td><td>F</td></tr><tr><td>10/90 beamsplitter</td><td>Chroma</td><td>21012</td><td>BS</td></tr><tr><td>PBS</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>190 nm fluorescent nanoparticles</td><td>Bangs laboratories</td><td>FC02F/9942</td><td></td></tr><tr><td>110 nm fluorescent nanoparticles</td><td>Bangs laboratories</td><td>FC02F/10617</td><td></td></tr><tr><td>Coverslip</td><td>Fisher Scientific</td><td>12-545A</td><td></td></tr><tr><td>Powermeter</td><td>Thorlabs</td><td>PM100D</td><td></td></tr><tr><td>CMOS</td><td>Thorlabs</td><td>DCC1545M</td><td></td></tr><tr><td>Iris</td><td>Thorlabs</td><td>SM1D12D</td><td></td></tr><tr><td>Microscope</td><td>Mad City Labs</td><td>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 ...

a-protocol-for-real-time-3d-single-particle-tracking

Research

JoVE Journal

Bioengineering

14.8K 次观看.  Duke University. 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。

视频: A Protocol for Real-time 3D Single Particle Tracking

Three-dimensional Particle Tracking Velocimetry for Turbulence Applications: Case of a Jet Flow

3D-PTV is a quantitative flow measurement technique that aims to track the Lagrangian paths of a set of particles in three dimensions using stereoscopic recording of image sequences. The basic components, features, constraints and optimization tips of a 3D-PTV topology consisting of a high-speed camera with a four-view splitter are described and discussed in this article. The technique is applied to the intermediate flow field (5 &#60;x/d &#60;25) of a circular jet at Re &#8776; 7,000. Lagrangian flow features and turbulence quantities in an Eulerian frame are estimated around ten diameters downstream of the jet origin and at various radial distances from the jet core. Lagrangian properties include trajectory, velocity and acceleration of selected particles as well as curvature of the flow path, which are obtained from the Frenet-Serret equation. Estimation of the 3D velocity and turbulence fields around the jet core axis at a cross-plane located at ten diameters downstream of the jet is compared with literature, and the power spectrum of the large-scale streamwise velocity motions is obtained at various radial distances from the jet core.

A three-dimensional particle tracking velocimetry (3D-PTV) system based on a high-speed camera with a four-view splitter is described here. The technique is applied to a jet flow from a circular pipe in the vicinity of ten diameters downstream at Reynolds number Re &#8776; 7,000.

湍流应用三维粒子跟踪测速:一喷流病例

3D-PTV is a quantitative flow measurement technique that aims to track the Lagrangian paths of a set of particles in three dimensions using stereoscopic ...

科研

工程学

Preparation and 3D Tracking of Catalytic Swimming Devices

We report a method to prepare catalytically active Janus colloids that "swim" in fluids and describe how to determine their 3D motion using fluorescence microscopy. One commonly deployed method for catalytically active colloids to produce enhanced motion is via an asymmetrical distribution of catalyst. Here this is achieved by spin coating a dispersed layer of fluorescent polymeric colloids onto a flat planar substrate, and then using directional platinum vapor deposition to half coat the exposed colloid surface, making a two faced "Janus" structure. The Janus colloids are then re-suspended from the planar substrate into an aqueous solution containing hydrogen peroxide. Hydrogen peroxide serves as a fuel for the platinum catalyst, which is decomposed into water and oxygen, but only on one side of the colloid. The asymmetry results in gradients that produce enhanced motion, or "swimming". A fluorescence microscope, together with a video camera is used to record the motion of individual colloids. The center of the fluorescent emission is found using image analysis to provide an x and y coordinate for each frame of the video. While keeping the microscope focal position fixed, the fluorescence emission from the colloid produces a characteristic concentric ring pattern which is subject to image analysis to determine the particles relative z position. In this way 3D trajectories for the swimming colloid are obtained, allowing swimming velocity to be accurately measured, and physical phenomena such as gravitaxis, which may bias the colloids motion to be detected.

A method to prepare catalytically active Janus colloids that can "swim" in fluids and determine their 3D trajectories is presented.

催化游泳器件的制备及其三维跟踪

We report a method to prepare catalytically active Janus colloids that "swim" in fluids and describe how to determine their 3D motion using fluorescence ...

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

单分子跟踪显微镜 - 确定细胞分子扩散状态的工具

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

生物化学

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

生物工程

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

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

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.

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 ...

Controlling Flow Speeds of Microtubule-Based 3D Active Fluids Using Temperature

We present a method for using temperature to tune the flow speeds of kinesin-driven, microtubule-based three-dimensional (3D) active fluids. This method allows for tuning the speeds in situ without the need to manufacture new samples to reach different desired speeds. Moreover, this method enables the dynamic control of speed. Cycling the temperature leads the fluids to flow fast and slow, periodically. This controllability is based on the Arrhenius characteristic of the kinesin-microtubule reaction, demonstrating a controlled mean flow speed range of 4&#8211;8 &#181;m/s. The presented method will open the door to the design of microfluidic devices where the flow rates in the channel are locally tunable without the need for a valve.

The goal of this protocol is to use temperature to control the flow speeds of three-dimensional active fluids. The advantage of this method not only allows for regulating flow speeds in situ but also enables dynamic control, such as periodically tuning flow speeds up and down.

使用温度控制基于微管的3D活性流体的流速

We present a method for using temperature to tune the flow speeds of kinesin-driven, microtubule-based three-dimensional (3D) active fluids. This method ...

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take place, are inherently difficult to assess with standard biophysical methods. The introduction of mass-sensitive particle tracking (MSPT) constitutes an important step toward a thorough quantitative characterization of such processes. Technically, this was made possible through the advent of interferometric scattering microscopy (iSCAT)-based mass photometry (MP). When the background removal strategy is optimized to reveal the two-dimensional motion of membrane-associated particles, this technique allows the real-time analysis of both diffusion and molecular mass of unlabeled macromolecules on biological membranes. Here, a detailed protocol to perform and analyze mass-sensitive particle tracking of membrane-associated systems is described. Measurements performed on a commercial mass photometer achieve time resolution in the millisecond regime and, depending on the MP system, a mass detection limit down to 50 kDa. To showcase the potential of MSPT for the in-depth analysis of membrane-catalyzed macromolecule dynamics in general, results obtained for exemplary protein systems such as the native membrane interactor annexin V are presented.

This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls.

质量敏感颗粒跟踪，用于表征膜相关的高分子动力学

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take ...

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using the analysis of cell membrane receptor clusters as a model, we present a detailed protocol for this image analysis task using Fiji (ImageJ) and Matlab routines to: 1) define regions of interest and design masks adapted to these regions; 2) track the particles in fluorescence microscopy videos; 3) analyze the diffusion and intensity characteristics of selected tracks. The quantitative analysis of the diffusion coefficients, types of motion, and cluster size obtained by fluorescence microscopy and image processing provides a valuable tool to objectively determine particle dynamics and the consequences of modifying environmental conditions. In this article we present detailed protocols for the analysis of these features. The method described here not only allows single-molecule tracking detection, but also automates the estimation of lateral diffusion parameters at the cell membrane, classifies the type of trajectory and allows complete analysis thus overcoming the difficulties in quantifying spot size over its entire trajectory at the cell membrane.

Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy.

用荧光显微镜分析膜受体扩散和簇簇的图像处理协议

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using ...

免疫与感染

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. &#160;To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.&#160;

<meta charset="utf8"></head><body>用于果蝇树突和神经肌肉接头突触微管动力学的无创体内分析的 3D 粒子跟踪

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

用于果蝇树突和神经肌肉接头突触微管动力学的无创体内分析的 3D 粒子跟踪

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. ...

一种实时3D 单粒子跟踪协议

摘要

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用于果蝇树突和神经肌肉接头突触微管动力学的无创体内分析的 3D 粒子跟踪