Name: visualizing diffusional dynamics of gold nanorods on cell membrane using single nanoparticle darkfield microscopy
Uploaded: 2021-03-05T15:00:00
Description: Watch this Scientific Journal Video about Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy at JoVE.com

这项工作得到了中国国家自然科学基金委员会的支持，赠款编号为21425519、91853105和21621003。

1. 细胞培养
<ol><li>通过添加胎儿牛血清（最终浓度10%）为U87MG细胞准备完整的介质青霉素-链霉素（最终浓度1%）到最低限度的基本介质（MEM）。使用塑料细胞培养皿进行细胞亚培养。</li>
	<li>通道单元每周2到3次。<ol>
<li>取出培养基，用杜尔贝科的磷酸盐缓冲盐水（D-PBS）冲洗细胞层2~3倍（80%~90%）。</li>
		<li>在细胞培养皿中加入1.0至2.0 mL的Trypsin-EDTA溶液，在倒置显微镜下观察细胞，直到细胞变圆?...

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所展示的协议用于研究细胞膜上的AuNR的动态。该协议由四个部分组成，包括微观成像、数据提取、动态参数计算和数据分析方法，每个部分都是灵活和通用的。因此，未来有许多可能的应用，例如，研究膜上NP相关膜分子的运动、NP标记受体的内分泌动力学、细胞内NP和囊泡涂层的NPs沿着微管运输的动态分析等。
基本步骤是使用DFM来映像细胞膜上的AuNR运动。目前，已开发出多...

纳米粒子（NPs）在膜上的动力学与细胞吸收过程密切相关，这对了解细胞功能、病毒或细菌感染以及人工纳米医学输送系统的发展至关重要。单粒子跟踪 （SPT） 技术是描述 NP3、4的异质行为的可靠工具。一般来说，细胞膜是流动的，这意味着蛋白质和脂质等成分可以在等离子膜平面5、6、7中横向移动。膜组织和结构的空间复杂性可能导致NP和膜之间相互作用的空间异质性。因此，在膜上直接可视化 NP 的运动需要高空间和时间分辨率。
单粒子跟踪显微镜监测活细胞中单个粒子的定位，空间分辨率为几十纳米，时间分辨率为几毫秒，已得到很好的开发，以研究NP或膜分子的动力学8，9。荧光显微成像技术已成为观测活细胞环境中的NPs/分子的宝贵工具。例如，总内部反射荧光显微镜，在具有高空间分辨率的基板/溶液接口上对样品的薄层（约100nm）进行成像，已广泛应用于膜分子动力学13、14的研究。然而，单荧光素的固有缺点，如低强度和快速不可逆的光出血降低了跟踪13的准确性和持续时间。因此，取代荧光探头的非荧光质子NP由于其独特的光学特性，在长期成像研究中越来越受到重视。基于质粒NP探针的散射信号，利用多种光学显微成像技术研究生物过程的机理，如暗场显微镜（DFM）16、干涉学散射（iSCAT）显微镜17和微分干扰对比显微镜（DICM）18。此外，AuNR的运动和旋转动态可以使用DFM和DICM 18、19、20、21、22获得。通常，在SPT实验中，物体的运动由光学显微镜记录，然后通过SPT分析方法3进行分析。单个NP产生的定时轨迹和定向角度通常是随机的和异质的，因此有必要用各种分析方法呈现丰富的动态信息。
在这里，我们提供了一个集成协议，使用DFM监控细胞膜上的AuNR的动态，用ImageJ和MATLAB提取AuNR的位置和方向，并用SPT分析方法描述AuNR的扩散。作为演示，我们在这里展示如何使用SPT协议在U87MG细胞膜上可视化未改性AuNR（CTAB-AuNR，由溴化铵分子作为保护剂合成）的动态。研究表明，CTAB-AuNR可以在生物环境中吸收蛋白质，在细胞膜上移动，然后进入细胞2，20，22。U87MG细胞是中枢神经系统最常见、最恶性的肿瘤，其膜受体表达异常。膜受体可以与AuNR上的蛋白质相互作用，影响AuNR的动态。我们的协议通常适用于生物学领域的其他SPT实验。

<table>
	<tbody>
		<tr>
			<td>CTAB coated gold nanorods(CTAB-AuNRs)</td>
			<td>Nanoseedz</td>
			<td>NR-40-650</td>
			<td>85 nm * 40 nm</td>
		</tr>
		<tr>
			<td>Color CMOS camera</td>
			<td>Olympus</td>
			<td>DP74</td>
			<td>Japan</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Citoglas</td>
			<td>z10212222C</td>
			<td>22*22 mm</td>
		</tr>
		<tr>
			<td>Dark-field microscopy</td>
			<td>Nikon</td>
			<td>80i</td>
			<td>upright microscope</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>10099141</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>National Institutes of Health</td>
			<td>2.0.0-rc-69/1.52 p</td>
			<td>a distribution of ImageJ</td>
		</tr>
		<tr>
			<td>Grooved glass slide</td>
			<td>Sail brand</td>
			<td>7103</td>
			<td>Single concave</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>National Institutes of Health</td>
			<td>1.52 j</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>R2019b</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB Code</td>
			<td></td>
			<td></td>
			<td>https://github.com/fenggeqd/JOVE-2020</td>
		</tr>
		<tr>
			<td>Minimum essential medium (MEM)</td>
			<td>Gibco</td>
			<td>10-010-CVR</td>
			<td>with phenol red</td>
		</tr>
		<tr>
			<td>Minimum essential medium (MEM)</td>
			<td>Gibco</td>
			<td>51200038</td>
			<td>no phenol red</td>
		</tr>
		<tr>
			<td>Origin</td>
			<td>OriginLab</td>
			<td>Origin Pro 2018C</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cell culture dishes</td>
			<td>Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cell culture dishes</td>
			<td>Falcon</td>
			<td>353001</td>
			<td>35*10 mm</td>
		</tr>
		<tr>
			<td>U87 MG cell</td>
			<td>American Type Culture Collection</td>
			<td>ATCC HTB-14</td>
			<td>a human primary glioblastoma cell line</td>
		</tr>
	</tbody>
</table>

visualizing diffusional dynamics of gold nanorods on cell membrane using single nanoparticle darkfield microscopy

分析纳米粒子在细胞膜上的扩散动力学，对于更好地了解细胞吸收过程具有重要作用，为纳米药物输送的合理设计提供了理论依据。单粒子跟踪（SPT）分析可以探测单个纳米粒子在细胞膜上的位置和方向，并揭示其转化和旋转状态。在这里，我们展示了如何使用传统的暗场显微镜来监测活细胞膜上的金纳米棒（AuNR）的动态。我们还展示了如何使用 ImageJ 和 MATLAB 提取 AuNR 的位置和方向，以及如何描述 AuNR 的扩散状态。对数百个粒子的统计分析表明，单个AuNR在U87MG细胞膜表面执行布朗运动。然而，单个长轨迹分析表明，AuNR在膜上有两种截然不同的运动状态，即远程传输和有限区域禁闭。我们的SPT方法可用于研究不同生物细胞的表面或细胞内粒子扩散，并可以成为研究复杂细胞机制的有力工具。

在协议中，使用了未经修改的 40 x 85 nm CTAB-AuNR。如图2B所示，其纵向质离子最大值为 650 nm（红色区域），横向共振为 520 nm（绿色区域）。以往的文献显示，质子AuNR的光学特性（如LSPR强度）将发生显著变化，其直径为20，22。在图2C中，U87细胞膜上CTAB-AuNR的散射强度显示了典型的高斯分布，宽度狭窄，与玻璃?...

Watch this Scientific Journal Video about Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy at JoVE.com

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

在这里，我们展示了使用传统的暗场显微镜来监测金纳米棒（AuNR）在细胞膜上的动态。使用 ImageJ 和 MATLAB 检测单个 AuNR 的位置和方向，AuNR 的扩散状态以单个粒子跟踪分析为特征。

使用单纳米粒子暗场显微镜可视化细胞膜上金纳米棒的扩散动力学

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

我们的协议使用单个粒子检查分析来监测位置和方向的动态，并描述金纳米棒在细胞膜上的扩散。利用这种方法，可以获取金纳米棒的转化和旋转动力学，并综合分析和呈现金纳米棒的动态。该协议有可能用于研究其他类型的复杂生物系统。首先在火焰上燃烧乙醇浸玻璃盖片，将盖片放入一个35至10毫米的细胞培养皿中，其中含有两毫升细胞培养介质，不含苯酚红色。在盖片上加入50微升的细胞悬浮液，轻轻摇动盘来回，左右均匀分布细胞。将菜放入细胞培养孵化器约12小时，直到细胞达到20%至40%的结合度，然后再在培养皿中加入20微升CTAB涂层金纳米棒。轻轻摇晃后，将纳米棒均匀地分散在盘子上，将菜放在潮湿的氛围中五分钟。在孵化结束时，缓慢地将100微升的超纳坦从盘子中转移到玻璃滑梯的凹槽中，并小心地将盖片细胞侧向下放置到滑动槽上，然后用指甲油密封盖片的边缘，让指甲油干燥，然后将滑梯放到暗场显微镜阶段。对于通过暗地显微镜跟踪的单个粒子，将一滴油放在油浸入暗场冷凝器上，然后转动旋钮，直到冷凝器接触玻璃滑动。将一滴油放在盖玻璃顶部，转动对焦旋钮，直到 60 倍的油浸入目标接触油。打开光源，稍微打开对焦旋钮以聚焦成像平面。然后单击显微镜软件中的相机图标，使用彩色 CMOS 相机记录样品散射光图像时间系列，并以 TIFF 格式保存图像。要提取单个长期轨迹，请打开 ImageJ 中的图像，然后单击图像、类型和 8 位，将图像从 RGB 模式转换为 8 位模式。要调整对比度，请单击图像、调整亮度对比度并设置参数。选择目标粒子并使用控制 X 切断时间串背景。单击插件、粒子跟踪器经典和粒子跟踪器以打开粒子检测和粒子链接窗口，并将半径设置为 6，截止到零，百分位数设置为 0.01%， 将链接范围设置为 10，位移为 10，然后单击 OK 打开粒子跟踪器结果窗口以查看结果。单击可视化所有轨迹以检查生成的轨迹。如果软件生成的轨迹与金纳米棒的移动轨迹匹配，单击"保存完整报告"以保存结果。如果软件生成的轨迹与金纳米棒的移动轨迹不匹配，请单击重新链接粒子，将检测到的粒子与不同的链接范围和百分位数参数重新链接。要根据 XY 坐标在每帧中查找金纳米棒的中心像素坐标，请使用 xy 协调。m函数。要划定三乘三像素矩阵，请提取红色或绿色通道的九个散射强度值并计算平均值，使用 rgextraction。m函数。然后使用极地角。m 函数使用双通道差分法计算极角。要使用表中的公式计算动态参数，运行两个分析脚本。要对轨迹进行视觉分析，请将 X 坐标设置为 X，Y 坐标为 Y，时间设置为 Y，然后单击绘图、散射和颜色图，设置图形参数，并添加颜色条。要生成均值方位移时间间隔图，请将时间间隔设置为 X，将平均方位移设置为 Y.Click 绘图、散射、分析、拟合、非线性曲线拟合和打开对话，并设置图形参数。对于多粒子统计分析，将感兴趣的动态参数设置为 Y，然后单击绘图和直方图。双击直方图以设置除法大小或分区数，然后单击应用。然后添加一个列并设置图形参数。对于时间系列分析，将时间设置为 X，将时间系列参数设置为 Y.Click 绘图、多窗格和堆叠。在弹出窗口中，选择行和确定。然后设置图形的参数。纵向质离子最大40乘85纳米CTAB涂层金纳米棒约为650纳米，横向共振为520纳米。CTAB 涂层金纳米棒在 U87 细胞膜上的散射强度显示了典型的高斯分布，其宽度与玻璃上的 CTAB 涂层金纳米棒的宽度一致，表明本实验中跟踪的 CTAB 涂层金纳米棒分布良好。如图所示，500多个CTAB涂层的金纳米棒轨迹可以通过暗场显微镜进行跟踪，并可分为远距离扩散轨迹和密闭扩散轨迹。在此代表性分析中，所有 500 个轨迹的回旋半径显示小值分布，平均回旋半径为 0.5 微米，最大位移在小值下分布得更多。正如在此合奏时间平均方位移分析中所示，CTAB 涂层的金纳米棒通常以大约一个字母的字母扩散。然而，扩散系数和从所有轨迹中获得的α的密度分布表明，金纳米棒的动态呈现出具有超扩散、布朗尼和亚扩散运动的异质分布。此外，单粒子统计分析和时间系列参数分析可用于描述两个具有代表性的长期限制和移动轨迹。有必要从单个粒子跟踪分析读出的数据中总结和提取单一的新解释，因为通常有一些难以做出的权衡。

Research

JoVE Journal

Biochemistry

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

使用SPHIRE的电子冷冻显微镜图像的高分辨率单粒子分析

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

科研

生物化学

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

原子力显微镜成像在核细胞结构和动力学研究中的应用

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

电化学和荧光显微镜在质子泵膜酶研究中的单脂质体测定

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Evaluation of ProteinÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“Protein Interactions using an On-Membrane Digestion Technique

使用膜内消化技术评估蛋白质与蛋白质相互作用

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

重建线粒体膜动力学的模型膜平台

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

单分子鱼的转录活性等位基因单细胞分析

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...