作者感谢的土地，巴登-符腾堡州和Europäische联盟的Europäischer全宗献给模具REGIONALE Entwicklung -资金ZAFH光子N，Bundesministerium献给教化和Forschung（BMBF）资助的研究经费。 1792C08和巴登 - 符腾堡州基金会有限公司为融资项目"Aurami"的。

1。播种和培养细胞<ol><li>种单个细胞， 例如 U373-MG或U-251-MG的成胶质细胞瘤细 ​​胞在一个典型的密度为100个细胞/ mm 2的载玻片上，并成长为4872小时，在培养基中（ 例如与10％胎牛血清的RPMI 1640培养基和抗生素）使用的培养箱，在37℃和5％CO 2的 。 </li><li>孵育细胞的膜标记物， 例如 6-十二烷酰基-2-二甲基氨基萘（laurdan）60分钟施加在8μM（培养液中）4，或细胞质标记的浓度， 例如:钙黄绿素acetomethlyester申请在40分钟浓度为5μM3，或相关联的膜光敏剂， 例如，原卟啉IX诱导后，培养4小时，其前体5 -氨基乙酰丙酸（5-ALA，1毫摩尔）4,5。 </li><li>洗涤细胞用培养基或磷酸盐缓冲盐水（PBS）在荧光显微镜之前&lt;/ P&gt; </ol> 2。 VA-TIRFM <ol><li>使用一个直立的的显微镜， 例如蔡司Axioplan，并更换冷凝器的照明设备，按文献。 3具有半球形或半圆筒状的玻璃棱镜光学耦合到的对象滑动（参见图1）。 </li><li>使用的平行准直的激光束成像后的玻璃棱镜和进一步的光学元件（ 例如凹面镜）再次结果的样品的表面上的平行光束（远心光瞳射线）。照顾，电场矢量垂直于入射平面的被极化。 </li><li>使用可调节的反射镜，位于成像的样品平面中，并按照可变入射角（对象射线）照亮样品内部的冷凝器。可调反射镜可以由步进电机驱动的，其中每个位置对应于一个良好定义的入射角，测角测量3所确定的</支持&gt;。 </li><li>校准可调反射镜的角位置，使用临界角Θ= 61.3°的玻璃水转变为一个固定的值。 Θc可以是可视化后，溶解于水中时的荧光染料（ 例如，1 mM的黄素单核苷酸，FMN）Θ的变化。镜子的位置在Θ=Θc被存储在镜定位程序。 </li><li>设置的摄像头（背照式EMCCD安道尔IXON，DV887DC，ANDOR科技，贝尔法斯特，英国6）参数，包括温度，增益，记录时间和换挡速度。 </li><li>使用适当的倍率的物镜和优先温和的数值孔径（ 例如 40×/ 0.75，或63×/ 0.90水浸泡）和本地化的对象在显微镜滑动。使用浸油，以保证样品与玻璃棱镜的光学接触。变化的记录时相同的样品的荧光图像照明角度（Θ≥Θc与θC对应于64.5°的小区-玻璃界面）。建议为0.5°或1°66°-74°的角度范围内的步骤。使用适当的长通或带通滤波器，用于荧光检测。 </li></ol> 3。数据分析<ol><li>阅读的图像（ 例如以ASCII格式）记录在不同的角度Θ，以及相机设置（背景歧视），激发波长和对玻璃的折射率（n 1个 = 1.52）和细胞（n 2个 = 1.37）到评估程序（LabView的纳米单元）。对于每组参数的渗透深度d（Θ），透射系数T（Θ）的自动计算。 </li><li>选择用于评价细胞 - 基底距离Δ根据等式5（膜标记）或公式4（细胞质标记），个人的图像不均匀的图像照明（揭示E，G条纹）可能会被排除在外。 </li><li>计算图像的的细胞基质拓扑结构，选择适当的颜色代码显示。期间可以被显示，如在图2中所示的各个像素的评价公司。作为拟合的质量的指标，可以使用这些配置文件。 </li><li>存储的评价协议。 </li></ol> 4。代表性的成果与基因工程U251-MG胶质母细胞瘤细胞的1月莫伦豪尔，分子肿瘤学系教授，南丹麦大学，欧登塞提供的代表进行了实验。在两个亚克隆那些U251细胞的肿瘤抑制基因TP53或PTEN过度表达用的Tet-诱导的表达系统，例如，这些细胞显示出降低的致瘤性，并可能被视为恶性程度较低。高度恶性的U251-MG的抑制基因细胞，而不被用作对照，和U251-MG野生型细胞用于进一步比较。发射准连续系列的皮秒脉冲（LDH400与驱动程序PDL 800-B，Picoquant，柏林，德国，波长:391纳米，脉冲能量:12 PJ;重复率:40兆赫）的激光二极管作为激发源使用。 在图3中TIRFM U251-MG与活化的TP53抑癌基因和与laurdan（8μM，60分钟）温育的成胶质细胞瘤细 ​​胞的图像的图6-3描述了入射角为66.0°，69.0°和73.0°的穿透深度相应于瞬态电磁场为约140 nm，75 nm和60 nm处，分别随着降低穿透深度荧光强度减小（见尺度）和来源于更为肤浅的站点的细胞（ 例如，它们的边缘上的接触斑）。电池基片的距离（ 图三维可视化的范围从0到600纳米的颜色代码）强烈变化的细胞之间的上LY几个纳米（白色，黄色）和250-300纳米（暗红色）， 即接触斑和更大的电池基片的距离都在酒店附近。这同样适用于U251-MG的成胶质细胞瘤细 ​​胞活化的抑制基因PTEN（ 图4d），而细胞-基底距离U251-MG的控制和野生型细胞与约80-100 nm的典型值（黄色， 图4b是相当恒定的）和150-200纳米（橙红色; 图4a）。 <img alt="图1" src="/files/ftp_upload/4133/4133fig1.jpg" /> 图1。VA-TIRFM在直立显微镜的照明装置。由步进电动机驱动的可调节的反射镜允许±0.25°的角分辨率。 <img alt="图2" src="/files/ftp_upload/4133/4133fig2.jpg" /> 图2。评价一个像素的档案EL（示意图）。当LN [ⅠF / T（Θ）]作为1次/ d（Θ）的函数作图，斜率表示细胞衬底距离Δ。此斜率通常测定的精度为±10％左右。从评估的价值之一 - 产生不均匀的亮度 - 已标记和排除。 <img alt="图3" src="/files/ftp_upload/4133/4133fig3.jpg" /> 图3。TIRFM技术的图片，U251-MG的成胶质细胞瘤细 ​​胞与活化TP53孵育laurdan（8μM，60分钟）记录在不同的照明角度（交流）时抑制基因;所示细胞基板在0-600纳米的范围内的距离通过颜色代码（D）;激发波长为391纳米，检测:≥420 nm的图像尺寸:210微米×210微米。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/4133/4133fig3large.jpg" target="_blank">点击此处查看大图</a> 。 （四） 图4。细胞基底的距离的U251-MG glioblatoma的细胞在0-600纳米的范围内显示由一个彩色码（一）野生型（b）控制;（三）细胞活化的抑制基因TP53;细胞激活抑癌基因PTEN，激发波长为391纳米，检测:≥420纳米，图像大小:210微米×210微米。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/4133/4133fig4large.jpg" target="_blank">点击此处查看大图</a> 。

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没有利益冲突的声明。

描述了一种方法，用于测量细胞衬底与纳米精度的距离。目前，超高分辨率显微镜的方法， 例如 ，基于结构照明7或单分子检测8-10以及受激发射损耗（STED）显微镜是相当大的兴趣。然而，没有这些技术允许的轴向分辨率低于50nm。此外，需要相当高的为50100 W / cm 2的辐照度为单分子的方法和超过3000瓦/厘米2的受激发射损耗显微镜。这超过太阳辐照度（约100毫...

当光束传播通过的介质的折射率n 1，满足的第二介质的折射率n 2 &lt;n 1的一个接口，全内反射发生的所有角度，这是大于临界角的入射角ΘΘ=反正弦（N 2 / N 1）。尽管被全反射入射光束的唤起的瞬态电磁场，渗入所述第二介质和呈指数衰减，与从界面的垂直距离z根据I = I 0（z）的易通/天（Θ）。 I（z）的对应的电磁场和d（Θ）的强度，所给出的在波长λ的穿透深度 D（θ）=（λ/4π）（1 2罪2Θ - N 2 2）-1 / 2 据报道在文献1,2中 ，I（0） </sUB&gt; 站 |电子乘以传输系数T（Θ），比n 2 / n 1个与入射光的强度相对应。如果该光束的电场矢量的偏振垂直于入射平面的（ 即所跨越的入射和反射光束的平面），该传输系数由下式给出 T（Θ）= 4余弦2Θ/ [1 - （n 2个 / nL的1）2] 对于检测到的荧光强度在TIRFM测量中，光吸收具有集成的样品的所有层和乘以有关染料的荧光量子收率η，以及检测与立体角Ω。正如以前报道的3，这种强度可以通过以下进行说明 我F（Θ）= AT（Θ）OC（Z）E-Z / D（Θ）DZ 如果所有这些因素是独立的f<html>的ROM的入射角Θ和坐标z均包括在实验常数A.等式3可以解析解，如果荧光基团的浓度c（z）的被认为是几乎恒定的 -或者， 例如 ，在所有坐标Z≥Δ的细胞质内的细胞，从该接口具有的距离Δ。在这种情况下，积分计算从z =Δ到z =∞导致 I F = A C T（Θ）D（Θ）E-Δ/ D（Θ） -或之间的Z =Δ -吨/ 2和z =Δ+ t / 2只， 即一层薄薄的厚度t内， 例如位于从界面的距离Δ的细胞膜。在这种情况下，可以计算出的荧光强度为 I F =是C T（Θ）TE-Δ/ D（Θ） 如果tΔ比较小。 耳鼻喉科"&gt;从方程（4）和（5）细胞-基底距离Δ，如果可以计算出的荧光强度I F的图像被记录为可变角度Θ，如果In（I F / T d的）（公式4）或ln（I F / T）（公式5）被评价为1次/ d的函数的这些角度，对于本文中所用的膜标记laurdan（第下文）根据公式5进行评价。 正如以前报道的3，图像采集和评估要求 - 在样本激发光的均匀分布的， -有效性的2层模型（与折光率n 1和n 2分别为在基板和细胞质，分别）。这认为，如果等离子体膜是非常薄的，如果在细胞外介质中的层（细胞和衬底之间）与入射光的波长相比是很小的。 此外，温和的数值孔径（通常为A≤0.90）的显微镜物镜是有利的，以避免由于辐射的各向异性，在近场中的电介质界面亮度偏差。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 类型 </td><td> 评论 </td></tr><tr><td>恒温箱</td><td> Nunc公司</td><td> Nunc公司蜂星 QM1300SVBA </td><td></td></tr><tr><td>层流</td><td>霍尔滕安全</td><td> S2010 1.2 EN GG 1LN </td><td></td></tr><tr><td> DMEM +10％FCS + 1％青霉素/链霉素</td><td> BIOCHROM </td><td></td><td>栽培介质</td></tr><tr><td>玻璃对象的幻灯片</td><td>马林费尔德</td><td>纯白色的玻璃</td><td>使用了特殊的清洗程序</td></tr><tr><td> 6-十二烷酰基-2-二甲基氨基萘（laurdan） </td><td>分子探针</td><td></td><td>荧光标记（储备液:2 mm的乙醇） </td></tr><tr><td>显微镜</td><td>卡尔·蔡司</td><td> Axioplan 1 </tD&gt; <td></td></tr><tr><td>激光二极管</td><td> PicoQuant </td><td> LDH 400与驱动程序PDL 800-B </td><td>波长:391纳米</td></tr><tr><td>单模光纤系统</td><td>点源</td><td> kineFlex </td><td>可用于准直光学器件</td></tr><tr><td> EMCCD相机</td><td>安道​​尔</td><td> DV887DC </td><td>背照式摄像头</td></tr></tbody></table>

nanotopology of cell adhesion upon variable-angle total internal reflection fluorescence microscopy (va-tirfm)

表面的拓扑结构， 例如，在衬底上生长的细胞中，确定由可变角度总内部反射荧光显微镜（VA-TIRFM）与纳米精度。将细胞培养在透明的幻灯片，并与荧光标记物均匀地分布在它们的细胞膜中孵育。照明发生由一个平行的激光束根据变量的总内部反射（TIR）的角度，用不同的穿透深度的瞬态电磁场。荧光图像后，在约10个不同的角度照射的记录允许，计算细胞衬底的距离的精度为几纳米。这样确定的各种细胞株， 例如，肿瘤细胞和恶性细胞少，密合性的差异。此外，可能发生的变化时的细胞粘附化学或光动力治疗可以进行检查。与其他方法相比，超分辨率显微镜的光照射保持活细胞的非常小，并且没有损坏预期发生。

Watch this Scientific Journal Video about Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM at JoVE.com

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM

细胞粘附在基板上的拓扑结构是由可变角度的总内部反射荧光显微镜（VA-TIRFM）与纳米精度测量。

Nanotopology细胞粘附在可变角度的全内反射荧光显微镜（VA-TIRFM）

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence ...

nanotopology-cell-adhesion-upon-variable-angle-total-internal

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

10.1K 次观看.  Institut für Angewandte Forschung. 细胞粘附在基板上的拓扑结构是由可变角度的总内部反射荧光显微镜（VA-TIRFM）与纳米精度测量。

视频: Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

调查单分子粘附的原子力谱

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single ...

科研

工程学

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

生物工程

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins.

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. ...

生物化学

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

通过全内反射荧光（TIRF）显微镜 在体外 可视化肌动蛋白和微管耦合动力学

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

使用水凝胶图案化技术控制细胞粘附，用于牵引力显微镜的应用

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

通过总内部反射荧光显微镜与数量和亮度分析相结合，活细胞中细胞表面受体的显微化动力学

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...

生物学

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

通过粘附足迹测定法对细胞滚动中的分子粘附进行成像

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

Tension Gauge Tether Probes for Quantifying Growth Factor Mediated Integrin Mechanics and Adhesion

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate multiple functions, including adhesion, proliferation, migration, and differentiation. Mechanical forces can be transmitted from the cell via the adhesion receptor integrin to ligands in the ECM. The amount and spatial organization of these cell-generated forces can be modulated by growth factor receptors, including epidermal growth factor receptor (EGFR). The tools currently available to quantify crosstalk-mediated changes in cell mechanics and relate them to&#160;focal adhesions, cellular morphology, and signaling are limited. DNA-based molecular force sensors known as tension gauge tethers (TGTs) have been employed to quantify these changes. TGT probes are unique in their ability to both modulate the underlying force threshold and report piconewton scale receptor forces across the entire adherent cell surface at diffraction-limited spatial resolution. The TGT probes used here rely on the irreversible dissociation of a DNA duplex by receptor-ligand forces that generate a fluorescent signal. This allows quantification of the cumulative integrin tension (force history) of the cell. This article describes a protocol employing TGTs to study the impact of EGFR on integrin mechanics and adhesion formation. The assembly of&#160;the TGT mechanical sensing platform is systematically detailed and the procedure to image forces, focal adhesions, and cell spreading is outlined. Overall, the ability to modulate the underlying force threshold of the probe, the adhesion ligand, and the type and concentration of growth factor employed for stimulation make this a robust platform for studying the interplay of diverse membrane receptors in regulating integrin-mediated forces.

TGT surface is an innovative platform to study growth factor-integrin crosstalk. The flexible probe design, specificity of the adhesion ligand, and precise modulation of stimulation conditions allow robust quantitative assessments of EGFR-integrin interplay. The results highlight EGFR as a 'mechano-organizer' tuning integrin mechanics, influencing focal adhesion assembly and cell spreading.

用于量化生长因子介导的整合素力学和附着力的张力计系绳探头

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate ...

TIRF Microscopy to Visualize Actin and Microtubule Coupling Dynamics

Source: Henty-Ridilla, J.L., <a href="https://appjove.unicartagenaproxy.elogim.com/v/64074">Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy.</a> J. Vis. Exp. (2022)
This video describes TIRF, a total internal reflection microscopy-based technique to visualize actin and microtubule polymerization dynamics. The method allows the high-resolution imaging of actin and microtubule coupling dynamics in real-time, which is essential for understanding cellular crosstalks.

TIRF 显微镜可视化肌动蛋白和微管耦合动力学

Source: Henty-Ridilla, J.L., Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy. J. ...

Nanotopology细胞粘附在可变角度的全内反射荧光显微镜（VA-TIRFM）

摘要

探索更多视频

此视频中的章节

调查单分子粘附的原子力谱

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

通过全内反射荧光（TIRF）显微镜在体外可视化肌动蛋白和微管耦合动力学

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

使用水凝胶图案化技术控制细胞粘附，用于牵引力显微镜的应用

通过总内部反射荧光显微镜与数量和亮度分析相结合，活细胞中细胞表面受体的显微化动力学

通过粘附足迹测定法对细胞滚动中的分子粘附进行成像

用于量化生长因子介导的整合素力学和附着力的张力计系绳探头

TIRF 显微镜可视化肌动蛋白和微管耦合动力学

Nanotopology细胞粘附在可变角度的全内反射荧光显微镜（VA-TIRFM）

摘要

探索更多视频

此视频中的章节

调查单分子粘附的原子力谱

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

通过全内反射荧光（TIRF）显微镜 在体外 可视化肌动蛋白和微管耦合动力学

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

使用水凝胶图案化技术控制细胞粘附，用于牵引力显微镜的应用

通过总内部反射荧光显微镜与数量和亮度分析相结合，活细胞中细胞表面受体的显微化动力学

通过粘附足迹测定法对细胞滚动中的分子粘附进行成像

用于量化生长因子介导的整合素力学和附着力的张力计系绳探头

TIRF 显微镜可视化肌动蛋白和微管耦合动力学

通过全内反射荧光（TIRF）显微镜在体外可视化肌动蛋白和微管耦合动力学