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://www.jove.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://www.jove.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

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

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. ...

科研

生物工程

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

珠聚集测定假定的细胞粘附分子的表征

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

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 ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

湿化学和肽固定在聚四氟乙烯为改善细胞粘附

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

3D 基质中哺乳动物细胞分裂的定量共焦反射显微镜观察

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

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 ...