我们感谢他的技术支持和香，李Layland（两个弗劳恩霍夫IGB斯图加特）斯特芬·科赫，他的手稿的有益的建议。这项工作是由财政支持的弗劳恩霍夫协会的BMBF（KS-L的）。吸引程序。

1。生物样品的制备<ol><li>活细胞的制备<ol><li>贴壁细胞的制备<ol><li>种子在体外培养或新鲜分离的细胞上的玻璃底菜（格雷纳BioOne /德国），并于37°C和5％的CO 2细胞附着，直到完成。 </li><li>去除培养基，轻轻洗脸用PBS事先测量三次。保持与PBS或细胞培养液覆盖在整个测量过程中的细胞。 </li></ol></li><li>悬浮细胞的制备<ol><li>根据共同协议（如胰蛋白酶EDTA，细胞刮） 在体外培养细胞的分离，离心细胞，并重新挂起得到细胞沉淀，PBS或细胞培养液中。 </li><li>细胞悬液100μL（浓度:最大10万个细胞/毫升）转移到玻璃底菜。 </li></ol></li></ol></li><li>准备的NAT香港专业教育学院组织<ol><li>收获后的组织，他们转移到无菌，冰冷的PBS不再让他们在4°C或上冰前测量超过12小时。以前的实验表明，延长贮存时间（冷缺血时间&gt; 12小时在4°C）由于自然降解过程的光谱变化。 </li><li> PBS或中等覆盖，以避免由于组织干燥ECM蛋白和细胞的损害组织必须执行测量。 </li></ol></li></ol> 2。拉曼光谱仪我们定制的拉曼光谱仪结合的拉曼光谱仪，使亮场的拉曼光谱和荧光图像直接比较标准的荧光显微镜（奥林巴斯IX 71）。基本设置包括1 784 nm的激光二极管（TOPTICA光电公司/德国），陷波滤波器的拉曼散射光激发光分离，在显微镜ND A摄谱仪，电荷藕合器件（CCD相机）优化光谱信息检测（软成像系统/德国的F-查看）（凯泽光学系统公司，安阿伯/美国）。 3。控制功能的激光<ol><li>启动软件索利斯安道尔（安道尔/英国）和CCD相机设置温度-60°C至最大限度地减少相机的热感应电流所造成的噪音。 </li><li>校准过程硅片放置在显微镜阶段。 </li><li>关闭激光和设置功率85兆瓦。 </li><li>使用的软件单元B（奥林巴斯/德国）聚焦到晶圆的激光，直到出现的XY。 </li><li>以1秒的单一集成使用一个60X空中目标的时间测量硅片。 </li><li>改变从像素数安道尔索利斯软件的拉曼位移（厘米-1）x轴的单位。 </li><li>因在520厘米-1硅峰激光聚焦收集的频谱，以便找到这拉曼光谱的强度最大。计数的最低金额必须超过11,000高，有一个成功的校准。 </li></ol> 4。拉曼光谱测量所有测量均在室温下进行。 <ol><li>基本设置<ol><li>使用数值孔径为1.2 1 60倍水浸泡目标（奥林巴斯/德国），收集样本的频谱。 </li><li>改变收购设置集成/ 10秒10，共100秒，每测量。 </li></ol></li><li>贴壁细胞的测量<ol><li>采取与细胞玻璃底菜，放在显微镜舞台上。 </li><li>为了获得更好的信号，并确保可重复性，集中在细胞核的激光，把显微镜的光，并开始收集的频谱。 </li><li>衡量一个背景e的参考谱非常10光谱通过旁边的细胞移动激光焦点。重要的是要考虑改变焦点时，一个新的背景下，必须为每个重点深入收集。 </li></ol></li><li>细胞悬浮液中的测量<ol><li> 100μL细胞悬液转移到玻璃底菜放在显微镜舞台上。 </li><li>集中于细胞中心的激光，把显微镜的光，并开始收集的频谱。 </li><li>通过旁边的细胞移动激光聚焦的背景参考谱每10光谱测量。当改变焦点，一个新的背景下，必须收集新焦点深度。 </li></ol></li><li>本地组织的测量<ol><li>采取样本，并放入一个玻璃底部菜。面临底部的菜，应该是面向感兴趣区域（ROI）的。 </li><li>有足够的PBS填充盘覆盖样品。 </li><li>样品置于玻璃盖，以避免任何MOV在测量过程中样品ement。 </li><li>激光聚焦到利益格局的设置（深度分辨率是激光和组织依赖），并开始收集的光谱。 </li><li>收集整个组织区移动激光聚焦的背景参考谱每10光谱。当改变焦点，一个新的背景下，必须收集新焦点深度。 </li></ol></li><li>免疫荧光（IF）的测量标记的冰冻切片<ol><li>节新鲜，冷冻单元的组织样本，使用标准cryotom和二氧化硅涂层的盖玻片上安装。 </li><li>染色后，从业人员只有很短的固定步骤（最多10分钟，4％多聚甲醛）和使用适当的检测感兴趣的蛋白质抗体的常规协议的冰冻切片。 </li><li>执行拉曼光谱测量，荧光发生在该地区为重点。 </li></ol></li><li>弹性蛋白降解实验<ol><li>解放军CE ventricularis的解剖猪主动脉瓣叶面临着底部的玻璃底菜（富弹性，血液流入心脏瓣叶层侧）。 </li><li>作为"非孵化控制"，在整个组织表面的纤维状结构为重点的30个随机点测量的本地组织。 </li><li>分为3个部分样品放入他们单独充满弹性蛋白酶溶液（5 U / ml的，沃辛顿/德国）2毫升2.5毫升的Eppendorf管。 </li><li>孵育15或30分钟的组织，在37°C。 </li><li>潜伏期为15或30分钟后，取出从Eppendorf管组织，并仔细用PBS冲洗，以完全停止酶反应。 </li><li>测量每个样品在30个随机点，重点在纤维状结构。 </li></ol></li></ol> 5。数据处理和分析<ol><li>拉曼光谱处理 治疗前的产生的光谱进行的OPUS软件（布鲁克OPTIK公司/德国）。 <ol><li>为了减少玻璃和中等以及干扰信号，以避免在测量过程中重点的变化引起的变化，从收集到的光谱减去相应的背景光谱。 </li><li>减少光谱波数区域之间400-1800厘米-1，它提供的信息的最高金额。 </li><li>如果需要的话，正常化的光谱的最大峰值。出的强度波动和系统故障，简化了检测样品的光谱中的结构性变化，正常化的因素。 </li><li>执行基线校正，以增加不同的实验之间的可比性。 </li></ol></li><li>拉曼光谱的分析 使用PCA与理瓶机软件（迷彩/挪威），拉曼光谱进行了分析。这多元的分析检测范围内的光谱数据的异同套。每收集计数每拉曼位移为基础的多维空间中的单点谱绘制。每个主成分（PC）的描述一定数量的原始数据中的总信息。第一台PC是一个包含最高的变异来源。每个下面的PC，以包含，低于前一个信息。每个变量有每一台PC上的得分和装载。通过绘制电脑（=分数），可以接触重要的样本相关。负荷描述每个PCA的分析变量的贡献。 <ol><li>标记每个组通过创建每个样本组连续范围的测量。 </li><li>使用PCA的下列基本设置:NIPALS算法，交叉验证，无旋转，并开始分析。这些设置是光谱依赖。 </li><li>执行的PCA。 </li></ol></li></ol> 6。代表结果拉曼SP贴壁细胞产生ectra往往揭示了低信号噪声比和较低的整体信号强度（ 图1）。激光焦点附近的玻璃底，干扰的影响的事实，由于11玻璃信号是相当高，造成实际的采样信号屏蔽。因此，采样信号可能会被最小化甚至消除在随后的背景减法。因此，我们倾向于使用悬浮细胞拉曼光谱的分析，因为它们允许更详细的光谱信息检测。然而，贴壁和悬浮细胞的光谱表现出相同的只有在不同强度的主峰。 对于不同类型的细胞内悬挂的特性，没有预先处理是必需的。平均拉曼光谱和人成纤维细胞，间充质干细胞（MSCs），软骨细胞和角质细胞的悬浮液中测量的标准偏差图2描绘。所有拉曼光谱类似的结构，峰从典型的生物分子，如蛋白质，核酸和脂类（ 见表1）12。对于这些类型的细胞，谱区之间的600和1800厘米-1包含最相关的光谱信息，其中明显的差异可检测到不同类型的细胞（ 图2A）之间。模范，我们强调一个谱区（1280年至1350年厘米-1）显示明显的结构性差异，这是分配的胶原蛋白和脂类分子振动。相比之下，形态分析是不适合大多数细胞的识别和区分（ 图2B-Ⅰ）。虽然软骨细胞和皮肤细胞之间的差异是可观的（ 图2D，H对B，F和E，我 ），成纤维细胞和骨髓间充质干细胞是难以分开使用纯粹明亮的领域micrsocopy（ 图2B，相比，F，C，G）13。 本地组织，特别是对ECM蛋白，拉曼光谱分析的要求，投资回报率可以由明场成像的可视化，以便能够专注于各自的结构。对于一个特定的指纹图谱蛋白质的分配，我们产生了市售的纯蛋白质和immunohistologically染色冰冻切片的拉曼光谱。在这里，我们确定了指纹图谱的比较冻干弹性和免疫荧光染色冰冻切片，采用对弹性蛋白的抗体，在本地组织的弹性纤维。然而，由于弹性具有很高的自体荧光，这是反映在拉曼光谱，数据分析是具有挑战性（ 图3A）。由于特定的属性，如自体荧光，适当的处理的数据集的样本，以减少系统的故障是至关重要的。在我们的数据alyses，我们使用正常化消除纯弹性蛋白的显着更高的信号强度，因此，我们能够产生类似的拉曼光谱（ 图3B）。弹性是一个最稳定的ECM蛋白在体内，因此很难降解。在我们的实验设置14，我们诱导健康的猪主动脉瓣叶弹性退化执行酶解。运用多元分析，我们确定了拉曼光谱的酶处理样品和本地控制（ 图3C）之间的显着性差异。这些光谱差异，观察在861，1003和1664 cm -1处的载荷谱。预期中含有弹性蛋白纤维由于曝光时间延长到弹性蛋白酶的结构性变化显示哈特的染色（ 图4），这也反映在更独特的可分离得分集群（ 图3C）。 <img alt="图1" src="/files/ftp_upload/3977/3977fig1.jpg" /> 图1。平均拉曼光谱分离和贴壁的成纤维细胞。 <img alt="图2" src="/files/ftp_upload/3977/3977fig2.jpg" /> 图2（a）平均拉曼光谱和4个不同的主隔离类型的细胞（成纤维细胞，间充质干细胞，软骨细胞和角质）的标准偏差。框架强调谱区1280 - 1350厘米-1的结构性差异。 （BE）明场图像的分离（二）成纤维细胞（C），骨髓间充质干细胞（D）软骨细胞和（五）角质。比例尺等于20微米。 （FI）的明场图像壁（女），成纤维细胞，（G）的骨髓间充质干细胞，（高）的软骨细胞和（我）角质。比例尺等于200微米。 <img alt="图3" src="/files/ftp_upload/3977/3977fig3.jpg" /> （一）未经lyophili正常化的拉曼光谱图3。ZED弹性（蓝线），免疫荧光（IF）标记的冰冻切片（橙色线）和本地主动脉瓣叶内测量的弹性纤维（红线）。高的冻干弹性的信号强度是造成自体荧光。 （二）拉曼光谱正常化后，为了消除系统故障。 （三）成绩和负荷之间的非治疗对照组（红色）和酶降解（蓝色和绿色）在本地组织的弹性纤维比较。 <img alt="图4" src="/files/ftp_upload/3977/3977fig4.jpg" /> 图4。HART&#39;s染猪主动脉瓣叶。弹性纤维，是黑色的可视化。 （A）和（二）非治疗控制和（C）及（D）描绘组织样品暴露30分钟的弹性蛋白降解酶，弹性蛋白酶。 <table border="1"><tbody><tr><td> 在厘米-1波数 </strong&gt; </td><td colspan="2"> 转让12 </td></tr><tr><td> 717-719 </td><td>点数</td><td>磷脂</td></tr><tr><td> 785-788 </td><td> DNA / RNA的基地， OPO的骨干</td><td> DNA / RNA的</td></tr><tr><td> 1003 - 1005 </td><td>苯丙氨酸</td><td>蛋白质</td></tr><tr><td> 1220至1280年</td><td>酰胺三</td><td>蛋白质</td></tr><tr><td> 1445年至1447年</td><td>通道2 </td><td>蛋白质/油脂</td></tr><tr><td> 1655年至1680年</td><td>酰胺的IC = C的</td><td>蛋白脂质</td></tr></tbody></table> 表1。拉曼带内的所有类型的细胞（成纤维细胞，间充质干细胞，软骨细胞和角质）光谱检测。

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Biophotonics. 1, 451-462 (2008).</li></ol>

没有利益冲突的声明。

拉曼光谱是一个合适的工具来分析生物样本，如在体外培养的细胞和细胞外基质蛋白以及细胞内原生组织。11,15,16在这里，我们表明，这种非接触，无标签技术允许歧视不同类型的细胞和基质蛋白降解，完全基于对这些生物样本的内在生物分子组成的检测。 拉曼光谱的主要优点是，以非侵入性的量化的样品，其产生的拉曼光谱生化指纹的能力。拉曼光谱在红外光谱，产生类似的信息，可以收集水样，从水的拉曼散射是薄弱。此外，拉曼光谱只检测的单色光散射的基础上，因此没有来样加工，需要事先测量。这些属性使镭人谱了体内成像应用的潜力有前途的替代。在这方面，相干反斯托克斯拉曼光谱（CARS）是一个非常有趣的技术，因为它实现了更快和更敏感的收购在我们的实验中使用相同的振动信号为基础的数据。15,16其他的替代方法，包括多光子诱导自体荧光二次谐波成像，此前已被证明是适合非最小侵入性监测生物样品17。然而，这些成像成本很高，并仅限于自体荧光产生的分子。此外，拉曼光谱仪很容易结合，与传统的光学显微镜。这些特点使拉曼光谱研究在生理环境生物样品一个有价值的工具。 我们的拉曼光谱的电流限制设立相对较小的激光聚焦（250 nm的全宽半最高（FWHM）的横向和700 nm的半高宽轴向）是由高数值孔径的物镜（NA = 1.2）创建。虽然高数值孔径允许覆盖发出的拉曼光在高信号的信噪比产生了良好的数额，高NA产生只有一个小的集合内的样本，通常是比细胞小的焦点。为了比较不同细胞的拉曼光谱，收集有代表性的频谱是至关重要的，这是很难获得一个小的重点领域。为了解决这个问题，我们正在努力的过程自动化在细胞内的不同点（=拉曼光谱映射），在频谱平均和有代表性的频谱产生的信号采集。此外，这项技术将提供概述具体拉曼带的分布，细胞内蛋白质分布的实例。 生物学ogical样本是非常复杂和异构有助于收集的拉曼光谱的生物分子混合物组成。因此，频谱模式是非常复杂的一个单一类型分子在拉曼光谱监测是很难完成的，不同的分子信号重叠。此外，样品的固有荧光可能掩盖拉曼信号较弱的有价值的信息。有趣的是，在我们以前的一些研究中，我们发现在拉曼光谱的自体荧光区分细胞类型，使用适当的分析工具（间充质干细胞和成纤维细胞）的主要因素。13我们还发现，在整体的拉曼信号强度的变化可以作为ECM主动脉瓣叶内的胶原蛋白和胶原纤维的状态指示灯。9然而，分析这些组织的弹性状态时，我们不能够探测到类似的结果。正如前面提到的结果s节，我们只能够检测弹性蛋白酶处理过的样品的具体拉曼带改建时相比，本机的控制。我们没有看到一个整体如预期中的酶处理样品的拉曼信号的减少。这些观测结果中的得分没有透露在以往的研究中看到一个明确的集群形成的阴谋。9相反，酶处理的影响，在PCA结果检出。我们假设之间的两个ECM蛋白，弹性蛋白和胶原蛋白，这些差异的基础上的形态差异和不同的酶降解过程:在主动脉瓣叶，富含胶原蛋白的区（fibrosa）是一个连续的层变得松动由于酶处理，即弹性含有区（ventricularis）的暴露后弹性蛋白酶（ 图4）分散的网络配置出现。单点测量，因此不拨款吃这样的小破裂，检测范围内的弹性网络。在这里，一个组织的拉曼光谱，将有助于识别网络故障。 生物样品的拉曼光谱中的一个进一步的挑战是减少测量时间。解决办法之一是增加激光功率，这是适合生物样品只要不影响光损伤。我们目前的实验研究证明的原则，侧重于基础研究;然而，我们的总体目标是实现临床应用，包括再生医学（如组织工程产品的质量控制），移植前移植监测和拉曼光谱癌症诊断。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>弹性蛋白酶</td><td>沃辛顿</td><td> LS006363 </td><td></td></tr><tr><td>抗弹性蛋白抗体</td><td>西格玛</td><td> HPA018111 </td><td> 1:75;柠檬酸缓冲</td></tr><tr><td> PBS </td><td>龙沙</td><td> 17-512F </td><td></td></tr><tr><td>玻璃底菜</td><td>格雷纳BioOne </td><td> 627860 </td><td></td></tr><tr><td>该理瓶机</td><td>迷彩</td><td></td><td></td></tr><tr><td>电视剧</td><td>布鲁克</td><td></td><td></td></tr></tbody></table>

non-contact, label-free monitoring of cells and extracellular matrix using raman spectroscopy

监察细胞和组织培养的非破坏性，非接触式和自由标签技术在生物医学研究领域的需要。1-5，但目前可用常规方法需要处理的步骤和改变样品的完整性。拉曼光谱仪是一种快速的方法，使测量生物样品，而不需要作进一步的处理步骤。这种基于激光技术检测非弹性散射的单色光，由于每个化学振动被分配到一个特定的拉曼光谱（单位:cm -1波数），每一个生物样品具有典型的光谱模式，由于其固有的生化组成7。 -拉曼光谱范围内，峰值强度相关金额与目前的分子键。1光谱数据集的异同和分歧，可以采用多变量分析（如主成分分析（PCA））检测10。 </suP&gt; 在这里，我们执行的活细胞和本地组织的拉曼光谱。细胞接种于玻璃底菜是悬浮液中保持正常的细胞培养条件下（37℃，5％CO 2）测量前。土著组织解剖和磷酸盐存储在4℃事先测量缓冲液（PBS）。根据我们的实验装置，我们则侧重于细胞核或细胞外基质（ECM），如弹性蛋白和胶原蛋白。对于所有的研究中，最小的30格或30随机点在ECM内的利益衡量。数据处理步骤包括背景减法和正常化。

Watch this Scientific Journal Video about Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy at JoVE.com

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

拉曼光谱是一种适用于非接触式技术，无标记的活细胞，组织工程的结构和本地组织的分析。源特定的光谱指纹可以生成和分析，采用多元分析。

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

non-contact-label-free-monitoring-cells-extracellular-matrix-using

Research

JoVE Journal

Bioengineering

16.9K 次观看.  Eberhard Karls University, Tübingen. 拉曼光谱是一种适用于非接触式技术，无标记的活细胞，组织工程的结构和本地组织的分析。源特定的光谱指纹可以生成和分析，采用多元分析。

视频: Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

细胞外基质刚性三维肿瘤模型的纵向测量用粒子跟踪Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

科研

生物工程

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

无标记技术的单细胞分泌物的时空成像

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

无标记单分子检测使用微型环光学谐振器

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

细胞外基质蛋白纤维的布里渊谱的制备

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

PIP-on-a-chip:蛋白质 - 磷酸肌醇相互作用的无标记研究

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

肾皮质细胞外基质衍生水凝胶的制备

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used method, but has the drawback that it requires fluorescent labeling, which can interfere with the activity of the molecules. Also, photobleaching and photodamage are concerns. In the case of microtubules, we have found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM). This suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro. In this paper, we show how a fluorescence microscope can be modified simply to obtain IRM images. IRM is easier and considerably cheaper to implement than other contrast techniques such as differential interference contrast microcopy or interferometric scattering microscopy. It is also less susceptible to surface defects and solution impurities than darkfield microscopy. Using IRM, together with the image analysis software described in this paper, the field of view and the frame rate is limited only by the camera; with a sCMOS camera and&#160;wide-field&#160;illumination&#160;microtubule length can be measured with precision up to 20 nm with a bandwidth of 10 Hz.&#160;

This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays.

实现干扰反射显微镜,实现无标签、高速微管成像

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...