作者要感谢加拿大基因组和基因组不列颠哥伦比亚省为平台的资金和支持。我们也感谢博士卡罗尔E.帕克的稿件和编辑援助的严格审查。 CHL也感谢不列​​颠哥伦比亚省蛋白质组学网络支持。

1。组织切片<ol><li>闪光冻结问题的标本，一次采收，使用液氮，把它们运干冰上（如需要运费），并将其存储于-80℃直至组织切片。 （如果商业样品的使用，保证了样品制备以这种方式。） </li><li>剪下机关管理的大小以适合MALDI靶。修剪过的器官任何不需要的部分。在这项研究中这里所描述，小牛镜头采用组织切片之前，先前描述的方法35解封装。 </li><li>从-80℃冰箱中取出整个器官和解决这些问题上的低温组织切割阶段。要修复与小牛的镜头，放置在低温恒温器的组织切段一两滴的水。迅速将透镜在水中凝固之前。可选地，最佳切割温度（OCT）化合物也可用于修复组织到切割阶段。如果OCT化合物时，微型OCT的正常量应该被应用，并应小心，以确保切的组织切片不会与十月化合物可与电离和检测分析物5,36,37的干扰污染。 </li><li>使低温恒温器内的温度平衡至-18℃。寒冷或温暖的温度，可用于较软或较硬的组织，分别为。然后切组织平展到20微米厚的切片。用10-15微米厚的薄片对于大多数组织;，因为牛晶状体组织的脆弱性，然而，20微米厚的切片使用。牛镜头纸，舍弃前组织切片，并只用切片，其中接近或在赤道平面。 </li><li>如果目镜组织进行拍摄，使用1.5微升甲酸（98％的纯度，LC-MS级），以预湿的铟锡氧化物（ITO）涂覆的玻璃载片的表面上。 </li><li>组织切片小心转移至ITO的共同的表面低温恒温器内ATED玻璃显微镜幻灯片。组织切片将迅速解冻，将成为紧紧地贴在滑动面。通常，多个组织切片可以安装到这样一个相同的ITO膜的滑动。 </li><li>冻干幻灯片的MALDI基质申请前15分钟。 </li><li>对于矩阵测试，溶于适当溶剂中的各个矩阵。手动发现1微升每个矩阵解决方案到组织切片。此外，发现一个小分子校准标准到组织核实MALDI灵敏度。 </li><li>加3教学马克在ITO涂覆的玻璃载片的记录对与校正液笔在ITO涂覆的玻璃载片上的不导电的表面上。采取使用平板扫描仪的组织切片的光学图像并将其保存在一个合适的格式，如TIFF或JPG格式。 </li></ol> 2。矩阵涂层 2.1。自动化矩阵涂层<ol><li>美联社帘布层基质溶液，其中含有乙腈或混合的乙腈/水作为溶剂，自动使用Bruker的ImagePrep或类似的电子矩阵喷雾器中的组织切片的表面。 </li><li>用胶带贴在带ITO膜的玻璃载片的前表面的边缘，以使基质不涂层的滑动的边缘。这确保了可用于组织切片对准的相反表面上的教学痕。不覆盖矩阵滑动的边缘，因为它们被用​​作接触点维持ITO涂覆载片的导电性。 </li><li>涂层通过使用矩阵涂层（2 - 仲丁基喷雾，30秒温育，和60秒的干燥时间为每个周期）的20个循环的载玻片上。 </li></ol> 2.2。人工基质涂层<ol><li>如果有机溶剂（ 例如氯仿和乙酸乙酯），它们与电子矩阵喷雾器的制造材料是不兼容的，地磁红色，使用一个气动辅助喷枪喷涂应用矩阵。所制备的基质溶液加入到喷枪枪的溶剂储存并应用加压氮气水流平缓，以最优惠的喷雾。 </li><li>覆盖ITO涂布的载玻片用磁带的前表面的边缘，以使基质不涂层的滑动件的边缘。这确保了可用于组织切片对准的相反表面上的教学痕。不覆盖矩阵滑动的边缘，因为它们被用​​作接触点维持ITO涂覆载片的导电性。 </li><li>经过稳定和精细喷雾已经观察到，手动喷矩阵，使其完全覆盖在组织切片。应用的每个周期期间，以勉强润湿表面，以防止可能的分析物的离域所需的基质溶液的最小量。在一般情况下，使用约10个循环矩阵喷雾涂覆的组织切片;周期数是依赖于组织类型和基质组合物。 </li></ol> 3。 MALDI质谱<ol><li>水（含0.1％甲酸的最终混合物）:通过稀释"ES调谐混合"标准由1:200的因子在60:40异丙醇溶液中制备一个质量校准溶液。 </li><li>引入2微升/稀释的"ES调谐混合"溶液到双模式电喷雾电离（ESI）/ MALDI离子源上的FTICR质谱计，从ESI侧最小。 </li><li>操作的FTICR仪器中的正离子ESI模式，随着宽带检测和1024字节/秒的一个数据采集的大小。典型ESI参数是毛细电电压，3900伏;喷屏蔽电压，3600伏;雾化气体（N 2）流量，2升/分钟;干气（N 2）流量和温度，4升/分钟和200℃;撇油器1电压，15伏;的飞行（TOF）的时间，0.009秒;碰撞气（Ar）流量0.4升/秒;源离子累积时间，0.1秒;和碰撞池的离子累积时间，0.2秒。调，以便最大限度地在质量范围从M / Z 200至1400的分析灵敏度，同时保持良好的时域的自由感应衰减（FID）信号的FTICR操作的参数。通常情况下，ICR操作参数搭档电压8伏; Sidekick的失调电压，8 V，10励磁放大;激励脉冲时间，0.01-0.015秒;前面的活板电压，1.5伏;回来的活板电压，1.6 V和分析器入口电压，-4 V后一组FTICR运行参数已经确定，收购ESI质谱和使用参考群众的标准化合物中的"ES调音混音"的解决方案校准仪器。 </li><li>要调整仪器用于MALDI操作，溶解混合特非那定和利血平标准溶液中在每个1微米的浓度基质溶液的几个1微升等份，并发现这些解决方案直接到布甲之一Ë部分（ 即一个测试组织切片），已安装在涂有ITO的幻灯片。将ITO涂层滑入一个组织切片转接器（ 即一种特殊的MALDI靶），并从电离侧加载适配器插入双ESI / MALDI离子源。优化激光功率和激光发射的数目为每个质量扫描MALDI信号积累等适当的MALDI操作参数典型的MALDI操作参数包括:激光枪，50和300 V. MALDI板上电压</li><li>调谐后，校准和优化工具用于MALDI-MSI的实验中，使组织切片与成像软件中的记录的光学图像进行成像的物理位置。使用三点三角测量方法使用三个"校正流体"标记，这先前已经提上了ITO膜的滑动面的相反侧（步骤1.9），此对齐。 </li><li>执行同步ESI和MALDI操作，使每个质谱图包含了收购后的内部质量校准的"ES调音混合"解决方案的参考质量峰。这将导致在MALDI MS的最准确的质量测定。要做到这一点，首先通过降低毛细管电压，直到电离信号主宰谱，而ESI校准信号仍然不够高，内部质量校准衰减ESI信号。 </li><li>接下来，建立一个自动化的光栅扫描方法，激光照射。定义组织区域进行成像并设置适当的激光光栅步长。需要注意的是较小的栅格步长提供更高的分辨率组织图像，但需要一个较长的显著质谱采集时间和更多的数据存储空间。该图像的像素的数目是依赖于激光光栅的步长来设置和组织的大小。对于一个典型的牛晶状体具有1cm 2的面积的组织中，组织图像通常由约的。 </em&gt;的，如果为200微米的激光光栅的步长是用在一个FTICR仪器5000像素。使用一个"随机点"的分析，因为这可以防止基于位置的偏差是由于渐进信号的衰减在实验过程中。 </li></ol> 4。数据分析<ol><li>使用内部校准的初始比较来校准MALDI质谱和选择的MS / MS峰。德同位素，并选择如前所述的单一同位素峰，使用自定义的VBA脚本38。 </li><li>导出所得到的单一同位素峰列表和输入测量的m / z值到METLIN 39和/或用于质量匹配的库条目的HMDB 40代谢组数据库。在数据库检索数据考虑（M + H）+，（M + Na）的+和（M + K）+离子，具有±1ppm的可允许的误差质量。 </li><li>生成图像的MALDI所有在用IMAG整个组织切片检测脂质实体电子分析软件，具有1 ppm在峰顶一个质量过滤器的宽度。 </li><li>一旦图像已生成匹配数据库中的条目，所有m / z值，生成图像的所有其他峰，以及寻找可以在以后调查独特的分布格局。 </li></ol> 5。成像的脂质中的身份确认<ol><li>确认的高丰度的脂质，其具有可以利用的FTICR仪器（ 如 184.073为磷脂）进行检测，通过MALDI-MS/MS特征碎片离子的身份。使用碰撞诱导解离（CID）直接对组织进行MALDI-MS/MS。 </li><li>对于不能由MALDI-MS/MS直接证实了这些脂质物种中，使用耦合到Q-TOF质谱仪34 UPLC系统。 <ol><li>手动解剖含有〜10从其中所关注的物种是局部的区域毫克组织的等分试样。把这些组织等分成2毫升离心管中。 </li><li>均质组织各等分在250微升的水，用搅拌机磨用两个5毫米的不锈钢金属球。 </li><li>加入1毫升氯仿 - 甲醇溶液（1:3，V / V），和涡流管。接着，离心分离用离心，12,000 xg离心10分钟，在试管中。 </li><li>收集上清液并擦干他们在一个转速真空浓缩。 </li><li>溶解残留物中加入100μl30:70异丙醇:水。注入10微升等分到UPLC色谱柱使用梯度洗脱分离。 </li><li>使用的色谱条件为在线脂质LC-MS/MS，先前已公布的34。 </li><li>产生提取的离子电流（XIC）色谱图使用的理论m / z值，为±百万分之50左右的理论质量的一个窗口。 </li><li>如果可用正宗的化合物对于那些血脂，搭配地道的化合物的保留时间与那些对应的从组织样本对应XIC峰。如果化合物是相同的，保留时间和MS / MS谱图应该匹配。 </li></ol></li><li>如果一个地道的化合物是不可用的，使用检测脂质碎片模式匹配从代谢组数据库，如METLIN或HMDB标准的MS / MS谱。用从头质谱解释，确定一个可能的结构脂质。 </li></ol>

我们什么都没有透露。

最重要的考虑因素，成功MALDI微星是:1）组织准备; 2）基质的选择; 3）矩阵的应用，以及4）数据的解释和分析。当样品和基质被适当地制备时，MS数据采集是自动化的。从这种类型的实验的数据分析是相当费力。 适当的组织准备是成功的MALDI微星实验的关键。组织的来源和处理可以对最终的分析有很大的影响。样品必须立刻闪在液氮中冷冻并储存在-80℃，并且它们不应被存储?...

质谱成像（MSI）是一种分析技术，用于确定组织部分1,2的表面上的化合物的空间定位和分布模式。基质辅助激光解吸/电离（MALDI）微星多肽和蛋白质的分析已经使用了十多年，也有过在样品制备，检测灵敏度，空间分辨率，可重复性和数据处理方法3,4很大的改进。从组织学染色切片和MSI实验相结合的信息，病理学家能够具体化合物的分布与病理生理学有趣的功能5相关。 小分子，包括外源性药物6,7及其代谢物8-10的分布格局也被审问MALDI-MS组织成像11。脂类也许是最广泛研究共轭亚油酸SS与MALDI成像化合物，无论是在12-17的MS和MS / MS 18模式。为小分子成像中使用的MALDI MSI的已经被限制由几个因素:1）的MALDI基质本身是小分子（通常为M / Z &lt;500），其产生大量的离子信号。这些丰富的信号可以抑制小分子分析物的电离，干扰他们的检测19,20。无溶剂基质涂料21，矩阵升华22，以及矩阵预涂MALDI MS 23，除其他外，已经开发了改进的小分子的MSI。 新的矩阵，可以提高低分子量化合物的分析是在小分子微星极大的兴趣。这些矩阵应提供增加的信号分析与降低矩阵信号。在正离子模式下，2,5 -二羟基苯甲酸（DHB）和α-氰基-4 -羟基肉桂酸（CHCA）是两种常用的MALDI MS矩阵MSI 24 </SUP&gt;。理想的基质会形成小晶体，从而保留分析物的空间定位。 DHB容易形成较大的晶体，因此施加利用升华的基质已经开发出来，部分地克服这一问题，并允许使用该矩阵的对磷脂22,25的敏感成像。 9 -氨基吖啶已经用于质子分析物的MSI在正离子模式26和用于核苷酸和磷脂在负离子模式26-29。 2 -巯基苯并已发现，得到高效的MALDI检测脂类30的，并已被用于小鼠脑的成像神经节苷脂31。傅立叶变换的超分辨率变换离子回旋共振（FTICR）质谱仪可以通过从矩阵信号32解决分析物的信号有所缓解这个问题。利用FTICR-MS法的另一个优点是，亚稳的矩阵簇的强度是红眼版33，这也降低这些干扰27。 使用地蒽酚的（DT; 1,8 -二羟基-9,10 -二氢蒽-9 -酮）作为MALDI基质用于组织成像先前已报道34。在当前的工作中，一个详细的协议提供了一种用于对牛晶状体组织切片的表面上使用DT的内源性脂质的MSI，在正离子模式。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Rat Liver</td>
			<td>Pel-Freez Biologicals</td>
			<td>56023-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Calf Lens</td>
			<td>Pel-Freez Biologicals</td>
			<td>57114-2</td>
			<td>Sample should be decapsulated29 before use</td>
		</tr>
		<tr>
			<td>Dithranol (DT)</td>
			<td>Sigma-Aldrich</td>
			<td>10608</td>
			<td>MALDI Matrix</td>
		</tr>
		<tr>
			<td>&#945;-Cyano-4-hydroxy-cinnamic Acid (CHCA)</td>
			<td>Sigma-Aldrich</td>
			<td>70990</td>
			<td>MALDI Matrix</td>
		</tr>
		<tr>
			<td>2,5-Dihydroxybenzoic Acid (DHB)</td>
			<td>Sigma-Aldrich</td>
			<td>85707</td>
			<td>MALDI Matrix</td>
		</tr>
		<tr>
			<td>Reserpine</td>
			<td>Sigma-Aldrich</td>
			<td>83580</td>
			<td></td>
		</tr>
		<tr>
			<td>Terfenadine</td>
			<td>Sigma-Aldrich</td>
			<td>T9652</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>14265</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Formate</td>
			<td>Sigma-Aldrich</td>
			<td>14266</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>320145</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic Acid (TFA)</td>
			<td>Sigma-Aldrich</td>
			<td>302031</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma-Aldrich</td>
			<td>39253</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>34967</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>34972</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>34965</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>366927</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>34850</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Commercial Alcohols</td>
			<td></td>
			<td>95%</td>
		</tr>
		<tr>
			<td>ES Tuning Mix</td>
			<td>Agilent Technologies</td>
			<td>G2431A</td>
			<td></td>
		</tr>
		<tr>
			<td>ITO Coated Glass Slides</td>
			<td>Hudson Surface Technology</td>
			<td>PSI1207000</td>
			<td>Ensure that samples are placed on the electrically conductive side</td>
		</tr>
		<tr>
			<td>Wite-Out Shake-N-Squeeze Correction Pen</td>
			<td>Bic</td>
			<td>WOSQP11</td>
			<td></td>
		</tr>
		<tr>
			<td>Airbrush Sprayer</td>
			<td>Iwata</td>
			<td>Eclipse HP-CS</td>
			<td></td>
		</tr>
		<tr>
			<td>ImagePrep</td>
			<td>Bruker</td>
			<td>249500-LS</td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI adapter</td>
			<td>Bruker</td>
			<td>235380</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dithranol as a matrix for matrix assisted laser desorption/ionization imaging on a fourier transform ion cyclotron resonance mass spectrometer

质谱成像（MSI）确定的组织切片的表面上的化合物的空间定位和分布模式，主要使用MALDI（基质辅助激光解吸/电离）为基础的分析技术。需要新的矩阵为小分子的MSI，从而可以提高低分子量（MW）的化合物的分析。这些矩阵应提供增加分析物的信号，同时降低MALDI背景信号。另外，使用超高清晰度的仪器，如傅立叶变换离子回旋共振（FTICR）质谱仪，具有从矩阵信号解析分析物信号的能力，这可以部分地克服与来自MALDI背景始发相关的许多问题矩阵。在亚稳态矩阵集群由FTICR MS的强度的降低也有助于克服一些与其他乐器基质峰相关联的干扰。高分辨率文书，如FTICR质谱仪是有利的，因为他们可以同时产生许多化合物的分布模式，同时还提供信心的化学鉴定。蒽三酚（DT; 1,8 - 二羟基-9,10 - 二氢蒽-9 - 酮）先前已被报道作为MALDI基质用于组织成像。在这项工作中，一个协议的使用DT的用于MALDI成像从哺乳动物组织切片表面的内源性脂的，由正离子的MALDI-MS，对超高清晰度混合四极FTICR仪器已经提供。

<html>被切片，解冻安装在ITO镀膜载玻片组织样本应完好，无明显的撕裂。对于许多组织，直接组织解冻安装到的ITO镀膜玻璃幻灯片是可以接受的。对于一些特定的组织如牛晶状体中，组织的广泛撕裂常常看到时直接解冻安装时（ 图1a）。用乙醇或甲酸在ITO玻璃载玻片的预涂（ 图1b）有助于保持组织切片的组织安装时的完整性。 矩阵两者的选择和溶剂?...

Watch this Scientific Journal Video about Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer at JoVE.com

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

蒽三酚（DT; 1,8 - 二羟基-9,10 - 二氢蒽-9 - 酮）先前已被报道作为MALDI基质为小分子的组织成像;对于使用DT的内源性脂质的对的MALDI成像协议这里所提供的组织切片由正离子的MALDI-MS在一个超高分辨率四极-FTICR仪器表面。

地蒽酚作为傅立叶的基质辅助激光解吸/离子化成像矩阵变换离子回旋共振质谱仪

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

dithranol-as-matrix-for-matrix-assisted-laser-desorptionionization

Research

JoVE Journal

Biology

14.1K 次观看.  University of Victoria. 蒽三酚（DT; 1,8 - 二羟基-9,10 - 二氢蒽-9 - 酮）先前已被报道作为MALDI基质为小分子的组织成像;对于使用DT的内源性脂质的对的MALDI成像协议这里所提供的组织切片由正离子的MALDI-MS在一个超高分辨率四极-FTICR仪器表面。 

视频: Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

使用丙烯酰胺水凝胶的细胞功能的研究矩阵刚度的影响

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Quantifying Mixing using Magnetic Resonance Imaging

Mixing is a unit operation that combines two or more components into a homogeneous mixture. This work involves mixing two viscous liquid streams using an in-line static mixer. The mixer is a split-and-recombine design that employs shear and extensional flow to increase the interfacial contact between the components. A prototype split-and-recombine (SAR) mixer was constructed by aligning a series of thin laser-cut Poly (methyl methacrylate) (PMMA) plates held in place in a PVC pipe. Mixing in this device is illustrated in the photograph in Fig. 1. Red dye was added to a portion of the test fluid and used as the minor component being mixed into the major (undyed) component. At the inlet of the mixer, the injected layer of tracer fluid is split into two layers as it flows through the mixing section. On each subsequent mixing section, the number of horizontal layers is duplicated. Ultimately, the single stream of dye is uniformly dispersed throughout the cross section of the device.
 Using a non-Newtonian test fluid of 0.2% Carbopol and a doped tracer fluid of similar composition, mixing in the unit is visualized using magnetic resonance imaging (MRI). MRI is a very powerful experimental probe of molecular chemical and physical environment as well as sample structure on the length scales from microns to centimeters. This sensitivity has resulted in broad application of these techniques to characterize physical, chemical and/or biological properties of materials ranging from humans to foods to porous media 1, 2. The equipment and conditions used here are suitable for imaging liquids containing substantial amounts of NMR mobile 1H such as ordinary water and organic liquids including oils. Traditionally MRI has utilized super conducting magnets which are not suitable for industrial environments and not portable within a laboratory (Fig. 2). Recent advances in magnet technology have permitted the construction of large volume industrially compatible magnets suitable for imaging process flows. Here, MRI provides spatially resolved component concentrations at different axial locations during the mixing process. This work documents real-time mixing of highly viscous fluids via distributive mixing with an application to personal care products.

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

量化混合使用磁共振成像

Mixing is a unit operation that combines two or more components into a homogeneous mixture.  This work involves mixing two viscous liquid streams using an ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

提高蛋白质结晶的成功率随机Microseed矩阵筛选

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

从富集组织及消化细胞外基质蛋白的成肽质谱分析

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

激光辅助显微切割（LAM）作为个体细胞类型的转录谱的工具

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become available for biologists. These systems are primarily designed for multi-channel two-photon fluorescence microscopy. However, without any modification, complementary non-linear optical microscopy such as second-harmonic generation (SHG) or third harmonic generation (THG) can also be performed with this set-up, allowing label-free imaging of structured molecules or aqueous medium-lipid interfaces. These techniques are well suited for in-vivo observation, but are limited in chemical specificity. Chemically selective imaging can be obtained from inherent vibration signals based on Raman scattering. Confocal Raman microscopy provides 3D spatial resolution, but it requires high average power and long acquisition time. To overcome these difficulties, recent advances in laser technology have permitted the development of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS). CARS microscopy has therefore emerged as a powerful tool for biological and live cell imaging, by chemically mapping lipids (via C-H stretch vibration), water (via O-H stretch vibrations), proteins or DNA. In this work, we describe the implementation of the CARS technique on a standard OPO-coupled multiphoton laser scanning microscope. It is based on the in-time synchronization of the two laser lines by adjusting the length of one of the laser beam path. We present a step-by-step implementation of this technique on an existing multiphoton system. A basic background in experimental optics is helpful and the presented system does not require expensive supplementary equipment. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodent, and we show that this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.

Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser.

蓝宝石OPO基于激光的标准激光扫描显微镜:在TI相干反斯托克斯拉曼散射（CARS）系统的实现

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.